Studies on the Probiotic, Adhesion and Induction Properties of Artisanal Lactic Acid Bacteria: To Customize a Gastrointestinal Niche to Trigger Anti-Obesity Functions

Studies on the Probiotic, Adhesion and Induction Properties of Artisanal Lactic Acid Bacteria: To Customize a Gastrointestinal Niche to Trigger Anti-Obesity Functions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Studies on the Probiotic, Adhesion and Induction Properties of Artisanal Lactic Acid Bacteria: To Customize a Gastrointestinal Niche to Trigger Anti-Obesity Functions Aslihan Kamber, Cisem Bulut Albayrak, Hayriye Sebnem Harsa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535093/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 9 You are reading this latest preprint version Abstract Aims: The primary goals of this work are to explore the potential of probiotic LAB's mucin/mucus layer thickening properties and to identify anti-obesity candidate strains that improve appropriate habitat for use with the Akkermansia group population in the future. Methods and Results: The HT-29 cell binding, antimicrobial properties, adhesion to the mucin/mucus layer, growth in the presence of mucin, stability during in vitro gastrointestinal (GI) conditions, biofilm formation, and mucin/mucus thickness increment abilities were all assessed for artisanal LAB strains. Sixteen LAB strains out of 40 were chosen for further analysis based on their ability to withstand GI conditions. Thirteen strains maintained their vitality in the simulated intestinal fluid, whereas the majority of the strains displayed high viability in the gastric juice simulation. Furthermore, 35.2-65.4% of those 16 bacteria adhered to the mucin layer. Besides, different lactate levels were produced, and Streptococcus thermophilus UIN9 exhibited the highest biofilm development. Also, with a 50 mM lactate concentration and the presence of mucin that had been injected originally, Lacticaseibacillus casei NRRL-B 441 had the maximum amount of mucin (321.6 µg/mL). Conclusions: Two isolates of olive bacteria from Lactiplantibacillus plantarum were chosen as the anti-obesity prototype candidates; these strains did not consume mucin sugars. Significance and Impact of the Study : Probiotic LAB's attachment to the colonic mucosa and its ability to stimulate HT-29 cells to secrete mucus are critical mechanisms that may support the development of Akkermansia. Lactic acid bacteria HT-29 cell culture mucin/mucus layer anti-obesity probiotic Akkermansia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction According to the World Health Organization [ 1 ], obesity is one of the major global health issues and has become an epidemic. Because obesity is strongly correlated with several chronic illnesses, including diabetes, osteoarthritis, cardiovascular disease, liver disease, and several types of cancer, it is a severe health concern [ 2 , 3 ]. Over the past 50 years, the global obesity epidemic has grown; in 1975, the typical adult's obesity rate was three times higher than it is today [ 4 ]. Around 650 million people have been impacted globally, and 13% of the world's population now considers it to be a serious issue [ 5 ]. If the global obesity data continues to show upward trends by 2030, 20% of adult people worldwide are expected to be obese [ 6 ]. As a result, methods to lessen the obesity issue are becoming more and more crucial. It has recently been shown that the gut microbiota can affect weight gain or reduction in a variety of ways [ 7 ] and that "modulation of the microbiota" [ 6 , 8 ] can help combat obesity [ 9 ]. Studies on humans and animals have demonstrated a correlation between obesity and a high relative ratio of bacteria (Firmicutes (F)) to bacteria (Bacteroidetes (B), Actinobacteria, and Proteobacteria) (F/B) [ 6 , 10 – 13 ]. The original sample of A. muciniphila was firstly obtained from a healthy patient [ 14 ]. According to Kim et al. [ 15 ], there is an inverse correlation between the number of Akkermansia muciniphila in the GIT and several disease states, such as obesity and associated metabolic issues. Since its initial isolation from the stool of a healthy patient in 2004, this bacterium has been recognized as an efficient combatant against obesity [ 16 – 18 ]. Over the past fifteen years, research has focused on the roles that A. muciniphila , a member of the next-generation of probiotics (NGP) class [ 19 – 21 ], plays in the gastrointestinal system [ 17 , 22 , 23 ]. These roles include lowering insulin resistance, regulating intestinal immunity, enhancing intestinal barrier function, and reducing obesity, diabetes, and inflammation [ 24 – 27 ]. A. muciniphila is a common member of the gastrointestinal tract (GIT) and a Gram-negative, non-sporulating, strict anaerobic bacterium that belongs to the phylum Verrucomicrobia [ 28 – 31 ]. It is an obligatory chemoorganotroph that breaks down mucin and uses mucus as its only source of energy, carbon, and nitrogen. It can produce propionate, acetate, succinate, ethanol, and 1,2-propanediol in smaller amounts by using these [ 14 , 24 , 32 ]. Other good bacteria can improve mucus structure, which in turn can stimulate the population of Akkermansia in the intestinal area. For its spread, several probiotic LAB can encourage the environment's mucin/mucus thickening. The two most prevalent probiotic strains, Lactobacillus and Bifidobacterium , adhere to the intestinal mucosa and competitively eliminate pathogens [ 33 – 37 ]. Probiotic LAB's mucin-promoting qualities, for example, may be correlated with the population of Akkermansia . In this regard, an in vitro intestinal model system was used to investigate the impact of probiotic mixtures on A. muciniphila [ 38 ]. The results demonstrated that probiotic supplementation led to a gradual increase in the colon's short-chain fatty acid (SCFA) concentration and encouraged the thickening of mucin. It was mentioned that A. muciniphila , which breaks down mucin, develops more readily in such an environment. As a result, methods that promote a mucin/mucus environment have gained importance for maintaining intestinal health, managing the Akkermansia population, and reducing obesity. Mucus plays a role in defense, conservation, and partner-host homeostasis. Bacteria that encourage the production of mucus strengthen the immune system because inflammatory diseases are caused by a decrease in mucus thickness. According to Devi and Halami [ 39 ], goblet cells facilitate the dynamic process of mucus formation in the gut, which is then maintained by transcription factors like KLF4. The primary constituents of epithelial mucus layers are mucins, which are extracellular, high molecular weight glycoproteins (0.5–20 MDa) [ 40 ]. Because it is made up of oligosaccharides and amino acids, mucin is utilized as a food source by gut bacteria. Mucins and microflora interact in ways that are crucial to normal function. Numerous diseases involve modifications to mucins, which may result from either excessive mucin peptides or glycosylation [ 41 ]. Thus, two crucial mechanisms that aid in the development of Akkermansia are the adhesion of probiotic LAB to the mucosa in the colon and the stimulation of mucus secretion by intestinal epithelial cells (HT-29 cells). In addition, some bacteria, such as S. thermophilus , which have poor adhesion to mucus, produce lactate by breaking down lactose and thus support the mucus pathway, regulate the mucus environment, stimulate the mucus layer, and may also be a signal that can modulate the colonic epithelium [ 42 , 43 ]. Additionally, lactate's antimicrobial property helps to fight pathogens in the intestine [ 44 ] and maintain Akkermansia 's improved mucin/mucus environment. Furthermore, lactate has a multitude of advantageous effects that include nourishing gut microbial communities, reducing inflammatory processes, and fortifying the gut barrier by producing mucus [ 42 , 45 ]. For example, Nishiyama et al. [ 46 ] found that elevated lactate levels were linked to an increase in lactobacilli in the intestine and that the amount of Akkermansia was positively impacted by the increasing lactate concentration. According to research on epithelial cell cultures, probiotic LAB can help form mucin and mucus by adhering to the colon's mucosa. In vitro experiments are conducted to investigate this phenomenon using HT-29 cells [ 47 – 51 ]. In accordance with numerous studies [ 52 – 54 ], lactobacilli stimulate mucin expression and secretion. According to Dudik et al. [ 55 ], pre-incubating lactobacilli with mucin increases their adhesion to HT-29 cells in vitro , which may improve their bioavailability in the gut during in vivo conditions. In the majority of these studies, probiotic LAB strains (e.g., L. plantarum 299v, L. rhamnosus GG, and L. acidophilus A4) have been shown to enhance the expression of MUC2 and MUC3 genes (mucin) in HT-29 colon cell cultures, thereby potentially supporting barrier function and inhibiting pathogens. While there are a few studies that demonstrate the mucin/mucus-promoting abilities of probiotic LAB [ 54 ], our study may be the first to look into the possibility of probiotics supporting the population of Akkermansia by analyzing lactate production, mucus thickening properties, antimicrobial effect, and other factors to suggest probiotic candidates that may be anti-obesity. The majority of Lactobacillus species, including strains of L. plantarum , L. rhamnosus , L. casei , L. acidophilus , L. reuteri , L. paracasei , L. lactis , L. brevis , and L. delb. ssp. bulgaricus , have an anti-obesity effect, even though some of these species are linked to weight gain [ 56 – 59 ]. Probiotic and/or symbiotic preparations promoting the Akkermansia group can be developed to support the microbiota that fights obesity. The primary goal of this work is to characterize the resistance to gastrointestinal conditions, mucin degradation abilities, adhesion properties, mucin/mucus thickening role, and other aspects of the Akkermansia population to investigate the anti-obesity probiotic potential of LAB isolated from food environments. The underlying premise of this study was that probiotic-containing LAB could alter the microbiota or the environment in which microorganisms of the Akkermansia group grow. 2. Materials and methods Materials The experiments used a LAB containing forty strains, including isolated and reference strains. The ARS Culture Collection (NRRL, USA) provided reference strains of Lactiplantibacillus plantarum NRRL-B 4496, Lactobacillus acidophilus NRRL-B 1910, Lacticaseibacillus casei NRRL-B 441, Lacticaseibacillus rhamnosus NRRL-B 442, and Lactiplantibacillus pentosus NRRL-B 227. The German Collection of Microorganisms and Cell Cultures GmbH (Germany) and the Czech Collection of Microorganisms (CCM) provided Lactiplantibacillus plantarum DSM 1954 and Streptococcus thermophilus CCM 4757, respectively. According to their published reports, reference strain species were chosen primarily for their high lactate production [ 60 – 62 ], adhesion ability to mucin [ 63 , 64 ], and anti-obesity features [ 65 , 66 ]. The Izmir Institute of Technology's Department of Food Engineering's Molecular Food Microbiology Laboratory (IZTECH-FED-MFMLCC) culture collection contained LAB strains that were isolated from traditional fermented food environments, such as cheese, yogurt, table olives, pickles, and human milk. Furthermore, the cell culture laboratory of Assoc. Prof. Dr. Olcay BOYACIOGLU kindly provided HT-29 cell culture. Growth of bacterial cultures and their stability in simulated gastrointestinal conditions The stabilities of 33 isolated and 7 references LAB strains in the gastrointestinal environment were investigated. Two rounds of 18–24 h of subculturing were used to revive these strains. The Lactobacillus, Lactiplantibacillus, Lacticaseibacillus , and lactococcal LAB strains were cultured at 37°C in MRS and M17 medium, respectively. Strains of Streptococcus were cultured at 42°C in M17 medium. After an overnight culture of LAB of ten milliliters was centrifuged at 2500 x g and 4°C for 10 min, it was twice washed with an equivalent volume of phosphate-buffered saline (PBS, 0.1 M, pH 7, 0.85% NaCl). They were then resuspended in 1.5 mL of PBS to prepare the cell suspension (approximately 10 9 -10 10 CFU/mL). With minor adjustments, simulations of the gastric and intestinal fluid solutions were made by the methods of Guo et al. [ 67 ] and Bengoa et al. [ 68 ]. For simulated gastric juice, 1 N HCl was used to bring the pH of the PBS medium down to 3.0, and it was then sterilized at 121°C for 15 min. After the autoclaving, filter (pore size: 0.22 µm) sterilized pepsin solution (3 g/L) was added. Nine milliliters of PBS (pH 3.0) supplemented with pepsin were mixed with one milliliter of cell suspension, and the mixture was incubated at 37°C for 1.5 h After incubation, the total number of viable cells was determined by the pour plate method, and the survival rates were calculated using the following Eq. 2.1: Survival rate (%) = (log CFU N 1 /log CFU N 0 ) × 100 ( Eq. 2.1) (N 1 = total number of viable LAB after gastric juice passage, CFU/mL; N 0 = total number of viable LAB before implementation, CFU/mL) For simulated intestinal juice, 0.1% w/v pancreatin and 0.15% w/v bovine bile salts were added to PBS (pH 7.2-8) and sterilized using a filter (0.22 µm). One milliliter of the culture suspension obtained from the gastric passage experiment was transferred into 9 mL of simulated intestinal juice. Then, they were incubated at 37°C for 3 h which represents intestinal passage conditions. Afterward, total viable LAB was determined by pour plate cultivation using MRS & M17 agar at 37°C for 48 h, and survival rates were calculated using the following Eq. 2.2: Survival rate (%) = (log CFU N 2 /log CFU N 0 ) × 100 (Eq. 2.2) (N 2 = total number of viable LAB after intestinal juice passage, CFU/mL; N 0 = total number of viable LAB before implementation, CFU/mL) Mucin degradation ability of LAB strains For five reference and seven isolated LAB strains, the mucin degradation property was examined. The trial was conducted by Turpin et al. [ 69 ]. In summary, modified MRS was combined with glucose (20.00 g/L) and porcine/large pig gastric mucin-type III (MUCIN, 3.00 g/L). Five µL of the overnight bacterial cultures were applied as a spot on modified MRS media and plates were incubated at 37°C for 72 h. After that, they were stained for 30 min with Amido black (3 g/L) in 3.5 M acetic acid. Following the staining, plates were cleaned with 1.2 M acetic acid until the colorless halo, or mucin lysis site was visible. Colorless ring surrounding the spotting bacterial cultures was considered as indication of mucin lysis. Growth ability in the presence of mucin The experiment was carried out as described by Turpin et al. [ 69 ]. 2% (v/v) of overnight LAB culture was transferred into modified MRS broth containing 20.0 g/L glucose and porcine gastric mucin (3.00 g/L) at 37°C for 24 h. After incubation, the absorbance values were measured at 600 nm. Results were expressed by using the following equation: A 1 : the absorbance values obtained in modified MRS medium without fermentable carbohydrates (A 1 ) (Sugar-free-control) A 2 : the absorbance values obtained in modified MRS containing glucose or mucin (A 2 ) A M/G : Growth ability in the presence of mucin/glucose (A 2 -A 1 ) Bacterial adhesion to mucin Adhesion to mucin potential of LAB strains was assessed, as previously reported by Bengoa et al. [ 68 ] and Carasi et al. [ 70 ] with a few minor revisions. In summary, 100 µL of a 3 g MUCIN/L solution was added to each well of 96 well sterile polystyrene plates after MUCIN had been dissolved in PBS at a pH of 7.0. The plates were incubated at 37°C for 1 h, followed by an overnight incubation period at 4°C. After subsequent incubation at 37°C for 2 h, 200 µL of PBS was used to wash the wells twice. To ascertain the adhesion of the strain under analysis, four parallels were used. As previously mentioned, LAB cell concentrations were adjusted to about 10 9 CFU/mL in PBS. Each well received 100 µL of the ready-made bacterial cell suspensions. After the plates were incubated at 37°C for 2 h, the wells were washed 6 times with 200 µL of sterile PBS to remove unbound bacteria, and then they were treated with 200 µL of a 5 mL/L Triton X-100 solution at 37°C for 30 min to desorb the adherent bacteria. 100 µL of bacterial suspension that was taken from each well and the number of bound LAB was ascertained by pour plating the serial dilutions using MRS and M17 agar. The viable cell number was counted at 37°C after 48 h. The adhesion percentage of mucin was calculated using the following equation: Adhesion to mucin%= Adherent number of bacteria/Initial number of bacteria Biofilm formation ability LAB strains with a high capacity for biofilm formation can thicken mucin. The methods outlined by Couvigny et al. [ 71 ] were used to determine this property. One isolated LAB strain and five reference strains were employed in the experiments. To do this, 200 µL of MRS/M17 broth was added to each well of 96-well cell culture plates. Next, 10 µL of overnight grown bacterial cultures were added and the plates were incubated at 37°C for 18 h. After incubation, plates were inverted and emptied. Then, 50 µL of 1% crystal violet (CV) was added to each well and incubated at room temperature for 15 min. The remaining dye and unbound cells were removed by three washes by 200 µL of deionized water. Then, the microtiter plate wells were dried at 37°C for 1 h. After that, 200 µL of EtOH was used to dissolve the CV dye, and an h or so was needed for the dye to dissolve. Three independent replicates and four parallel runs were conducted for these experiments. The optical density (OD) values that were obtained through absorbance measurements at 590 nm were used to express the results. Sugar fermentation tests and microbial metabolic activity The fermentation of carbohydrates in LAB strains was examined, as mentioned by Fernandez et al. [ 42 ]. These studies also looked into the LAB strains' capacity to break down mucin sugars. Various carbon sources were used, including N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose, lactose, glucose, sucrose, xylose, fructose, rhamnose, and fucose. Five milliliters of overnight grown LAB cultures were centrifuged at 5000 rpm and 4°C for 10 min. Following that, bacterial pellets were twice washed in 5 mL of modified MRS (pH: 6.2–6.6, using Bromocresol Purple (BCP) in place of sugar). They were suspended in 5 mL of modified MRS. Then, 40 µL of 10% sugar solutions was added to each well of the 96-well plate and 160 µL of LAB cell suspension was mixed within. The experiment's positive control was glucose, and the negative control was modified MRS medium. The observation of a yellow color at 37°C after 24 h of incubation was regarded as a positive reaction. Determination of lactate production Using High-Pressure Liquid Chromatography (HPLC) equipment, the amount of lactate in the culture supernatant was determined [ 72 ]. One milliliter of LAB strains cultured overnight was centrifuged at 14000 x g and 4°C for 15 min. Next, before being injected, 1 mL of supernatant was combined with 4 mL of dH 2 O and run through a 0.20 µm filter. The Perkin Elmer HPLC system (Perkin Elmer (PE), Boston, MA, USA) was used for the analyses. It came with an interface (PE Series 900), a pump (PE Series 200), and a refractive index detector (PE Series 200). Software called Turbochrom Navigator was in charge of the system. The Aminex HPX-87H ion exclusion column (300 x 7.8 mm) from Bio-Rad Laboratories in Hercules, California, USA, was used to achieve the separation. 5 mM H 2 SO 4 was used as the mobile phase, and it was pumped at 0.6 mL/min. The temperature of the column oven (Metatherm, Lake Forest, CA, USA) was 65°C, and the analysis period was 15 min. The study's findings were presented as a g/L sample at the conclusion. Antimicrobial properties of LAB strains Using the agar spot test, the antimicrobial activity of five references and one isolated LAB was ascertained [ 73 ]. The pathogenic microorganisms used in the experiments were E. coli NRRL-B 3008, E. coli RSHM 4024 (ATCC 25922), L. innocua NRRL-B 33314, S. aureus RSKK 1009, and B. cereus ATCC 14579. In the experiments, pathogen concentrations were set to approximately 10 5 -10 6 CFU/mL. The growth medium used was TSB (Tryptone Soy Broth). 5 mL of LAB strain overnight cultures were spotted on MRS/M17 agar and incubated at 37°C/42°C for 24 h. An overnight-grown pathogen culture (roughly 10 7 CFU/mL) was mixed with TSB soft agar (0.6% (w/v)) at a ratio of 1/100 and applied to the MRS/M17 agar spot. Following the plates' solidification, they were incubated at 37°C/42°C for 24 h. The clear zones around the spots were indication of the antibacterial property and the diameters were measured in millimeters. Mucin/mucus thickening properties of LAB strains The ability of LAB strains to withstand simulated gastrointestinal conditions was assessed, and the strains' ability to thicken mucin and mucus was examined. In the experiments, 16 LAB strains with high viability (above 10 4 CFU/mL) were employed. Various factors were examined, including the initial mucin concentration (50 µg/mL) and its presence or absence, co-incubation with LAB strains, and the concentration of lactate (20 mM & 50 mM). Cultivation of HT-29 cell culture with LAB strains According to Dudik et al. [ 55 ], HT-29 cell line cultures were grown alongside bacterial cultures to examine the mucin-thickening effect of LAB strains. 1 mL of overnight bacterial cultures were centrifuged at 4900 x g and 4°C for 5.5 min. 200 µL of PBS was added to create a cell suspension after the cells were twice washed with 1 mL of PBS (pH 7.2). 12 well plates containing 4.5 mg glucose mL-1, 1000 U penicillin mL − 1 , 1000 µL streptomycin mL − 1 , 10% fetal bovine serum, and 0.3 mg glutamine mL − 1 were used to cultivate HT-29 cells in Dulbecco's Modified Eagle Medium (DMEM) medium at 37°C in a 5% CO 2 atmosphere. Each well was initially seeded with roughly 1.2 x 10 5 /1.12 cm 2 (80% confluence) of cells. After that, the experiments were completed at 100% confluency. Over 14 days, the culture medium was refreshed every two days. The medium's antibiotics were eliminated 24 h before the experiments. During the experiment, any excess medium was removed from the plates and the cells were washed twice by using 500 µL of PBS. Subsequently, 10 µL of LAB cell suspensions (corresponding to roughly 10 8–9 CFU/mL) were combined with 500 µL of penicillin-free DMEM and added to each well. HT-29 cells and LAB cell suspensions were co-cultured in 12-well plates at 37°C for 1.5 h. There were three parallel runs of the experiments. Determination of the mucin layer The quantity and thickness of mucin generated by HT-29 cells with and without LAB were investigated using spectrophotometric (Quantitative Determination) and microscopic (Periodic acid-Schiff Staining) methods. Periodic acid-Schiff (PAS) staining The staining protocol used to investigate mucin glycoproteins under light microscopy was modified from the descriptions provided by Ning et al. [ 74 ]. After removing the cell culture media from the 12-well plates, the cells were treated for 5 min with 0.5% periodic acid and then 5 min with distilled water washing. The cells were then immersed in Schiff's reagent for 10 min and then given another 5 min of dH 2 O washing. Following washing, 5 min were spent washing with dH 2 O after applying counterstain with Harris hematoxylin for 1 min. They were then kept for 1 min in 1% acid alcohol. Following the application of 26% ammonia water until the samples' color turned blue, the cells were rinsed in dH 2 O for 5 min. Lastly, they were dried using ethanol concentrations of 70%, 95%, and 99.9%. Under a light microscope, mucin glycoproteins were thus visible as purple-pink (magenta) in color. Quantitative determination By modifying Schiff's reagent method, the amount of mucin glycoprotein was also quantitatively determined [ 75 , 76 ]. Following the removal of the cell culture medium from the 12-well plates, the cells were lysed using PBS containing 1% Triton X-100 in 1 mL. Three parallel transfers of the cell lysates into 96-well plates were made, and they were treated with 0.5% periodic acid. In this application, 10 µL of 50% periodic acid was mixed with 10 mL of 7% acetic acid to create a periodic acid solution. Following a 2-h incubation period at 37°C, 100 µL of Schiff's reagent was introduced into each well, and the samples were left in a dark room for 30 min. Using a microplate reader, the absorbance at 555 nm was used to calculate the amount of mucin. A calibration curve with a linear range of 10–600 µg/mL was produced using the porcine stomach mucin-type III. Statistical analysis In duplicates, the results were presented as mean ± standard deviation. The differences in mean mucin amounts were examined using one-way analysis of variance (ANOVA) and the Tukey test (p > 0.05) by the statistical software MINITAB 21.2.0.0. 3. Results Growth of bacterial cultures and their stability in simulated gastrointestinal conditions Out of the 40 LAB strains, eight LAB species were chosen and the starting LAB strain counts in the pre-screening investigations ranged from 10 8 to 10 10 CFU/mL. In the intestinal fluid, 13 of the 40 LAB strains had viability greater than 10 5 –10 6 CFU/mL, and 3 of them had viability greater than 10 4 (Table 1). In the simulated gastric juice, fifteen of the sixteen bacteria persisted in being viable within the range of 10 6 –10 8 CFU/mL, while thirteen of the sixteen bacteria persisted in being viable within the range of 10 5 –10 7 CFU/mL in the simulated intestinal fluid. All the bacteria displayed viability in the range of 10 5 –10 6 CFU/mL during the simulated gastric fluid passage, but some of them did not show enough viability following the intestinal fluid passage (below 10 4 CFU/mL). Even though some LAB strains ( L. lactis A19, O. AK6, and O. AK8) had lower viability, they were nonetheless used as a comparison in the subsequent experiments. Additionally, those bacteria may possess certain traits that promote the thickness of mucin and mucus. On the log base, the viable numbers for 16 LAB strains obtained under simulated gastric and intestinal conditions are displayed (Fig. 1a). The initial logarithmic values of the bacteria ranged from 9.09 to 10.13, and after passing through the stomach, they dropped to between 5-8.85. The logarithmic number of bacteria in the simulated intestinal fluid ranged from 4.18 to 7.78. The percent viability data of LAB strains (Fig. 1b) provide a better understanding of tolerance to gastrointestinal passage. As a result, after simulating gastric fluid, the percentage values for 16 bacteria varied from 55 to 89.4%. The range of percentage values for these bacteria after simulated intestinal fluid is 46-78.6%. Determination of mucin degradation ability of isolates and growth ability in the presence of mucin Mucin degradation ability of LAB strains The ability of five reference and seven isolated LAB strains to degrade mucin was tested. The reference strains were L. plantarum NRRL-B 4496, L. acidophilus NRRL-B 1910, L. rhamnosus NRRL-B 442, L. casei NRRL-B 441, and L. plantarum DSM 1954. The isolate strains were L. bulgaricus UZ22, L. bulgaricus DT54, L. bulgaricus DT62B, L. bulgaricus UIIN24, L. bulgaricus UIIN26, L. bulgaricus UZ12, and L. bulgaricus UIN42. In the mucin-containing media for those 12 LAB strains, there was no evidence of a lysis zone-a clear area surrounding the colonies. Growth ability in the presence of mucin The absorbance values for 12 different species of bacteria, including L. plantarum , L. acidophilus , L. rhamnosus , L. casei , and L. bulgaricus , when mucin and glucose were present in the medium are displayed in Fig. 2. According to the expected growth ability behavior, absorbance values varied between 0.72 and 1.74 in the presence of glucose. Extremely low absorbance values (0-0.08) for mucin were found, and the results indicated that those strains had either no growth at all or very little. Bacterial adhesion to mucin For sixteen LAB strains, the amount of bacterial adhesion on mucin was measured (Fig. 3a, Fig. 3b, and Table 1). The experiment's starting bacteria numbers ranged from 10 9 to 10 10 CFU/mL. As a result, the initial values varied between 9.15 and 10.23 log, and the adhesion test values varied between 3.36 and 5.54 log. The percent adhesion values for these bacteria varied from 35.2 to 65.4% in Figure 3b. The species with the highest adhesion ability are L. plantarum DSM 1954 (65.4%), S. thermophilus UIN9 (63.9%), and L. lactis A19 (61.1%), while O. AK6 (35.2%) has the lowest adhesion ability (Fig. 3b). Biofilm formation ability Fig. 4 and Table 1 show the biofilm formation properties of several LAB strains. The highest biofilm formation property was exhibited by S. thermophilus UIN9 (3.26 absorbance value), while the absorbance values of other LAB strains were lower than the control. This strain of S. thermophilus had the highest viability in the simulated gastrointestinal conditions out of 15 strains (Table 1). Sugar fermentation tests and microbial metabolic activity Table 2 displays the sugar fermentation profiles of 16 LAB strains. All LAB strains used the tested sugars’ glucose, galactose, mannose, sucrose, fructose, and N-acetylglucosamine; a color shift from purple to yellow indicated the fermentation-induced production of lactate. Among those sugars were N-acetylglucosamine, N-acetylgalactosamine, and fucose, which are mucin sugars. N-acetylglucosamine was fermented by all of those bacteria, and they used the sugar for metabolic processes. Some LAB strains (O. AK6, O. AK8, O. AK16, O. AK22, O. AK23, and O. AK50) that were isolated from olive environments did not use both fucose and N-acetylgalactosamine. Determination of lactate production The highest lactate producers in a 24 h were identified after the lactate production capacities of LAB strains were examined (Fig. 5). Additionally, the amounts of glucose that persisted in the media were calculated, as was the amount of glucose consumed. Glucose and lactate had retention times of 8.2 and 13.25 min, respectively. Lactate production capacity was highest in L. plantarum NRRL-B 4496 (18.2 g/L). The other five bacteria produced varying amounts of lactate (11.3–18.2 g/L). As predicted, there was a negative correlation between the amount of lactate produced and the remaining glucose concentration. The remaining concentrations of glucose ranged from 3.4 to 9.5 g/L. Since lactose served as the primary carbon source in the S. thermophilus growth medium, glucose was excluded, and there is no information regarding glucose in Fig. 5. Antimicrobial properties of LAB strains The antimicrobial activities of LAB strains against five pathogens were tested. The ranges of the LAB inhibition zones were as follows: 0-19 mm for T. UIN9, 10-40 mm for P. 4496, 4-20 mm for A. 1910, 24-35 mm for C. 441, 25-46 mm for R. 442, and 25-48 mm for DSM 1954 (Fig. 6 and Table 1). S. thermophilus UIN9 only inhibited the L. innocua NRRL-B 33314 pathogen bacteria, whereas all Lactobacillus strains demonstrated an inhibitory effect against tested pathogen bacteria. L. acidophilus NRRL-B 1910 and S. thermophilus UIN9 showed the lowest growth inhibitions among the tested LAB strains. Mucin/mucus thickening properties of LAB strains Initial tests revealed that the percentage of LAB strain adhesion on HT-29 cell cultures ranged from 7.07 to 21.15% in different proportions. Determination of the mucin layer by periodic acid-Schiff (PAS) staining The inclusion of LAB in cell culture studies increased in the intensity of the purple-pink (magenta) color, which indicates an increased amount of mucin. This is demonstrated in Figures 7a and 7b. When 20 mM lactate was added to the HT-29 cell culture, mucin observation improved. Moreover, when LAB was added to the same experiments, increased mucin production was visible. When mucin was added to the experiments, even at low lactate concentrations (20 mM), LAB addition showed increased mucin production (Fig. 7c and 7d). A notable rise in mucin was observed at a 50 mM lactate concentration (Fig. 7e and 7f). Mucin concentration increased when the HT-29 cell line and LAB (about 10 8 CFU/mL) were co-incubated. Furthermore, representative images demonstrated that LAB produced higher amounts of mucin when mucin was included in the experiments (Fig. 8a and Fig. 8b). More significant mucin structures were produced when lactate and mucin coexisted than when they did not (Fig. 8c and Fig. 8d). The presence of mucin at a high lactate concentration (50 mM) resulted in the highest amount of mucin density, which was further enhanced by the addition of LAB (Fig. 8e and Fig. 8f). As a result, every experiment demonstrated that the presence of lactate, LAB, and mucin supported the thickness of mucin and mucus. Quantitative determination of the mucin layer As shown in Fig. 9a, most of the tested strains had higher mucin levels when LAB strains were added to the experiments. This demonstrated that the intestinal environment's presence of LAB improves the thickness of the mucin and mucus layers. The mucin thickness of all 14 LAB appears to be greater than that of the control. The isolated olive (O. AK8) had the maximum amount of mucin (287.79 µg/mL). The mucin thickness was less in L. rhamnosus NRRL-B 442 and S. thermophilus UIN9 than in the control, respectively. In the same experiments, 11 LAB exhibited thicker mucin than the control when 20 mM lactate was added. Likewise, the isolate with the highest mucin thickness (257.57 µg/mL) was O. AK8. Then, as shown in Fig. 9b, the mucin thickness of five LAB strains ( L. lactis A19, S. thermophilus UIN9, L. rhamnosus NRRL-B 442, L. plantarum C47, and O. AK22) was less than that of the control. Even in control experiments, mucin thickness was greater than the 20 mM lactate concentration when the lactate concentration was raised to 50 mM. This demonstrated that the 50 mM lactate concentration had a significant impact on the HT-29 cell line culture's ability to secrete mucin. In a similar vein, the majority of the LAB strains (15/16) exhibited thicker mucin than the control. Once more, the isolated O. AK8 exhibited the maximum mucin thickness (332.55 µg/mL), which was also the highest value in all of these tests. The mucin thickness of only one LAB strain (O. AK22) was less than that of the control (Fig. 9c). Eleven LAB seem to have mucin thicknesses that are thicker overall than the control experiment (Fig. 10a). L. acidophilus NRRL-B 1910 (308.62 µg/mL) had the highest amount of mucin among the LAB strains. The mucin thickness of certain strains ( L. lactis A19, O. AK8, L. rhamnosus NRRL-B 442, O. AK22, and L. plantarum D1) was found to be lower than that of the control. Variations in mucin thickness (156.25-233.29 µg/mL) were noted in the same experiments when 20 mM lactate was used (Fig. 10b). Thirteen of them had thicker mucin than the control group. L. plantarum NRRL-B 4496 was found to have the maximum mucin thickness (233.29 µg/mL). Mucin thickness was lower in only three LAB strains (O. AK8, O. AK22, and L. rhamnosus NRRL-B 442) compared to the control. It was evident that all experiments showed increased levels of mucin and mucus when there was a 50 mM lactate concentration present (Fig. 10c). Thirteen LAB in total showed thicker mucin than the control group. In that instance, the highest amount of mucin (321.59 µg/mL) was found in L. casei NRRL-B 441. The mucin thickness of only three LAB strains ( L. plantarum D1, L. plantarum C47, and L. rhamnosus NRRL-B 442) was less than that of the control. 4. Discussion The stability of LAB strains in simulated gastrointestinal conditions is one of the key factors for the selection of probiotic candidates. In the initial experiments, 40 LAB strains, which were composed of 8 LAB species chosen on their ability to produce high lactate, prevent obesity, adhere to the mucin/mucus layer, and not degrade mucin by considering recent literature as mentioned before. Tested LAB bacteria strains exhibited variable survival behavior in simulated gastric and intestinal fluids. Among them, most of the strains (24 strains) did not keep their viability therefore they were eliminated and 16 LAB strains were selected for their good viability potential. Although three of them had relatively lower viability than others, they were also used in the further experiments for their other potentials. After simulating intestinal fluid, the percentage values for 16 bacteria varied from is 46-78.6%. These results showed that all these strains can exhibit beneficial health effects when they reach in the intestinal environment. Similar experimental conditions were used to investigate the intestinal tolerance of L. paracasei , Lactobacillus , and Streptococcus species that were isolated from various sources, including yogurt, kefir, and newborn faces. According to their reports, the intestinal conditions had initial bacterial counts of 10 7 –10 8 CFU/mL and final bacterial counts of 10 3 –10 6 CFU/mL [ 68 , 77 , 78 ]. Comparable percentages of viability were reported in those studies to be between 37.6–79.8%. The goal of this study may require the selection of probiotic LAB strains that do not break down mucin to promote Akkermansia growth. Furthermore, knowing how to grow in the presence of mucin offers important knowledge for future research. All tested LAB strains did not have mucinolytic activity in the mucin containing media. Consequently, those strains lack mucinolytic activity and do not break down mucin. It is well known that mucin is necessary for the growth of Akkermansia species. Therefore, testing to confirm the negative mucinolytic activity can be a crucial step in choosing probiotics as anti-obesity LAB candidates. Moreover, only limited growth ability behavior (low absorbance values, 0-0.08) was observed for those strains in the presence of the mucin as a carbon source alone. Likewise, numerous investigations have demonstrated that distinct LAB strains lack mucinolytic activity [ 65 , 69 , 79 ]. For mucin/mucus thickness, choosing LAB strains with good adhesion behavior can be crucial. Variable degree of bacterial adhesion on mucin/mucus layer was obtained (35.2 to 65.4%, Fig. 3 b). High mucin binding properties (above 60%) for the Lactobacillus genus have been reported in numerous studies [ 68 , 70 , 80 ]. High adhesion qualities in strains that produce biofilms may be advantageous because of their capacity to stimulate the thickening of mucin and mucus. Strong biofilm formation was indicated by high absorbance values. The highest biofilm formation ability was obtained by S. thermophilus UIN9. Additionally, survival of bacteria on harsh conditions (acid, temperature etc.) can be improved by biofilm formation property. Also, attachment on the intestinal environment can be supported by biofilm production and growth of mucin/mucus layer can be triggered. While the ability of S. thermophilus species to form biofilms has been extensively studied, this trait varies depending on the strain, and similar findings have also been documented [ 71 ]. Once more, Gómez et al. [ 81 ] examined the capacity of eight LAB isolates to form biofilms and demonstrated that, following a 48-h incubation period, the isolates were capable of producing biofilms and 1.65, 1.38 and 1.10 absorbance values were obtained as three highest values. On the other hand, lower biofilm production values were also reported (0.34 and 0.43) after 48 hours of cultivation [82]. So, type of LAB strains, incubation time period (24h-48h), and wavelength (540–590 nm) in the measurements may have an impact on the outcome as well. Investigating sugar fermentation profile is an important issue to suggest suitable probiotic candidates that support mucin/mucus thickness. Moreover, it presents valuable data for designing growth media for specific cultures. Most of the sugars were used by tested LAB strains. Among mucin sugars, fucose and N-acetylgalactosamine were not used by all olive isolates. Therefore, these isolates can have more potential to support mucin/mucus layer. The selection of anti-obesity probiotic candidates may have the benefit of enhancing mucin/mucus thickness and creating an environment that is favorable for Akkermansia . Additionally, the following six bacteria's mucin-bacterial binding test results are listed, going from high to low: O. AK50, O. AK23, O. AK8, O. AK16, O. AK22, and O. AK6. Although these strains exhibit robust bacterial binding to mucin, they also aid in the growth of Akkermansia by failing to consume two of the three mucin sugars required by Akkermansia for the degradation of mucin. Fernandez et al. [ 42 ] reported similar investigations, which demonstrated that two S. thermophilus strains were not able to grow when the same mucin sugars were present. Furthermore, many LAB strains generally exhibit little to no fucose utilization [ 80 , 83 ]. Lactate production ability is another parameter for stimulation of mucus layer. According to Fernandez et al. [ 42 ], lactate production stimulates the mucus layer by sustaining the mucus pathway. Lactate can be viewed as a signal that can modulate the colonic epithelium. The highest lactate production was observed in L. plantarum NRRL-B 4496 (18.2 g/L) and varying degrees of lactate (11.3–18.2 g/L) was produced by other LAB strains. The strains of L. plantarum bacteria that were tested produced more lactate. Although some LAB strains bind to mucin slightly (e.g., R442 & AK6), they can also support the mucin/mucus pathway by producing lactate with sugar fermentations. Different strains of L. plantarum produced lactate to varying degrees, with reports of lactate amounts ranging from 6.08 to 29.6 g/L [84–87]. Different S. thermophilus strains were reported to produce lactate in similar amounts [ 42 , 83 ]. Understanding the antimicrobial properties of probiotic candidate strains can provide valuable data to develop suitable niche by inhibiting undesirable microorganisms therefore beneficial microorganisms can be more supported. The antimicrobial properties of LAB were studied to bolster the hypothesis that the production of lactate thickens the mucin/mucus layer and inhibits pathogenic microorganisms, thus promoting growth for the Akkermansia group. LAB can inhibit the growth of other microorganisms and produce a variety of antimicrobial metabolites, including hydrogen peroxide, organic acids, and bacteriocins. Most of the strains inhibited growth of the pathogenic microorganisms. The ranges of the LAB inhibition zones were 0–48 mm (Fig. 6 and Table 1 ). Similarly, various LAB strains were tested for antimicrobial activity against pathogenic bacteria [73, 88, 89]. Gaudana et al. [ 88 ], identified L. plantarum ATCC 8014 and L. rhamnosus CS25 as particularly potent against multiple pathogens, including E. coli S5. Arena et al. [ 73 ], found that 79 L. plantarum strains effectively countered diverse foodborne pathogens. Choi et al. [89], investigated LAB isolated from kimchi, noting bactericidal effects against pathogenic E. coli strains, especially by specific strains like Leuconostoc mesenteriodes (KCTC 13374) and L. plantarum (KCTC 33133). Majority of our strains had antimicrobial activity against tested pathogens and this can be another desirable criterion for the development of probiotics that support Akkermania for anti-obesity purposes. Moreover, inflammation can be a result of obesity and the anti-obesity bacterium Akkermansia lessens this effect depending on the selection of LAB that significantly inhibits pathogens [ 90 , 91 ]. Mucin plays a crucial role in defending against pathogens and supporting beneficial gut bacteria. The mucin/mucus thickening properties of specific probiotic LAB strains were identified by qualitative and quantitative techniques through in vitro cell culture studies. This demonstrated that there was enough LAB bound to the intestinal cell line. According to Dudik et al. [ 55 ], incorporating mucin (50 µg/mL) 24 h before the experiments can stimulate mucin expression and secretion. Relatively higher intensity of purple-pink color was observed for experiments with increased lactate concentration and mucin pre-addition. These results were also clarified and confirmed by quantitative analysis. For HT-29 cell line culture, the effects of initial mucin addition (50 µg/mL), co-incubation with LAB strains, and lactate concentration (20 mM & 50 mM) were examined (Fig. 9 and Fig. 10 ). Pre-addition of mucin was the subject of similar studies, the outcomes of which are shown in Fig. 10 . It was also recommended to add mucin initially before the experiments to increase mucin secretion [ 55 ]. In all experiments, the highest amount of mucin (321.59 µg/mL) was obtained in L. casei NRRL-B 441with initial mucin addition and 50 mM lactate concentration (Fig. 10 c). All of these studies demonstrated how LAB with additional mucin and 50 mM lactate affected the HT-29 cell culture's ability to produce more mucin. Similarly, probiotic Lactobacillus species induced MUC3 mucin expression on intestinal epithelial cells [ 92 ], enhanced mucin secretion and production, thus improved pathogen resistance and hindered the attachment of pathogens like E. coli , thereby protected against invasion [ 93 ]. More recently, L. rhamnosus GG acts as a probiotic, boosting mucin production to reinforce the intestinal barrier [ 76 ]. Furthermore, similar experiments were conducted for B. thetaiotaomicron by using HT29-MTX cells [ 94 ]. Increased mucus-related gene expression was reported and mucus production was supported. They showed that short-chain fatty acids, especially butyrate, stimulate in vitro mucin synthesis and production. In one of the in vitro recent studies, Limage et al. [ 76 ] investigated how the mucosubstance secretion is affected by commensal bacteria using the PAS staining method. They used intestinal epithelial cells of Caco-2 and HT29-MTX-E12 in the experiments. The highest average mucus thickness was obtained for digested Lactobacillus rhamnosus (14.38 µm) using HT29-MTX-E12 cells. When the cells exposed to L. rhamnosus and E. coli , an increased mucus thickness and changes in secretion of mucins were investigated. Although, there has been a limited number of in vitro research on how bacteria can affect mucin layer, those studies presented valuable data for better understanding the mechanisms in the intestinal area. Our results highlighted that LAB species and strain types, lactate concentration and initial mucin addition has significant effect on mucin/mucus thickness development. Further analysis of prototype probiotic candidates was performed by closer examination of a few strains. Consequently, six LAB strains with desired characteristics that were isolated from various sources (two reference cultures, two isolates of olives, one isolate of cheese, and one isolate of yogurt) were concentrated (Table 3 ). These bacteria exhibited good adhesion to mucin (4.52–6.54 log CFU/mL), resistance to simulated gastric and intestinal conditions (10 4 -10 6 CFU/mL), and higher mucin thickness than control in all cell culture experiments when mucin was initially included (Fig. 10 ). Thus, out of the 16 LAB strains, those strains have a higher chance of inducing mucin secretion. The results of the sugar fermentation test are another crucial factor, and only two olive isolates of these six LAB strains (O. AK16 and O. AK50) did not use the mucin sugars (N-acetylglucosamine and N-acetylgalactosamine). These characteristics may be crucial in choosing probiotic strains that are appropriate for promoting Akkermansia growth. Conclusion A few studies have been published in the literature that attempt to explain how the population of Akkermansia in the microbiota is modulated through probiotic effects and/or mechanisms that prevent obesity. Therefore, the purpose of this study is to make predictions about how to identify the characteristics of "Adhesion to Induction" to expand the variety of probiotics in the colon microbiota and lower the ratio of the microbial community associated with obesity. Understanding the significance of adhesion properties to the mucin/mucus layer and the effect of lactate on mucin/mucus layer thickening by in vitro HT-29 cell culture for LAB is necessary to predict the anti-obesity potential of probiotics. The study's findings suggest that two reference LAB strains ( L. acidophilus NRRL-B 1910 and L. plantarum DSM 1954), two isolates from olives (O. AK16 and O. AK50), one from cheese ( L. paracasei C15), and one from yogurt ( S. thermophilus UIN9) may serve as potential anti-obesity prototype candidates. These findings may also make a unique contribution to the body of scientific literature. As a result, a favorable niche for the novel probiotic Akkermansia muciniphila can be established. Furthermore, among the probiotics of the new generation, Bacteroides, Clostridium , and Faecalibacterium , may benefit from this environment created for Akkermansia. As a result, it becomes critical for the development of new generation super probiotics to combat diseases like cancer, autoimmune diseases, metabolic diseases, cardiovascular diseases, and gastrointestinal issues. Thus, in addition to being effective against obesity, these six LAB strains may also be beneficial for inflammatory conditions, cardiovascular diseases, and leaky gut syndrome. Furthermore, it is conceivable that advantageous bacteria might stick to the mucin layer and encourage the development of biofilms. Based on the information gathered from in vitro cell culture investigations, in vivo experiments with Akkermansia can be more effectively planned for future research. Additionally, a novel functional food ingredient containing probiotics and polyphenols to induce Akkermansia for the management of obesity can be developed. Abbreviations A 1 , The absorbance values obtained in modified MRS medium without fermentable carbohydrates (Sugar-free-control); A 2 , The absorbance values obtained in modified MRS containing glucose or mucin; A M/G , Growth ability in the presence of mucin/glucose (A 2 -A 1 ); ANOVA, Analysis of variance; ARS, Agricultural research service; ATCC, American type culture collection; BCP, Bromocresol purple; CCM, Czech collection of microorganisms; CFU, Colony forming unit; cm 2 , Centimeter power –2 ; CNT, Control; CO 2 , Carbon dioxide; CV, Crystal violet; DSM, Deutsche Saturn lung von mikroorganismen; dH 2 O, Deionized water; DMEM, Dulbecco's modified eagle medium; e.g, Exempli gratia (for example); eqn, Equation; etc, Et cetera; EtOH, Ethanol; g, The relative centrifugal force (RCF); g, Gram; GIT, Gastrointestinal tract; h, Hour; HCl, Hydrochloric acid; HPLC, High-pressure liquid chromatography; HT-29, Human colorectal adenocarcinoma cell line; H 2 SO 4 , Sulfuric acid; IZTECH-FED-MFMLCC, Izmir Institute of Technology, Department of Food Engineering, Molecular Food Microbiology Laboratory Culture Collection; L, Liter; LAB, Lactic acid bacteria; log, Logarithms; L20, 20 mM lactate; L50, 50 mM lactate; M, Molar; MDa, Megadalton; mg, Milligram; mL, Milliliter; mm, Millimeter; mM, Millimolar; min, Minute; MUCIN, Purified porcine gastric mucin-type III; MRS, De man, Rogosa and Sharpe; N, Normal; N 0 , Total number of viable LAB before implementation, CFU/mL; N 1 , Total number of viable LAB after gastric juice passage, CFU/mL; N 2 , Total number of viable LAB after intestinal juice passage, CFU/mL; NaCl, Sodium chloride; NGP, Next-generation of probiotics; nm, Nanometer; NRRL, Northern regional research lab; OD, Optical density; PAS, Periodic acid-Schiff; PBS, Phosphate buffered saline; PE, Perkin Elmer; pH, Quantitative measure of the acidity or basicity of aqueous or other liquid solutions (Power of hydrogen); rpm, Revolutions per minute; SCFA, Short-chain fatty acid; TSB, Tryptone soy broth; U, Units; v, Volume; w, Weight; %, Percent; °C, Centigrade degree; µg, Microgram; µL, Microliter; µm, Micrometer. Declarations Acknowledgement This research was supported by Scientific and Technological Research Council of Turkey (TUBITAK) (Project number:121S123) and Izmir Institute of Technology Scientific Research Project (BAP) (Project number:2020IYTE0084). The authors greatly appreciate to Assoc. Prof. Dr. Olcay BOYACIOĞLU, for providing HT-29 cell culture. Assist. Prof. Dr. Mürüvvet ABBAK and Lecturer Özgür OKUR were appreciated for their support during the cell culture experiments carried out together. Conflict of Interest The authors declare that they have no conflict of interest. Data availability All data generated and processed during this research are available from the corresponding author upon reasonable request. Funding This research was supported by Scientific and Technological Research Council of Turkey (TUBITAK) within the scope of TUBITAK 1002 Rapid Support Program (Project number:121S123) and Izmir Institute of Technology Scientific Research Project (BAP) with the project number 2020IYTE0084. Author contributions section All authors contributed to the realization of the research and the writing of the article. Aslihan Kamber: Conceptualization, data curation, investigation, methodology, validation, writing-original draft, writing-review & editing; Cisem Bulut Albayrak: Data curation, investigation, methodology, resources, supervision, writing-review & editing. H. Sebnem Harsa: Conceptualization, data curation, investigation, methodology, project administration, resources, supervision, writing – original draft, writing-review & editing. Declaration of competing interest The authors declare no conflict of interest. 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BMC Biol 11:1-13. https://doi.org/10.1186/1741-7007-11-61 Tables Table 1 Lactic acid bacteria strains used in the experiments and their viabilities during simulated gastrointestinal passage.[1] No Bacteria Source Code Number of Bacteria After Simulated Gastric Fluid (CFU/mL) Number of Bacteria After Simulated Intestinal Fluid (CFU/mL) 1 L. plantarum NRRL-B 4496 Reference P. 4496 2.45 x 10 8 2.09 x 10 7 2 L. acidophilus NRRL-B 1910 Reference A. 1910 1.82 x 10 7 7.25 x 10 5 3 L. casei NRRL-B 441 Reference C. 441 3.29 x 10 8 2.17 x 10 7 4 L. rhamnosus NRRL-B 442 Reference R. 442 4.32 x 10 7 1.08 x 10 6 5 L. pentosus NRRL-B 227 Reference P. 227 1.65 x 10 6 * 6 L. plantarum DSM 1954 Reference DSM 1954 3.49 x 10 8 2.8 x 10 7 7 S. thermophilus CCM 4757 Reference CCM 4757 * * 8 S. thermophilus AS95 Human breast milk T. AS95 * * 9 S. thermophilus C74 Yogurt T. C74 * * 10 S. thermophilus C77a Yogurt T. C77a * * 11 S. thermophilus C95-1 Yogurt T. C95-1 * * 12 S. thermophilus C95-2 Yogurt T. C95-2 * * 13 S. thermophilus C97-2 Yogurt T. C97-2 * * 14 S. thermophilus CTY24 Yogurt T. CTY24 * * 15 S. thermophilus CTY41 Yogurt T. CTY41 * * 16 S. thermophilus CTY44 Yogurt T. CTY44 1.4 x 10 6 * 17 S. thermophilus UN5 Yogurt T. UN5 * * 18 S. thermophilus UN9 Yogurt T. UN9 * * 19 S. thermophilus UN19 Yogurt T. UN19 1.1 x 10 6 * 20 S. thermophilus UIB31 Yogurt T. UIB31 1 x 10 5 * 21 S. thermophilus UIN9 Yogurt T. UIN9 1.19 x 10 7 7.1 x 10 5 22 L. paracasei C8 Cheese PR. C8 3 x 10 5 * 23 L. paracasei C15 Cheese PR. C15 9.5 x 10 6 4.95 x 10 6 24 L. plantarum C47 Cheese PL. C47 4.29 x 10 8 3.09 x 10 7 25 L. plantarum D1 Cheese P. D1 7.14 x 10 8 5.96 x 10 7 26 L. lactis A1 Cheese L. A1 5.5 x 10 5 * 27 L. lactis A19 Cheese L. A19 7.15 x 10 6 8.5 x 10 4 28 L. lactis A20 Cheese L. A20 1 x 10 5 * 29 L. lactis A22 Cheese L. A22 2 x 10 5 * 30 L. lactis A23 Cheese L. A23 * * 31 LAB. O. AK6 Olive O. AK6 3.25 x 10 6 3.5 x 10 4 32 LAB. O. AK8 Green Olive Brine O. AK8 1 x 10 5 1.5 x 10 4 33 LAB. O. AK16 Black Olive Brine O. AK16 2.4 x 10 8 5.4 x 10 5 34 LAB. O. AK22 Black Olive (Sele type) O. AK22 7.35 x 10 6 5.9 x 10 5 35 LAB. O. AK23 Black Olive (Sele type) O. AK23 ̴ 10 8 1.11 x 10 7 36 LAB. O. AK42 Black Olive Brine O. AK42 * * 37 LAB. O. AK50 Dark Green Olive Brine O. AK50 5 x 10 7 7.55 x 10 5 38 BT10 Pickle P. BT10 * * 39 BT14 Pickle P. BT14 * * 40 BT40 Pickle P. BT40 * * Table 2 Sugar fermentation abilities of lactic acid bacteria and identification of strains that do not use the mucin sugars.[2] Bacteria/Sugar Glucose Galactose Mannose Lactose Sucrose Xylose Fructose Rhamnose Fucose N-acetylglucosamine N-acetylgalactasomine L. plantarum NRRL-B 4496 + + + + + - (±) + - (±) - (±) + + (±) L. acidophilus NRRL-B 1910 + + + - (±) + - (±) + - (±) - (±) + + (±) L. casei NRRL-B 441 + + + + + - (±) + -(±) - (±) + + L. rhamnosus NRRL-B 442 + + + + + - + + - + + L. plantarum DSM 1954 + + + + + - (±) + + (±) - (±) + + (±) S. thermophilus UIN9 + + + + + - + - (±) - + + L. paracasei C15 + + + + + - + - (±) + + + L. plantarum C47 + + + + + - (±) + - (±) - (±) + + (±) L. plantarum D1 + + + + + - (±) + - (±) - (±) + + L. lactis A19 + + + + + - + - - + + LAB. O. AK6 + + + + + + + + (±) - + - (±) LAB. O. AK8 + + + + + - + + (±) - + - LAB. O. AK16 + + + + + - + + (±) - + - LAB. O. AK22 + + + + + + + - (±) - + - LAB. O. AK23 + + + + + + + + (±) - + - LAB. O. AK50 + + + + + + + + (±) - + - Table 3 Summary for mucin/mucus layer supportive properties of selected lactic acid bacteria strains for different lactate concentrations.[3] LAB Strains Mean mucin/mucus thickness for HT-29 + Mucin (µg/mL) Mean mucin/mucus thickness for HT-29 + Mucin + 20 mM Lactate (µg/mL) Mean mucin/mucus thickness for HT-29 + Mucin + 50 mM Lactate (µg/mL) L. acidophilus NRRL-B 1910 71.2±19 a 22±28.3 a 53.64±7.13 a L. plantarum DSM 1954 46.23±12.52 a 43.59±7.56 a 54.9±42.2 a O. AK16 36.08±5.06 a 15.63±13.85 a 47.6±57 a O. AK50 19.81±8.42 a 25.68±5.53 a 40.8±40.8 a L. paracasei C15 48.4±38.3 a 31.9±25.1 a 70.4±44.8 a S. thermophilus UIN9 19.2±21.2 a 15±17.8 a 58.4±48.8 a [1] *<10 5 for simulated gastric fluid and <10 4 for simulated intestinal fluid. [2] + (strong positive), + (±) (weak positive), - (±) (weak negative), - (strong negative). [3] Statistical analyzed were applied by one way analysis of variance (ANOVA) and Tukey test (p > 0.05) for the differences between mean mucin amounts of 6 lactic acid bacteria strains. The data sets present normal distribution and results were expressed as mean ± standard deviation in duplicates. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4535093","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317095651,"identity":"d1ccce01-0e74-40f0-ad69-94275312abbf","order_by":0,"name":"Aslihan Kamber","email":"","orcid":"","institution":"Izmir Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Aslihan","middleName":"","lastName":"Kamber","suffix":""},{"id":317095655,"identity":"13005177-af34-434f-90f7-43e0ad3f38cb","order_by":1,"name":"Cisem Bulut Albayrak","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Cisem","middleName":"Bulut","lastName":"Albayrak","suffix":""},{"id":317095656,"identity":"d4f94138-0313-4dbe-85a8-fe794f7740f1","order_by":2,"name":"Hayriye Sebnem Harsa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFACHhBxAEQwPkiACBkQrYXZIAGiGkgkEKeFTYKBGC3y7b3HHnzMuSNv3n/4WcXDtj9yDOzN2yQYf9zDqcXgzLl0w5nbnhnOuZFmdiOxzcCYgedYmQRDQjFuLRI5ZtK82w4zzpBgAGtJbACKALXgdpn8/Ddm0n+3HbafwX/8WwFQS32D/Bv8Whhu8JhJM247nDiDIceMAaglgUGCB78WgzN5aZK92w4nz5DIKZZIOGds2MaTVmyRkIbHYe1nj0n83HbYFuiwjR9/lMnJ87Mf3njjgw0eh6EARjZg7IAYxGoAgj/EKx0Fo2AUjIKRAwDB91LcrJpNzgAAAABJRU5ErkJggg==","orcid":"","institution":"Izmir Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Hayriye","middleName":"Sebnem","lastName":"Harsa","suffix":""}],"badges":[],"createdAt":"2024-06-05 15:21:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4535093/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4535093/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-024-10357-6","type":"published","date":"2024-10-09T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58948133,"identity":"b620446f-bbd7-4bf6-9f79-7eb602135cc6","added_by":"auto","created_at":"2024-06-24 13:20:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":362645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eLogarithmic values of viable lactic acid bacteria strains count during simulated gastric and intestinal passage\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Percent viability of lactic acid bacteria strains during simulated gastric and intestinal passage\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/2cd59fd95f29fa2944ff0b8c.jpg"},{"id":58948122,"identity":"aa5b1918-88e4-44a4-9fc3-f776f832f428","added_by":"auto","created_at":"2024-06-24 13:20:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63520,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth abilities of lactic acid bacteria strains in the presence of mucin \u0026amp; glucose by optical density measurements\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/f4583e4a9849eeb70c3adc1c.jpg"},{"id":58949555,"identity":"75e3bcfd-b99b-4740-ab40-04e98883f65e","added_by":"auto","created_at":"2024-06-24 13:36:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":296511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Logarithmic value of adhered lactic acid bacteria strains on mucin/mucus layer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Percent degree of adhered lactic acid bacteria strains on mucin/mucus layer\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/de73a69d6587ce9ade544fb5.jpg"},{"id":58948127,"identity":"08c93105-29ac-49c8-9901-f3d965f2fcd8","added_by":"auto","created_at":"2024-06-24 13:20:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39823,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm formation properties of lactic acid bacteria strains\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/46d5958ccd5ee5d8723ffc74.jpg"},{"id":58948927,"identity":"20e953cf-2e5b-45f1-a35c-31c76b6e4eed","added_by":"auto","created_at":"2024-06-24 13:28:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41538,"visible":true,"origin":"","legend":"\u003cp\u003eLactate and glucose concentrations after 24 h incubation in liquid medium for lactic acid bacteria strains\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/50eb3174b69f34f217561dd6.jpg"},{"id":58948929,"identity":"13cc8e26-b33f-439c-8d0c-0f33a23930de","added_by":"auto","created_at":"2024-06-24 13:28:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":51612,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial properties of lactic acid bacteria strains against pathogenic bacteria by agar spot test\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/8367f4b92a6fcb31fbae97ee.jpg"},{"id":58948129,"identity":"014fbb21-2e03-4562-bf4e-d3a23013102f","added_by":"auto","created_at":"2024-06-24 13:20:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":131548,"visible":true,"origin":"","legend":"\u003cp\u003eQualitative demonstration for reference lactic acid bacteriastrains to increase the mucin/mucus layer with different lactate concentrations (20 mM \u0026amp; 50 mM) on HT-29 cell culture using the periodic acid-Schiff (PAS) staining method. HT-29 cell (CNT-1) \u003cstrong\u003e(A),\u003c/strong\u003eHT-29 cell + \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003eNRRL-B 4496 \u003cstrong\u003e(B),\u003c/strong\u003e HT-29 cell + lactate (20 mM) (CNT-2) \u003cstrong\u003e(C),\u003c/strong\u003e HT-29 cell + lactate (20 mM) + \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e NRRL-B 441 \u003cstrong\u003e(D),\u003c/strong\u003e HT-29 cell + lactate (50 mM) (CNT-3) \u003cstrong\u003e(E),\u003c/strong\u003eHT-29 cell + lactate (50 mM) + \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NRRL-B 1910\u003cstrong\u003e (F)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/ee66371cdb5834a664c8b4ed.jpg"},{"id":58948124,"identity":"ff43c59b-a21d-4e61-9e33-644d88482391","added_by":"auto","created_at":"2024-06-24 13:20:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":125679,"visible":true,"origin":"","legend":"\u003cp\u003eQualitative demonstration for reference lactic acid bacteria strains to increase the mucin/mucus layer with initial mucin addition and different lactate concentrations (20 mM \u0026amp; 50 mM) on HT-29 cell culture using the periodic acid-Schiff (PAS) staining method.\u003cstrong\u003e \u003c/strong\u003eHT-29 cell + mucin (CNT-4) \u003cstrong\u003e(A),\u003c/strong\u003e HT-29 cell + mucin + \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NRRL-B 1910\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(B), \u003c/strong\u003eHT-29 cell + mucin + lactate (20 mM) (CNT-5) \u003cstrong\u003e(C),\u003c/strong\u003e HT-29 cell + mucin + lactate (20 mM) + \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e NRRL-B 4496 \u003cstrong\u003e(D), \u003c/strong\u003eHT-29 cell + mucin + lactate (50 mM) (CNT-6) \u003cstrong\u003e(E),\u003c/strong\u003e HT-29 cell + mucin + lactate (50 mM) + \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e NRRL-B 441 \u003cstrong\u003e(F)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/857cd5603f08115c54f49bd8.jpg"},{"id":58948130,"identity":"8e3a84b1-1bee-4f54-baad-724485e7a98a","added_by":"auto","created_at":"2024-06-24 13:20:01","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":443785,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative determination for reference and isolated lactic acid bacteria strains to increase the mucin/mucus layer with different lactate concentrations (20 mM \u0026amp; 50 mM) on HT-29 cell culture using the Schiff's reagent method and the results are expressed as mucin amount (µg/mL). HT-29 cell + lactic acid bacteria (µg/mL) \u003cstrong\u003e(A),\u003c/strong\u003e HT-29 cell (µg/mL) + lactate (20 mM) + lactic acid bacteria(µg/mL) \u003cstrong\u003e(B),\u003c/strong\u003e HT-29 cell + lactate (50 mM) + lactic acid bacteria (µg/mL) \u003cstrong\u003e(C)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/5ec8e811b2f1e2d3498e26c7.jpg"},{"id":58949556,"identity":"74f087e6-0fa9-4cc8-8acf-e473dacb2331","added_by":"auto","created_at":"2024-06-24 13:36:01","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":448483,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative determination for reference and isolated lactic acid bacteria strains to increase the mucin/mucus layer with initial mucin addition and different lactate concentrations (20 mM \u0026amp; 50 mM) on HT-29 cell culture using the Schiff's reagent method and the results are expressed as mucin amount (µg/mL). HT-29 cell + mucin (50 µg/mL) + lactic acid bacteria (µg/mL) \u003cstrong\u003e(A),\u003c/strong\u003eHT-29 cell (µg/mL) + mucin (50 µg/mL) + lactate (20 mM) + lactic acid bacteria (µg/mL) \u003cstrong\u003e(B),\u003c/strong\u003eHT-29 cell + mucin (50 µg/mL) + lactate (50 mM) + lactic acid bacteria (µg/mL) \u003cstrong\u003e(C)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/7c9d33ac5cdb840580b05a89.jpg"},{"id":66597107,"identity":"339d898a-752b-4917-8a55-46f67fe00c9c","added_by":"auto","created_at":"2024-10-14 16:07:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3589618,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/23351a83-8f58-444a-9739-fe86ca544e2d.pdf"},{"id":58948131,"identity":"403baa7a-8bd8-4a6c-bed7-a37a62647a2f","added_by":"auto","created_at":"2024-06-24 13:20:01","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2266843,"visible":true,"origin":"","legend":"","description":"","filename":"PAPGraphicalAbstract5.06.2024.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/5e00fc914d15e39ac39fa092.pptx"},{"id":58948930,"identity":"693bb53e-1e08-41ff-addc-7e0f0c700bcb","added_by":"auto","created_at":"2024-06-24 13:28:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":52225,"visible":true,"origin":"","legend":"","description":"","filename":"PAPHighlight5.06.2024.docx","url":"https://assets-eu.researchsquare.com/files/rs-4535093/v1/483675585374441bef6f607b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Studies on the Probiotic, Adhesion and Induction Properties of Artisanal Lactic Acid Bacteria: To Customize a Gastrointestinal Niche to Trigger Anti-Obesity Functions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the World Health Organization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], obesity is one of the major global health issues and has become an epidemic. Because obesity is strongly correlated with several chronic illnesses, including diabetes, osteoarthritis, cardiovascular disease, liver disease, and several types of cancer, it is a severe health concern [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Over the past 50 years, the global obesity epidemic has grown; in 1975, the typical adult's obesity rate was three times higher than it is today [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Around 650\u0026nbsp;million people have been impacted globally, and 13% of the world's population now considers it to be a serious issue [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. If the global obesity data continues to show upward trends by 2030, 20% of adult people worldwide are expected to be obese [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a result, methods to lessen the obesity issue are becoming more and more crucial.\u003c/p\u003e \u003cp\u003eIt has recently been shown that the gut microbiota can affect weight gain or reduction in a variety of ways [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and that \"modulation of the microbiota\" [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] can help combat obesity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies on humans and animals have demonstrated a correlation between obesity and a high relative ratio of bacteria (Firmicutes (F)) to bacteria (Bacteroidetes (B), Actinobacteria, and Proteobacteria) (F/B) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The original sample of \u003cem\u003eA. muciniphila\u003c/em\u003e was firstly obtained from a healthy patient [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to Kim et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], there is an inverse correlation between the number of \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e in the GIT and several disease states, such as obesity and associated metabolic issues.\u003c/p\u003e \u003cp\u003eSince its initial isolation from the stool of a healthy patient in 2004, this bacterium has been recognized as an efficient combatant against obesity [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Over the past fifteen years, research has focused on the roles that \u003cem\u003eA. muciniphila\u003c/em\u003e, a member of the next-generation of probiotics (NGP) class [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], plays in the gastrointestinal system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These roles include lowering insulin resistance, regulating intestinal immunity, enhancing intestinal barrier function, and reducing obesity, diabetes, and inflammation [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. muciniphila\u003c/em\u003e is a common member of the gastrointestinal tract (GIT) and a Gram-negative, non-sporulating, strict anaerobic bacterium that belongs to the phylum Verrucomicrobia [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It is an obligatory chemoorganotroph that breaks down mucin and uses mucus as its only source of energy, carbon, and nitrogen. It can produce propionate, acetate, succinate, ethanol, and 1,2-propanediol in smaller amounts by using these [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Other good bacteria can improve mucus structure, which in turn can stimulate the population of \u003cem\u003eAkkermansia\u003c/em\u003e in the intestinal area. For its spread, several probiotic LAB can encourage the environment's mucin/mucus thickening.\u003c/p\u003e \u003cp\u003eThe two most prevalent probiotic strains, \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, adhere to the intestinal mucosa and competitively eliminate pathogens [\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Probiotic LAB's mucin-promoting qualities, for example, may be correlated with the population of \u003cem\u003eAkkermansia\u003c/em\u003e. In this regard, an \u003cem\u003ein vitro\u003c/em\u003e intestinal model system was used to investigate the impact of probiotic mixtures on \u003cem\u003eA. muciniphila\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The results demonstrated that probiotic supplementation led to a gradual increase in the colon's short-chain fatty acid (SCFA) concentration and encouraged the thickening of mucin. It was mentioned that \u003cem\u003eA. muciniphila\u003c/em\u003e, which breaks down mucin, develops more readily in such an environment. As a result, methods that promote a mucin/mucus environment have gained importance for maintaining intestinal health, managing the \u003cem\u003eAkkermansia\u003c/em\u003e population, and reducing obesity. Mucus plays a role in defense, conservation, and partner-host homeostasis. Bacteria that encourage the production of mucus strengthen the immune system because inflammatory diseases are caused by a decrease in mucus thickness. According to Devi and Halami [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], goblet cells facilitate the dynamic process of mucus formation in the gut, which is then maintained by transcription factors like KLF4. The primary constituents of epithelial mucus layers are mucins, which are extracellular, high molecular weight glycoproteins (0.5\u0026ndash;20 MDa) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Because it is made up of oligosaccharides and amino acids, mucin is utilized as a food source by gut bacteria. Mucins and microflora interact in ways that are crucial to normal function. Numerous diseases involve modifications to mucins, which may result from either excessive mucin peptides or glycosylation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, two crucial mechanisms that aid in the development of \u003cem\u003eAkkermansia\u003c/em\u003e are the adhesion of probiotic LAB to the mucosa in the colon and the stimulation of mucus secretion by intestinal epithelial cells (HT-29 cells). In addition, some bacteria, such as \u003cem\u003eS. thermophilus\u003c/em\u003e, which have poor adhesion to mucus, produce lactate by breaking down lactose and thus support the mucus pathway, regulate the mucus environment, stimulate the mucus layer, and may also be a signal that can modulate the colonic epithelium [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, lactate's antimicrobial property helps to fight pathogens in the intestine [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and maintain \u003cem\u003eAkkermansia\u003c/em\u003e's improved mucin/mucus environment.\u003c/p\u003e \u003cp\u003eFurthermore, lactate has a multitude of advantageous effects that include nourishing gut microbial communities, reducing inflammatory processes, and fortifying the gut barrier by producing mucus [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For example, Nishiyama et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] found that elevated lactate levels were linked to an increase in lactobacilli in the intestine and that the amount of \u003cem\u003eAkkermansia\u003c/em\u003e was positively impacted by the increasing lactate concentration.\u003c/p\u003e \u003cp\u003eAccording to research on epithelial cell cultures, probiotic LAB can help form mucin and mucus by adhering to the colon's mucosa. \u003cem\u003eIn vitro\u003c/em\u003e experiments are conducted to investigate this phenomenon using HT-29 cells [\u003cspan additionalcitationids=\"CR48 CR49 CR50\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In accordance with numerous studies [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], lactobacilli stimulate mucin expression and secretion. According to Dudik et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], pre-incubating lactobacilli with mucin increases their adhesion to HT-29 cells \u003cem\u003ein vitro\u003c/em\u003e, which may improve their bioavailability in the gut during \u003cem\u003ein vivo\u003c/em\u003e conditions. In the majority of these studies, probiotic LAB strains (e.g., \u003cem\u003eL. plantarum\u003c/em\u003e 299v, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG, and \u003cem\u003eL. acidophilus\u003c/em\u003e A4) have been shown to enhance the expression of MUC2 and MUC3 genes (mucin) in HT-29 colon cell cultures, thereby potentially supporting barrier function and inhibiting pathogens. While there are a few studies that demonstrate the mucin/mucus-promoting abilities of probiotic LAB [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], our study may be the first to look into the possibility of probiotics supporting the population of \u003cem\u003eAkkermansia\u003c/em\u003e by analyzing lactate production, mucus thickening properties, antimicrobial effect, and other factors to suggest probiotic candidates that may be anti-obesity.\u003c/p\u003e \u003cp\u003eThe majority of \u003cem\u003eLactobacillus\u003c/em\u003e species, including strains of \u003cem\u003eL. plantarum\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. reuteri\u003c/em\u003e, \u003cem\u003eL. paracasei\u003c/em\u003e, \u003cem\u003eL. lactis\u003c/em\u003e, \u003cem\u003eL. brevis\u003c/em\u003e, and \u003cem\u003eL. delb.\u003c/em\u003e ssp. \u003cem\u003ebulgaricus\u003c/em\u003e, have an anti-obesity effect, even though some of these species are linked to weight gain [\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Probiotic and/or symbiotic preparations promoting the \u003cem\u003eAkkermansia\u003c/em\u003e group can be developed to support the microbiota that fights obesity. The primary goal of this work is to characterize the resistance to gastrointestinal conditions, mucin degradation abilities, adhesion properties, mucin/mucus thickening role, and other aspects of the \u003cem\u003eAkkermansia\u003c/em\u003e population to investigate the anti-obesity probiotic potential of LAB isolated from food environments. The underlying premise of this study was that probiotic-containing LAB could alter the microbiota or the environment in which microorganisms of the \u003cem\u003eAkkermansia\u003c/em\u003e group grow.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eThe experiments used a LAB containing forty strains, including isolated and reference strains. The ARS Culture Collection (NRRL, USA) provided reference strains of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e NRRL-B 4496, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NRRL-B 1910, \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e NRRL-B 441, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e NRRL-B 442, and \u003cem\u003eLactiplantibacillus pentosus\u003c/em\u003e NRRL-B 227. The German Collection of Microorganisms and Cell Cultures GmbH (Germany) and the Czech Collection of Microorganisms (CCM) provided \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e DSM 1954 and \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e CCM 4757, respectively.\u003c/p\u003e \u003cp\u003eAccording to their published reports, reference strain species were chosen primarily for their high lactate production [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], adhesion ability to mucin [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], and anti-obesity features [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Izmir Institute of Technology's Department of Food Engineering's Molecular Food Microbiology Laboratory (IZTECH-FED-MFMLCC) culture collection contained LAB strains that were isolated from traditional fermented food environments, such as cheese, yogurt, table olives, pickles, and human milk. Furthermore, the cell culture laboratory of Assoc. Prof. Dr. Olcay BOYACIOGLU kindly provided HT-29 cell culture.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth of bacterial cultures and their stability in simulated gastrointestinal conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe stabilities of 33 isolated and 7 references LAB strains in the gastrointestinal environment were investigated.\u003c/p\u003e \u003cp\u003eTwo rounds of 18\u0026ndash;24 h of subculturing were used to revive these strains. The \u003cem\u003eLactobacillus, Lactiplantibacillus, Lacticaseibacillus\u003c/em\u003e, and lactococcal LAB strains were cultured at 37\u0026deg;C in MRS and M17 medium, respectively. Strains of \u003cem\u003eStreptococcus\u003c/em\u003e were cultured at 42\u0026deg;C in M17 medium. After an overnight culture of LAB of ten milliliters was centrifuged at 2500 x g and 4\u0026deg;C for 10 min, it was twice washed with an equivalent volume of phosphate-buffered saline (PBS, 0.1 M, pH 7, 0.85% NaCl). They were then resuspended in 1.5 mL of PBS to prepare the cell suspension (approximately 10\u003csup\u003e9\u003c/sup\u003e-10\u003csup\u003e10\u003c/sup\u003e CFU/mL).\u003c/p\u003e \u003cp\u003eWith minor adjustments, simulations of the gastric and intestinal fluid solutions were made by the methods of Guo et al. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] and Bengoa et al. [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. For simulated gastric juice, 1 N HCl was used to bring the pH of the PBS medium down to 3.0, and it was then sterilized at 121\u0026deg;C for 15 min. After the autoclaving, filter (pore size: 0.22 \u0026micro;m) sterilized pepsin solution (3 g/L) was added. Nine milliliters of PBS (pH 3.0) supplemented with pepsin were mixed with one milliliter of cell suspension, and the mixture was incubated at 37\u0026deg;C for 1.5 h After incubation, the total number of viable cells was determined by the pour plate method, and the survival rates were calculated using the following Eq.\u0026nbsp;2.1:\u003c/p\u003e \u003cp\u003eSurvival rate (%) = (log CFU N\u003csub\u003e1\u003c/sub\u003e/log CFU N\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100 \u003cb\u003e(\u003c/b\u003eEq.\u0026nbsp;2.1)\u003c/p\u003e \u003cp\u003e(N\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;total number of viable LAB after gastric juice passage, CFU/mL; N\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;total number of viable LAB before implementation, CFU/mL)\u003c/p\u003e \u003cp\u003eFor simulated intestinal juice, 0.1% w/v pancreatin and 0.15% w/v bovine bile salts were added to PBS (pH 7.2-8) and sterilized using a filter (0.22 \u0026micro;m). One milliliter of the culture suspension obtained from the gastric passage experiment was transferred into 9 mL of simulated intestinal juice. Then, they were incubated at 37\u0026deg;C for 3 h which represents intestinal passage conditions. Afterward, total viable LAB was determined by pour plate cultivation using MRS \u0026amp; M17 agar at 37\u0026deg;C for 48 h, and survival rates were calculated using the following Eq.\u0026nbsp;2.2:\u003c/p\u003e \u003cp\u003eSurvival rate (%) = (log CFU N\u003csub\u003e2\u003c/sub\u003e/log CFU N\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100 (Eq.\u0026nbsp;2.2)\u003c/p\u003e \u003cp\u003e(N\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;total number of viable LAB after intestinal juice passage, CFU/mL; N\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;total number of viable LAB before implementation, CFU/mL)\u003c/p\u003e \u003cp\u003e \u003cb\u003eMucin degradation ability of LAB strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor five reference and seven isolated LAB strains, the mucin degradation property was examined. The trial was conducted by Turpin et al. [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In summary, modified MRS was combined with glucose (20.00 g/L) and porcine/large pig gastric mucin-type III (MUCIN, 3.00 g/L). Five \u0026micro;L of the overnight bacterial cultures were applied as a spot on modified MRS media and plates were incubated at 37\u0026deg;C for 72 h. After that, they were stained for 30 min with Amido black (3 g/L) in 3.5 M acetic acid. Following the staining, plates were cleaned with 1.2 M acetic acid until the colorless halo, or mucin lysis site was visible. Colorless ring surrounding the spotting bacterial cultures was considered as indication of mucin lysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth ability in the presence of mucin\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe experiment was carried out as described by Turpin et al. [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. 2% (v/v) of overnight LAB culture was transferred into modified MRS broth containing 20.0 g/L glucose and porcine gastric mucin (3.00 g/L) at 37\u0026deg;C for 24 h. After incubation, the absorbance values were measured at 600 nm. Results were expressed by using the following equation:\u003c/p\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e: the absorbance values obtained in modified MRS medium without fermentable carbohydrates (A\u003csub\u003e1\u003c/sub\u003e) (Sugar-free-control)\u003c/p\u003e \u003cp\u003eA\u003csub\u003e2\u003c/sub\u003e: the absorbance values obtained in modified MRS containing glucose or mucin (A\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003eA\u003csub\u003eM/G\u003c/sub\u003e: Growth ability in the presence of mucin/glucose (A\u003csub\u003e2\u003c/sub\u003e-A\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial adhesion to mucin\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAdhesion to mucin potential of LAB strains was assessed, as previously reported by Bengoa et al. [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] and Carasi et al. [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] with a few minor revisions. In summary, 100 \u0026micro;L of a 3 g MUCIN/L solution was added to each well of 96 well sterile polystyrene plates after MUCIN had been dissolved in PBS at a pH of 7.0. The plates were incubated at 37\u0026deg;C for 1 h, followed by an overnight incubation period at 4\u0026deg;C. After subsequent incubation at 37\u0026deg;C for 2 h, 200 \u0026micro;L of PBS was used to wash the wells twice. To ascertain the adhesion of the strain under analysis, four parallels were used.\u003c/p\u003e \u003cp\u003eAs previously mentioned, LAB cell concentrations were adjusted to about 10\u003csup\u003e9\u003c/sup\u003e CFU/mL in PBS. Each well received 100 \u0026micro;L of the ready-made bacterial cell suspensions. After the plates were incubated at 37\u0026deg;C for 2 h, the wells were washed 6 times with 200 \u0026micro;L of sterile PBS to remove unbound bacteria, and then they were treated with 200 \u0026micro;L of a 5 mL/L Triton X-100 solution at 37\u0026deg;C for 30 min to desorb the adherent bacteria. 100 \u0026micro;L of bacterial suspension that was taken from each well and the number of bound LAB was ascertained by pour plating the serial dilutions using MRS and M17 agar. The viable cell number was counted at 37\u0026deg;C after 48 h. The adhesion percentage of mucin was calculated using the following equation:\u003c/p\u003e \u003cp\u003eAdhesion to mucin%= Adherent number of bacteria/Initial number of bacteria\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm formation ability\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLAB strains with a high capacity for biofilm formation can thicken mucin. The methods outlined by Couvigny et al. [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] were used to determine this property. One isolated LAB strain and five reference strains were employed in the experiments. To do this, 200 \u0026micro;L of MRS/M17 broth was added to each well of 96-well cell culture plates. Next, 10 \u0026micro;L of overnight grown bacterial cultures were added and the plates were incubated at 37\u0026deg;C for 18 h. After incubation, plates were inverted and emptied. Then, 50 \u0026micro;L of 1% crystal violet (CV) was added to each well and incubated at room temperature for 15 min. The remaining dye and unbound cells were removed by three washes by 200 \u0026micro;L of deionized water. Then, the microtiter plate wells were dried at 37\u0026deg;C for 1 h. After that, 200 \u0026micro;L of EtOH was used to dissolve the CV dye, and an h or so was needed for the dye to dissolve. Three independent replicates and four parallel runs were conducted for these experiments. The optical density (OD) values that were obtained through absorbance measurements at 590 nm were used to express the results.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSugar fermentation tests and microbial metabolic activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe fermentation of carbohydrates in LAB strains was examined, as mentioned by Fernandez et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These studies also looked into the LAB strains' capacity to break down mucin sugars. Various carbon sources were used, including N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose, lactose, glucose, sucrose, xylose, fructose, rhamnose, and fucose. Five milliliters of overnight grown LAB cultures were centrifuged at 5000 rpm and 4\u0026deg;C for 10 min. Following that, bacterial pellets were twice washed in 5 mL of modified MRS (pH: 6.2\u0026ndash;6.6, using Bromocresol Purple (BCP) in place of sugar). They were suspended in 5 mL of modified MRS. Then, 40 \u0026micro;L of 10% sugar solutions was added to each well of the 96-well plate and 160 \u0026micro;L of LAB cell suspension was mixed within. The experiment's positive control was glucose, and the negative control was modified MRS medium. The observation of a yellow color at 37\u0026deg;C after 24 h of incubation was regarded as a positive reaction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of lactate production\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing High-Pressure Liquid Chromatography (HPLC) equipment, the amount of lactate in the culture supernatant was determined [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. One milliliter of LAB strains cultured overnight was centrifuged at 14000 x g and 4\u0026deg;C for 15 min. Next, before being injected, 1 mL of supernatant was combined with 4 mL of dH\u003csub\u003e2\u003c/sub\u003eO and run through a 0.20 \u0026micro;m filter. The Perkin Elmer HPLC system (Perkin Elmer (PE), Boston, MA, USA) was used for the analyses. It came with an interface (PE Series 900), a pump (PE Series 200), and a refractive index detector (PE Series 200). Software called Turbochrom Navigator was in charge of the system. The Aminex HPX-87H ion exclusion column (300 x 7.8 mm) from Bio-Rad Laboratories in Hercules, California, USA, was used to achieve the separation. 5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was used as the mobile phase, and it was pumped at 0.6 mL/min. The temperature of the column oven (Metatherm, Lake Forest, CA, USA) was 65\u0026deg;C, and the analysis period was 15 min. The study's findings were presented as a g/L sample at the conclusion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntimicrobial properties of LAB strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing the agar spot test, the antimicrobial activity of five references and one isolated LAB was ascertained [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The pathogenic microorganisms used in the experiments were \u003cem\u003eE. coli\u003c/em\u003e NRRL-B 3008, \u003cem\u003eE. coli\u003c/em\u003e RSHM 4024 (ATCC 25922), \u003cem\u003eL. innocua\u003c/em\u003e NRRL-B 33314, \u003cem\u003eS. aureus\u003c/em\u003e RSKK 1009, and \u003cem\u003eB. cereus\u003c/em\u003e ATCC 14579. In the experiments, pathogen concentrations were set to approximately 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e CFU/mL. The growth medium used was TSB (Tryptone Soy Broth). 5 mL of LAB strain overnight cultures were spotted on MRS/M17 agar and incubated at 37\u0026deg;C/42\u0026deg;C for 24 h. An overnight-grown pathogen culture (roughly 10\u003csup\u003e7\u003c/sup\u003e CFU/mL) was mixed with TSB soft agar (0.6% (w/v)) at a ratio of 1/100 and applied to the MRS/M17 agar spot. Following the plates' solidification, they were incubated at 37\u0026deg;C/42\u0026deg;C for 24 h. The clear zones around the spots were indication of the antibacterial property and the diameters were measured in millimeters.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMucin/mucus thickening properties of LAB strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe ability of LAB strains to withstand simulated gastrointestinal conditions was assessed, and the strains' ability to thicken mucin and mucus was examined. In the experiments, 16 LAB strains with high viability (above 10\u003csup\u003e4\u003c/sup\u003e CFU/mL) were employed. Various factors were examined, including the initial mucin concentration (50 \u0026micro;g/mL) and its presence or absence, co-incubation with LAB strains, and the concentration of lactate (20 mM \u0026amp; 50 mM).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCultivation of HT-29 cell culture with LAB strains\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAccording to Dudik et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], HT-29 cell line cultures were grown alongside bacterial cultures to examine the mucin-thickening effect of LAB strains. 1 mL of overnight bacterial cultures were centrifuged at 4900 x g and 4\u0026deg;C for 5.5 min. 200 \u0026micro;L of PBS was added to create a cell suspension after the cells were twice washed with 1 mL of PBS (pH 7.2). 12 well plates containing 4.5 mg glucose mL-1, 1000 U penicillin mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1000 \u0026micro;L streptomycin mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10% fetal bovine serum, and 0.3 mg glutamine mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were used to cultivate HT-29 cells in Dulbecco's Modified Eagle Medium (DMEM) medium at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Each well was initially seeded with roughly 1.2 x 10\u003csup\u003e5\u003c/sup\u003e/1.12 cm\u003csup\u003e2\u003c/sup\u003e (80% confluence) of cells. After that, the experiments were completed at 100% confluency. Over 14 days, the culture medium was refreshed every two days. The medium's antibiotics were eliminated 24 h before the experiments. During the experiment, any excess medium was removed from the plates and the cells were washed twice by using 500 \u0026micro;L of PBS. Subsequently, 10 \u0026micro;L of LAB cell suspensions (corresponding to roughly 10\u003csup\u003e8\u0026ndash;9\u003c/sup\u003e CFU/mL) were combined with 500 \u0026micro;L of penicillin-free DMEM and added to each well. HT-29 cells and LAB cell suspensions were co-cultured in 12-well plates at 37\u0026deg;C for 1.5 h. There were three parallel runs of the experiments.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDetermination of the mucin layer\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe quantity and thickness of mucin generated by HT-29 cells with and without LAB were investigated using spectrophotometric (Quantitative Determination) and microscopic (Periodic acid-Schiff Staining) methods.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003ePeriodic acid-Schiff (PAS) staining\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe staining protocol used to investigate mucin glycoproteins under light microscopy was modified from the descriptions provided by Ning et al. [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. After removing the cell culture media from the 12-well plates, the cells were treated for 5 min with 0.5% periodic acid and then 5 min with distilled water washing. The cells were then immersed in Schiff's reagent for 10 min and then given another 5 min of dH\u003csub\u003e2\u003c/sub\u003eO washing. Following washing, 5 min were spent washing with dH\u003csub\u003e2\u003c/sub\u003eO after applying counterstain with Harris hematoxylin for 1 min. They were then kept for 1 min in 1% acid alcohol. Following the application of 26% ammonia water until the samples' color turned blue, the cells were rinsed in dH\u003csub\u003e2\u003c/sub\u003eO for 5 min. Lastly, they were dried using ethanol concentrations of 70%, 95%, and 99.9%. Under a light microscope, mucin glycoproteins were thus visible as purple-pink (magenta) in color.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eQuantitative determination\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eBy modifying Schiff's reagent method, the amount of mucin glycoprotein was also quantitatively determined [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFollowing the removal of the cell culture medium from the 12-well plates, the cells were lysed using PBS containing 1% Triton X-100 in 1 mL. Three parallel transfers of the cell lysates into 96-well plates were made, and they were treated with 0.5% periodic acid. In this application, 10 \u0026micro;L of 50% periodic acid was mixed with 10 mL of 7% acetic acid to create a periodic acid solution. Following a 2-h incubation period at 37\u0026deg;C, 100 \u0026micro;L of Schiff's reagent was introduced into each well, and the samples were left in a dark room for 30 min. Using a microplate reader, the absorbance at 555 nm was used to calculate the amount of mucin. A calibration curve with a linear range of 10\u0026ndash;600 \u0026micro;g/mL was produced using the porcine stomach mucin-type III.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn duplicates, the results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The differences in mean mucin amounts were examined using one-way analysis of variance (ANOVA) and the Tukey test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) by the statistical software MINITAB 21.2.0.0.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003eGrowth of bacterial cultures and their stability in simulated gastrointestinal conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOut of the 40 LAB strains, eight LAB species were chosen and the starting LAB strain counts in the pre-screening investigations ranged from 10\u003csup\u003e8\u003c/sup\u003e to 10\u003csup\u003e10\u003c/sup\u003e CFU/mL. In the intestinal fluid, 13 of the 40 LAB strains had viability greater than 10\u003csup\u003e5\u003c/sup\u003e–10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCFU/mL, and 3 of them had viability greater than 10\u003csup\u003e4\u003c/sup\u003e (Table 1).\u003c/p\u003e\n\u003cp\u003eIn the simulated gastric juice, fifteen of the sixteen bacteria persisted in being viable within the range of 10\u003csup\u003e6\u003c/sup\u003e–10\u003csup\u003e8\u003c/sup\u003e CFU/mL, while thirteen of the sixteen bacteria persisted in being viable within the range of 10\u003csup\u003e5\u003c/sup\u003e–10\u003csup\u003e7\u003c/sup\u003e CFU/mL in the simulated intestinal fluid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the bacteria displayed viability in the range of 10\u003csup\u003e5\u003c/sup\u003e–10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCFU/mL during the simulated gastric fluid passage, but some of them did not show enough viability following the intestinal fluid passage (below 10\u003csup\u003e4\u003c/sup\u003e CFU/mL). Even though some LAB strains (\u003cem\u003eL. lactis\u003c/em\u003e A19, O. AK6, and O. AK8) had lower viability, they were nonetheless used as a comparison in the subsequent experiments. Additionally, those bacteria may possess certain traits that promote the thickness of mucin and mucus.\u003c/p\u003e\n\u003cp\u003eOn the log base, the viable numbers for 16 LAB strains obtained under simulated gastric and intestinal conditions are displayed (Fig. 1a). The initial logarithmic values of the bacteria ranged from 9.09 to 10.13, and after passing through the stomach, they dropped to between 5-8.85. The logarithmic number of bacteria in the simulated intestinal fluid ranged from 4.18 to 7.78. The percent viability data of LAB strains (Fig. 1b) provide a better understanding of tolerance to gastrointestinal passage. As a result, after simulating gastric fluid, the percentage values for 16 bacteria varied from 55 to 89.4%. The range of percentage values for these bacteria after simulated intestinal fluid is 46-78.6%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of mucin degradation ability of isolates and growth ability in the presence of mucin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMucin degradation ability of LAB strains\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe ability of five reference and seven isolated LAB strains to degrade mucin was tested. The reference strains were \u003cem\u003eL. plantarum\u003c/em\u003e NRRL-B 4496, \u003cem\u003eL. acidophilus\u003c/em\u003e NRRL-B 1910, \u003cem\u003eL. rhamnosus\u003c/em\u003e NRRL-B 442, \u003cem\u003eL. casei\u003c/em\u003e NRRL-B 441, and \u003cem\u003eL. plantarum\u003c/em\u003e DSM 1954. The isolate strains were \u003cem\u003eL. bulgaricus\u003c/em\u003e UZ22, \u003cem\u003eL. bulgaricus\u003c/em\u003e DT54, \u003cem\u003eL. bulgaricus\u003c/em\u003e DT62B, \u003cem\u003eL. bulgaricus\u003c/em\u003e UIIN24, \u003cem\u003eL. bulgaricus\u003c/em\u003e UIIN26, \u003cem\u003eL. bulgaricus\u003c/em\u003e UZ12, and \u003cem\u003eL. bulgaricus\u003c/em\u003e UIN42.\u0026nbsp;In the mucin-containing media for those 12 LAB strains, there was no evidence of a lysis zone-a clear area surrounding the colonies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGrowth ability in the presence of mucin\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe absorbance values for 12 different species of bacteria, including \u003cem\u003eL. plantarum\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. casei\u003c/em\u003e, and \u003cem\u003eL. bulgaricus\u003c/em\u003e, when mucin and glucose were present in the medium are displayed in Fig. 2.\u0026nbsp;According to the expected growth ability behavior, absorbance values varied between 0.72 and 1.74 in the presence of glucose. Extremely low absorbance values (0-0.08) for mucin were found, and the results indicated that those strains had either no growth at all or very little.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial adhesion to mucin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor sixteen LAB strains, the amount of bacterial adhesion on mucin was measured (Fig. 3a, Fig. 3b, and Table 1). The experiment's starting bacteria numbers ranged from 10\u003csup\u003e9\u003c/sup\u003e to 10\u003csup\u003e10\u003c/sup\u003e CFU/mL. As a result, the initial values varied between 9.15 and 10.23 log, and the adhesion test values varied between 3.36 and 5.54 log. The percent adhesion values for these bacteria varied from 35.2 to 65.4% in Figure 3b. The species with the highest adhesion ability are \u003cem\u003eL. plantarum\u003c/em\u003e DSM 1954 (65.4%), \u003cem\u003eS. thermophilus\u003c/em\u003e UIN9 (63.9%), and \u003cem\u003eL. lactis\u003c/em\u003e A19 (61.1%), while O. AK6 (35.2%) has the lowest adhesion ability (Fig. 3b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm formation ability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 4 and Table 1 show the biofilm formation properties of several LAB strains. The highest biofilm formation property was exhibited by \u003cem\u003eS. thermophilus\u003c/em\u003e UIN9 (3.26 absorbance value), while the absorbance values of other LAB strains were lower than the control. This strain of \u003cem\u003eS. thermophilus\u003c/em\u003e had the highest viability in the simulated gastrointestinal conditions out of 15 strains (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSugar fermentation tests and microbial metabolic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 2 displays the sugar fermentation profiles of 16 LAB strains. All LAB strains used the tested sugars’ glucose, galactose, mannose, sucrose, fructose, and N-acetylglucosamine; a color shift from purple to yellow indicated the fermentation-induced production of lactate. Among those sugars were N-acetylglucosamine, N-acetylgalactosamine, and fucose, which are mucin sugars. N-acetylglucosamine was fermented by all of those bacteria, and they used the sugar for metabolic processes. Some LAB strains (O. AK6, O. AK8, O. AK16, O. AK22, O. AK23, and O. AK50) that were isolated from olive environments did not use both fucose and N-acetylgalactosamine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of lactate production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest lactate producers in a 24 h were identified after the lactate production capacities of LAB strains were examined (Fig. 5). Additionally, the amounts of glucose that persisted in the media were calculated, as was the amount of glucose consumed. Glucose and lactate had retention times of 8.2 and 13.25 min, respectively.\u0026nbsp;Lactate production capacity was highest in \u003cem\u003eL. plantarum\u003c/em\u003e NRRL-B 4496 (18.2 g/L). The other five bacteria produced varying amounts of lactate (11.3–18.2 g/L). As predicted, there was a negative correlation between the amount of lactate produced and the remaining glucose concentration. The remaining concentrations of glucose ranged from 3.4 to 9.5 g/L. Since lactose served as the primary carbon source in the \u003cem\u003eS. thermophilus\u003c/em\u003e growth medium, glucose was excluded, and there is no information regarding glucose in Fig. 5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial properties of LAB strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activities of LAB strains against five pathogens were tested. The ranges of the LAB inhibition zones were as follows: 0-19 mm for T. UIN9, 10-40 mm for P. 4496, 4-20 mm for A. 1910, 24-35 mm for C. 441, 25-46 mm for R. 442, and 25-48 mm for DSM 1954 (Fig. 6 and Table 1).\u0026nbsp;\u003cem\u003eS. thermophilus\u003c/em\u003e UIN9 only inhibited the \u003cem\u003eL. innocua\u003c/em\u003e NRRL-B 33314 pathogen bacteria, whereas all \u003cem\u003eLactobacillus\u003c/em\u003e strains demonstrated an inhibitory effect against tested pathogen bacteria. \u003cem\u003eL. acidophilus\u003c/em\u003e NRRL-B 1910 and \u003cem\u003eS. thermophilus\u003c/em\u003e UIN9 showed the lowest growth inhibitions among the tested LAB strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMucin/mucus thickening properties of LAB strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitial tests revealed that the percentage of LAB strain adhesion on HT-29 cell cultures ranged from 7.07 to 21.15% in different proportions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDetermination of the mucin layer by periodic acid-Schiff (PAS) staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe inclusion of LAB in cell culture studies increased in the intensity of the purple-pink (magenta) color, which indicates an increased amount of mucin. This is demonstrated in Figures 7a and 7b. When 20 mM lactate was added to the HT-29 cell culture, mucin observation improved. Moreover, when LAB was added to the same experiments, increased mucin production was visible. When mucin was added to the experiments, even at low lactate concentrations (20 mM), LAB addition showed increased mucin production (Fig. 7c and 7d). A notable rise in mucin was observed at a 50 mM lactate concentration (Fig. 7e and 7f).\u0026nbsp;Mucin concentration increased when the HT-29 cell line and LAB (about 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) were co-incubated. Furthermore, representative images demonstrated that LAB produced higher amounts of mucin when mucin was included in the experiments (Fig. 8a and Fig. 8b). \u0026nbsp;More significant mucin structures were produced when lactate and mucin coexisted than when they did not (Fig. 8c and Fig. 8d). The presence of mucin at a high lactate concentration (50 mM) resulted in the highest amount of mucin density, which was further enhanced by the addition of LAB (Fig. 8e and Fig. 8f). As a result, every experiment demonstrated that the presence of lactate, LAB, and mucin supported the thickness of mucin and mucus.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuantitative determination of the mucin layer\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 9a, most of the tested strains had higher mucin levels when LAB strains were added to the experiments. This demonstrated that the intestinal environment's presence of LAB improves the thickness of the mucin and mucus layers. The mucin thickness of all 14 LAB appears to be greater than that of the control. The isolated olive (O. AK8) had the maximum amount of mucin (287.79 µg/mL). The mucin thickness was less in \u003cem\u003eL. rhamnosus\u003c/em\u003e NRRL-B 442 and \u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003eUIN9 than in the control, respectively.\u0026nbsp;In the same experiments, 11 LAB exhibited thicker mucin than the control when 20 mM lactate was added. Likewise, the isolate with the highest mucin thickness (257.57 µg/mL) was O. AK8. Then, as shown in Fig. 9b, the mucin thickness of five LAB strains (\u003cem\u003eL. lactis\u003c/em\u003e A19, \u003cem\u003eS. thermophilus\u003c/em\u003e UIN9, \u003cem\u003eL. rhamnosus\u003c/em\u003e NRRL-B 442, \u003cem\u003eL. plantarum\u003c/em\u003e C47, and O. AK22) was less than that of the control. Even in control experiments, mucin thickness was greater than the 20 mM lactate concentration when the lactate concentration was raised to 50 mM.\u0026nbsp;This demonstrated that the 50 mM lactate concentration had a significant impact on the HT-29 cell line culture's ability to secrete mucin. In a similar vein, the majority of the LAB strains (15/16) exhibited thicker mucin than the control. Once more, the isolated O. AK8 exhibited the maximum mucin thickness (332.55 µg/mL), which was also the highest value in all of these tests. The mucin thickness of only one LAB strain (O. AK22) was less than that of the control (Fig. 9c).\u003c/p\u003e\n\u003cp\u003eEleven LAB seem to have mucin thicknesses that are thicker overall than the control experiment (Fig. 10a). \u003cem\u003eL. acidophilus\u003c/em\u003e NRRL-B 1910 (308.62 µg/mL) had the highest amount of mucin among the LAB strains. The mucin thickness of certain strains (\u003cem\u003eL. lactis\u003c/em\u003e A19, O. AK8, \u003cem\u003eL.\u003c/em\u003e\u003cem\u003erhamnosus\u003c/em\u003e NRRL-B 442, O. AK22, and \u003cem\u003eL. plantarum\u003c/em\u003e D1) was found to be lower than that of the control. Variations in mucin thickness (156.25-233.29 µg/mL) were noted in the same experiments when 20 mM lactate was used (Fig. 10b). Thirteen of them had thicker mucin than the control group. \u003cem\u003eL. plantarum\u003c/em\u003e NRRL-B 4496 was found to have the maximum mucin thickness (233.29 µg/mL). Mucin thickness was lower in only three LAB strains (O. AK8, O. AK22, and \u003cem\u003eL. rhamnosus\u003c/em\u003e NRRL-B 442) compared to the control. It was evident that all experiments showed increased levels of mucin and mucus when there was a 50 mM lactate concentration present (Fig. 10c).\u0026nbsp;Thirteen LAB in total showed thicker mucin than the control group. In that instance, the highest amount of mucin (321.59 µg/mL) was found in \u003cem\u003eL. casei\u003c/em\u003e NRRL-B 441. The mucin thickness of only three LAB strains (\u003cem\u003eL. plantarum\u003c/em\u003e D1, \u003cem\u003eL. plantarum\u003c/em\u003e C47, and \u003cem\u003eL. rhamnosus\u003c/em\u003e NRRL-B 442) was less than that of the control.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe stability of LAB strains in simulated gastrointestinal conditions is one of the key factors for the selection of probiotic candidates. In the initial experiments, 40 LAB strains, which were composed of 8 LAB species chosen on their ability to produce high lactate, prevent obesity, adhere to the mucin/mucus layer, and not degrade mucin by considering recent literature as mentioned before.\u003c/p\u003e \u003cp\u003eTested LAB bacteria strains exhibited variable survival behavior in simulated gastric and intestinal fluids. Among them, most of the strains (24 strains) did not keep their viability therefore they were eliminated and 16 LAB strains were selected for their good viability potential. Although three of them had relatively lower viability than others, they were also used in the further experiments for their other potentials. After simulating intestinal fluid, the percentage values for 16 bacteria varied from is 46-78.6%. These results showed that all these strains can exhibit beneficial health effects when they reach in the intestinal environment. Similar experimental conditions were used to investigate the intestinal tolerance of \u003cem\u003eL. paracasei\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e species that were isolated from various sources, including yogurt, kefir, and newborn faces. According to their reports, the intestinal conditions had initial bacterial counts of 10\u003csup\u003e7\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e CFU/mL and final bacterial counts of 10\u003csup\u003e3\u003c/sup\u003e\u0026ndash;10\u003csup\u003e6\u003c/sup\u003e CFU/mL [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Comparable percentages of viability were reported in those studies to be between 37.6\u0026ndash;79.8%.\u003c/p\u003e \u003cp\u003eThe goal of this study may require the selection of probiotic LAB strains that do not break down mucin to promote \u003cem\u003eAkkermansia\u003c/em\u003e growth. Furthermore, knowing how to grow in the presence of mucin offers important knowledge for future research. All tested LAB strains did not have mucinolytic activity in the mucin containing media. Consequently, those strains lack mucinolytic activity and do not break down mucin. It is well known that mucin is necessary for the growth of \u003cem\u003eAkkermansia\u003c/em\u003e species. Therefore, testing to confirm the negative mucinolytic activity can be a crucial step in choosing probiotics as anti-obesity LAB candidates. Moreover, only limited growth ability behavior (low absorbance values, 0-0.08) was observed for those strains in the presence of the mucin as a carbon source alone. Likewise, numerous investigations have demonstrated that distinct LAB strains lack mucinolytic activity [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor mucin/mucus thickness, choosing LAB strains with good adhesion behavior can be crucial.\u003c/p\u003e \u003cp\u003eVariable degree of bacterial adhesion on mucin/mucus layer was obtained (35.2 to 65.4%, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). High mucin binding properties (above 60%) for the \u003cem\u003eLactobacillus\u003c/em\u003e genus have been reported in numerous studies [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh adhesion qualities in strains that produce biofilms may be advantageous because of their capacity to stimulate the thickening of mucin and mucus. Strong biofilm formation was indicated by high absorbance values. The highest biofilm formation ability was obtained by \u003cem\u003eS. thermophilus\u003c/em\u003e UIN9. Additionally, survival of bacteria on harsh conditions (acid, temperature etc.) can be improved by biofilm formation property. Also, attachment on the intestinal environment can be supported by biofilm production and growth of mucin/mucus layer can be triggered. While the ability of \u003cem\u003eS. thermophilus\u003c/em\u003e species to form biofilms has been extensively studied, this trait varies depending on the strain, and similar findings have also been documented [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Once more, G\u0026oacute;mez et al. [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e] examined the capacity of eight LAB isolates to form biofilms and demonstrated that, following a 48-h incubation period, the isolates were capable of producing biofilms and 1.65, 1.38 and 1.10 absorbance values were obtained as three highest values. On the other hand, lower biofilm production values were also reported (0.34 and 0.43) after 48 hours of cultivation [82]. So, type of LAB strains, incubation time period (24h-48h), and wavelength (540\u0026ndash;590 nm) in the measurements may have an impact on the outcome as well.\u003c/p\u003e \u003cp\u003eInvestigating sugar fermentation profile is an important issue to suggest suitable probiotic candidates that support mucin/mucus thickness. Moreover, it presents valuable data for designing growth media for specific cultures. Most of the sugars were used by tested LAB strains. Among mucin sugars, fucose and N-acetylgalactosamine were not used by all olive isolates. Therefore, these isolates can have more potential to support mucin/mucus layer. The selection of anti-obesity probiotic candidates may have the benefit of enhancing mucin/mucus thickness and creating an environment that is favorable for \u003cem\u003eAkkermansia\u003c/em\u003e. Additionally, the following six bacteria's mucin-bacterial binding test results are listed, going from high to low: O. AK50, O. AK23, O. AK8, O. AK16, O. AK22, and O. AK6. Although these strains exhibit robust bacterial binding to mucin, they also aid in the growth of \u003cem\u003eAkkermansia\u003c/em\u003e by failing to consume two of the three mucin sugars required by \u003cem\u003eAkkermansia\u003c/em\u003e for the degradation of mucin. Fernandez et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] reported similar investigations, which demonstrated that two \u003cem\u003eS. thermophilus\u003c/em\u003e strains were not able to grow when the same mucin sugars were present. Furthermore, many LAB strains generally exhibit little to no fucose utilization [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLactate production ability is another parameter for stimulation of mucus layer. According to Fernandez et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], lactate production stimulates the mucus layer by sustaining the mucus pathway. Lactate can be viewed as a signal that can modulate the colonic epithelium. The highest lactate production was observed in \u003cem\u003eL. plantarum\u003c/em\u003e NRRL-B 4496 (18.2 g/L) and varying degrees of lactate (11.3\u0026ndash;18.2 g/L) was produced by other LAB strains. The strains of \u003cem\u003eL. plantarum\u003c/em\u003e bacteria that were tested produced more lactate. Although some LAB strains bind to mucin slightly (e.g., R442 \u0026amp; AK6), they can also support the mucin/mucus pathway by producing lactate with sugar fermentations. Different strains of \u003cem\u003eL. plantarum\u003c/em\u003e produced lactate to varying degrees, with reports of lactate amounts ranging from 6.08 to 29.6 g/L [84\u0026ndash;87]. Different \u003cem\u003eS. thermophilus\u003c/em\u003e strains were reported to produce lactate in similar amounts [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding the antimicrobial properties of probiotic candidate strains can provide valuable data to develop suitable niche by inhibiting undesirable microorganisms therefore beneficial microorganisms can be more supported. The antimicrobial properties of LAB were studied to bolster the hypothesis that the production of lactate thickens the mucin/mucus layer and inhibits pathogenic microorganisms, thus promoting growth for the \u003cem\u003eAkkermansia\u003c/em\u003e group. LAB can inhibit the growth of other microorganisms and produce a variety of antimicrobial metabolites, including hydrogen peroxide, organic acids, and bacteriocins. Most of the strains inhibited growth of the pathogenic microorganisms. The ranges of the LAB inhibition zones were 0\u0026ndash;48 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly, various LAB strains were tested for antimicrobial activity against pathogenic bacteria [73, 88, 89]. Gaudana et al. [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e88\u003c/span\u003e], identified \u003cem\u003eL. plantarum\u003c/em\u003e ATCC 8014 and \u003cem\u003eL. rhamnosus\u003c/em\u003e CS25 as particularly potent against multiple pathogens, including \u003cem\u003eE. coli\u003c/em\u003e S5. Arena et al. [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], found that 79 \u003cem\u003eL. plantarum\u003c/em\u003e strains effectively countered diverse foodborne pathogens. Choi et al. [89], investigated LAB isolated from kimchi, noting bactericidal effects against pathogenic \u003cem\u003eE. coli\u003c/em\u003e strains, especially by specific strains like \u003cem\u003eLeuconostoc mesenteriodes\u003c/em\u003e (KCTC 13374) and \u003cem\u003eL. plantarum\u003c/em\u003e (KCTC 33133). Majority of our strains had antimicrobial activity against tested pathogens and this can be another desirable criterion for the development of probiotics that support \u003cem\u003eAkkermania\u003c/em\u003e for anti-obesity purposes. Moreover, inflammation can be a result of obesity and the anti-obesity bacterium \u003cem\u003eAkkermansia\u003c/em\u003e lessens this effect depending on the selection of LAB that significantly inhibits pathogens [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e91\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMucin plays a crucial role in defending against pathogens and supporting beneficial gut bacteria. The mucin/mucus thickening properties of specific probiotic LAB strains were identified by qualitative and quantitative techniques through \u003cem\u003ein vitro\u003c/em\u003e cell culture studies. This demonstrated that there was enough LAB bound to the intestinal cell line. According to Dudik et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], incorporating mucin (50 \u0026micro;g/mL) 24 h before the experiments can stimulate mucin expression and secretion. Relatively higher intensity of purple-pink color was observed for experiments with increased lactate concentration and mucin pre-addition. These results were also clarified and confirmed by quantitative analysis. For HT-29 cell line culture, the effects of initial mucin addition (50 \u0026micro;g/mL), co-incubation with LAB strains, and lactate concentration (20 mM \u0026amp; 50 mM) were examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Pre-addition of mucin was the subject of similar studies, the outcomes of which are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003e. It was also recommended to add mucin initially before the experiments to increase mucin secretion [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In all experiments, the highest amount of mucin (321.59 \u0026micro;g/mL) was obtained in \u003cem\u003eL. casei\u003c/em\u003e NRRL-B 441with initial mucin addition and 50 mM lactate concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). All of these studies demonstrated how LAB with additional mucin and 50 mM lactate affected the HT-29 cell culture's ability to produce more mucin.\u003c/p\u003e \u003cp\u003eSimilarly, probiotic \u003cem\u003eLactobacillus\u003c/em\u003e species induced MUC3 mucin expression on intestinal epithelial cells [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e92\u003c/span\u003e], enhanced mucin secretion and production, thus improved pathogen resistance and hindered the attachment of pathogens like \u003cem\u003eE. coli\u003c/em\u003e, thereby protected against invasion [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. More recently, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG acts as a probiotic, boosting mucin production to reinforce the intestinal barrier [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Furthermore, similar experiments were conducted for \u003cem\u003eB. thetaiotaomicron\u003c/em\u003e by using HT29-MTX cells [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Increased mucus-related gene expression was reported and mucus production was supported. They showed that short-chain fatty acids, especially butyrate, stimulate \u003cem\u003ein vitro\u003c/em\u003e mucin synthesis and production. In one of the \u003cem\u003ein vitro\u003c/em\u003e recent studies, Limage et al. [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] investigated how the mucosubstance secretion is affected by commensal bacteria using the PAS staining method. They used intestinal epithelial cells of Caco-2 and HT29-MTX-E12 in the experiments. The highest average mucus thickness was obtained for digested \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e (14.38 \u0026micro;m) using HT29-MTX-E12 cells. When the cells exposed to \u003cem\u003eL. rhamnosus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, an increased mucus thickness and changes in secretion of mucins were investigated. Although, there has been a limited number of \u003cem\u003ein vitro\u003c/em\u003e research on how bacteria can affect mucin layer, those studies presented valuable data for better understanding the mechanisms in the intestinal area. Our results highlighted that LAB species and strain types, lactate concentration and initial mucin addition has significant effect on mucin/mucus thickness development.\u003c/p\u003e \u003cp\u003eFurther analysis of prototype probiotic candidates was performed by closer examination of a few strains. Consequently, six LAB strains with desired characteristics that were isolated from various sources (two reference cultures, two isolates of olives, one isolate of cheese, and one isolate of yogurt) were concentrated (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These bacteria exhibited good adhesion to mucin (4.52\u0026ndash;6.54 log CFU/mL), resistance to simulated gastric and intestinal conditions (10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e CFU/mL), and higher mucin thickness than control in all cell culture experiments when mucin was initially included (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Thus, out of the 16 LAB strains, those strains have a higher chance of inducing mucin secretion. The results of the sugar fermentation test are another crucial factor, and only two olive isolates of these six LAB strains (O. AK16 and O. AK50) did not use the mucin sugars (N-acetylglucosamine and N-acetylgalactosamine). These characteristics may be crucial in choosing probiotic strains that are appropriate for promoting \u003cem\u003eAkkermansia\u003c/em\u003e growth.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA few studies have been published in the literature that attempt to explain how the population of \u003cem\u003eAkkermansia\u003c/em\u003e in the microbiota is modulated through probiotic effects and/or mechanisms that prevent obesity. Therefore, the purpose of this study is to make predictions about how to identify the characteristics of \"Adhesion to Induction\" to expand the variety of probiotics in the colon microbiota and lower the ratio of the microbial community associated with obesity. Understanding the significance of adhesion properties to the mucin/mucus layer and the effect of lactate on mucin/mucus layer thickening by \u003cem\u003ein vitro\u003c/em\u003e HT-29 cell culture for LAB is necessary to predict the anti-obesity potential of probiotics.\u003c/p\u003e \u003cp\u003eThe study's findings suggest that two reference LAB strains (\u003cem\u003eL. acidophilus\u003c/em\u003e NRRL-B 1910 and \u003cem\u003eL. plantarum\u003c/em\u003e DSM 1954), two isolates from olives (O. AK16 and O. AK50), one from cheese (\u003cem\u003eL. paracasei\u003c/em\u003e C15), and one from yogurt (\u003cem\u003eS. thermophilus\u003c/em\u003e UIN9) may serve as potential anti-obesity prototype candidates. These findings may also make a unique contribution to the body of scientific literature. As a result, a favorable niche for the novel probiotic \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e can be established. Furthermore, among the probiotics of the new generation, \u003cem\u003eBacteroides, Clostridium\u003c/em\u003e, and \u003cem\u003eFaecalibacterium\u003c/em\u003e, may benefit from this environment created for \u003cem\u003eAkkermansia.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAs a result, it becomes critical for the development of new generation super probiotics to combat diseases like cancer, autoimmune diseases, metabolic diseases, cardiovascular diseases, and gastrointestinal issues. Thus, in addition to being effective against obesity, these six LAB strains may also be beneficial for inflammatory conditions, cardiovascular diseases, and leaky gut syndrome.\u003c/p\u003e \u003cp\u003eFurthermore, it is conceivable that advantageous bacteria might stick to the mucin layer and encourage the development of biofilms. Based on the information gathered from \u003cem\u003ein vitro\u003c/em\u003e cell culture investigations, \u003cem\u003ein vivo\u003c/em\u003e experiments with \u003cem\u003eAkkermansia\u003c/em\u003e can be more effectively planned for future research. Additionally, a novel functional food ingredient containing probiotics and polyphenols to induce \u003cem\u003eAkkermansia\u003c/em\u003e for the management of obesity can be developed.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e,\u0026nbsp;The absorbance values obtained in modified MRS medium without fermentable carbohydrates (Sugar-free-control); A\u003csub\u003e2\u003c/sub\u003e, The absorbance values obtained in modified MRS containing glucose or mucin; A\u003csub\u003eM/G\u003c/sub\u003e, Growth ability in the presence of mucin/glucose (A\u003csub\u003e2\u003c/sub\u003e-A\u003csub\u003e1\u003c/sub\u003e); ANOVA, Analysis of variance;\u0026nbsp;ARS, Agricultural research service; ATCC, American type culture collection;\u0026nbsp;BCP,\u0026nbsp;Bromocresol purple; CCM,\u0026nbsp;Czech collection of microorganisms; CFU,\u0026nbsp;Colony forming unit; cm\u003csup\u003e2\u003c/sup\u003e,\u0026nbsp;Centimeter power\u003csup\u003e\u0026ndash;2\u003c/sup\u003e; CNT, Control; CO\u003csub\u003e2\u003c/sub\u003e, Carbon dioxide; CV,\u0026nbsp;Crystal violet;\u0026nbsp;DSM, Deutsche Saturn lung von mikroorganismen;\u0026nbsp;dH\u003csub\u003e2\u003c/sub\u003eO,\u0026nbsp;Deionized water; DMEM,\u0026nbsp;Dulbecco\u0026apos;s modified eagle medium; e.g,\u0026nbsp;Exempli gratia (for example); eqn,\u0026nbsp;Equation; etc,\u0026nbsp;Et cetera; EtOH,\u0026nbsp;Ethanol;\u0026nbsp;g,\u0026nbsp;The relative centrifugal force (RCF);\u0026nbsp;g,\u0026nbsp;Gram;\u0026nbsp;GIT,\u0026nbsp;Gastrointestinal tract;\u0026nbsp;h,\u0026nbsp;Hour;\u0026nbsp;HCl,\u0026nbsp;Hydrochloric acid;\u0026nbsp;HPLC,\u0026nbsp;High-pressure liquid chromatography; HT-29, Human colorectal adenocarcinoma cell line; H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, Sulfuric acid; IZTECH-FED-MFMLCC, Izmir Institute of Technology, Department of Food Engineering, Molecular Food Microbiology Laboratory Culture Collection; L, Liter; LAB, Lactic acid bacteria; log, Logarithms; L20, 20 mM lactate; L50, 50 mM lactate; M, Molar; MDa, Megadalton; mg, Milligram; mL, Milliliter; mm, Millimeter; mM, Millimolar; min, Minute; MUCIN, Purified porcine gastric mucin-type III; MRS, De man, Rogosa and Sharpe; N, Normal; N\u003csub\u003e0\u003c/sub\u003e, Total number of viable LAB before implementation, CFU/mL; N\u003csub\u003e1\u003c/sub\u003e, Total number of viable LAB after gastric juice passage, CFU/mL; N\u003csub\u003e2\u003c/sub\u003e, Total number of viable LAB after intestinal juice passage, CFU/mL; NaCl, Sodium chloride; NGP, Next-generation of probiotics; nm, Nanometer; NRRL, Northern regional research lab; OD, Optical density; PAS, Periodic acid-Schiff; PBS, Phosphate buffered saline; PE, Perkin Elmer; pH, Quantitative measure of the acidity or basicity of aqueous or other liquid solutions (Power of hydrogen); rpm, Revolutions per minute; SCFA, Short-chain fatty acid; TSB, Tryptone soy broth; U, Units; v, Volume; w, Weight; %, Percent; \u0026deg;C, Centigrade degree; \u0026micro;g, Microgram; \u0026micro;L, Microliter; \u0026micro;m, Micrometer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Scientific and Technological Research Council of Turkey (TUBITAK) (Project number:121S123) and Izmir Institute of Technology Scientific Research Project (BAP) (Project number:2020IYTE0084). The authors greatly appreciate to Assoc. Prof. Dr. Olcay BOYACIOĞLU, for providing HT-29 cell culture. Assist. Prof. Dr. Mürüvvet ABBAK and Lecturer Özgür OKUR were appreciated for their\u0026nbsp;support\u0026nbsp;during the cell culture experiments carried out together.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and processed during this research\u0026nbsp;are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Scientific and Technological Research Council of Turkey (TUBITAK) within the scope of TUBITAK 1002 Rapid Support Program (Project number:121S123) and Izmir Institute of Technology Scientific Research Project (BAP) with the project number 2020IYTE0084.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions section\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the realization of the research and the writing of the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAslihan Kamber: Conceptualization, data curation, investigation, methodology, validation, writing-original draft, writing-review \u0026amp; editing; Cisem Bulut Albayrak: Data curation, investigation, methodology, resources, supervision, writing-review \u0026amp; editing. H. Sebnem Harsa: Conceptualization, data curation, investigation, methodology, project administration, resources, supervision, writing – original draft, writing-review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization (WHO) (2000) Obesity: preventing and managing the global epidemic. https://apps.who.int/iris/handle/10665/42330. Accessed 17 December 2022\u003c/li\u003e\n\u003cli\u003eLim JJ, Jung AH, Suh HJ, Choi HS, Kim H (2022) \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e K8-based paraprobiotics prevents obesity and obesity-induced inflammatory responses in high fat diet-fed mice. 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J Dairy Sci 105(10):7865-7877. https://doi.org/10.3168/jds.2022-21877\u003c/li\u003e\n\u003cli\u003eGuo M, Lu M, Chen K, Xu R, Xia Y, Liu X, Liu Z, Liu, Q (2023) \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e ameliorate systemic lupus erythematosus by possibly regulating immune response and remodeling gut microbiota. mSphere 8(4):e00070-23. https://doi.org/10.1128/msphere.00070-23\u003c/li\u003e\n\u003cli\u003eMack DR, Ahrn\u0026eacute; S, Hyde L, Wei S, Hollingsworth MA (2003) Extracellular MUC3 mucin secretion follows adherence of \u003cem\u003eLactobacillus\u003c/em\u003e strains to intestinal epithelial cells \u003cem\u003ein vitro\u003c/em\u003e. Gut 52(6):827-833. https://doi.org/10.1136/gut.52.6.827\u003c/li\u003e\n\u003cli\u003eJung TH, Park JH, Jeon WM, Han KS (2015) Butyrate modulates bacterial adherence on LS174T human colorectal cells by stimulating mucin secretion and MAPK signaling pathway. Nutr Res Pract 9(4):343. https://doi.org/10.4162/nrp.2015.9.4.343 \u003c/li\u003e\n\u003cli\u003eWrzosek L, Miquel S, Noordine ML, Bouet S, Chevalier-Curt MJ, Robert V, Philippe C, Bridonneau C, Cherbuy C, Robbe-Masselot C, Langella P, Thomas M (2013) \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e and \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent. BMC Biol 11:1-13. https://doi.org/10.1186/1741-7007-11-61\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLactic acid bacteria strains used in the experiments and their viabilities during simulated gastrointestinal passage.[1]\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"723\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Bacteria After Simulated Gastric Fluid (CFU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Bacteria After Simulated Intestinal Fluid (CFU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 4496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. 4496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e2.45 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e2.09 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 1910\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eA. 1910\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1.82 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e7.25 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. casei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 441\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. 441\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e3.29 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e2.17 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. rhamnosus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 442\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eR. 442\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e4.32 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e1.08 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. pentosus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 227\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. 227\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1.65 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;DSM 1954\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDSM 1954\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e3.49 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e2.8 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eCCM 4757\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCCM 4757\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;AS95\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eHuman breast milk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. AS95\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. C74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C77a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. C77a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C95-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. C95-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eC95-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. C95-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C97-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. C97-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CTY24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. CTY24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CTY41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. CTY41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CTY44\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. CTY44\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1.4 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUN5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. UN5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. UN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUN19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. UN19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1.1 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUIB31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. UIB31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUIN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT. UIN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1.19 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e7.1 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. paracasei\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eC8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePR. C8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e3 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. paracasei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePR. C15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e9.5 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e4.95 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePL. C47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e4.29 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e3.09 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;D1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. D1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e7.14 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e5.96 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eL. A1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e5.5 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;A19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eL. A19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e7.15 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e8.5 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;A20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eL. A20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;A22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eL. A22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e2 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eA23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eL. A23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eOlive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e3.25 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e3.5 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eGreen Olive Brine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e1 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e1.5 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eBlack Olive Brine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e2.4 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e5.4 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eBlack Olive (Sele type)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e7.35 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e5.9 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eBlack Olive (Sele type)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e̴ 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e1.11 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eBlack Olive Brine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003eDark Green Olive Brine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e5 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e7.55 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBT10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003ePickle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. BT10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBT14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003ePickle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. BT14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.532503457814661%\"\u003e\n \u003cp\u003e\u003cstrong\u003e40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.52420470262794%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBT40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.225449515905947%\"\u003e\n \u003cp\u003ePickle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.309820193637622%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. BT40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.289073305670815%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.118948824343015%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSugar fermentation abilities of lactic acid bacteria and identification of strains that do not use the mucin sugars.[2]\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria/Sugar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGlucose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGalactose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMannose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLactose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSucrose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eXylose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFructose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRhamnose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eFucose\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eN-acetylglucosamine\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eN-acetylgalactasomine\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 4496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 1910\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. casei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 441\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-(\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. rhamnosus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 442\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;DSM 1954\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;UIN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. paracasei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;D1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. lactis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;A19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e- (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB. O. AK50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+ (\u0026plusmn;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSummary for mucin/mucus layer supportive properties of selected lactic acid bacteria strains for different lactate concentrations.[3]\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"620\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth\u003e\u003cbr\u003e\u003c/th\u003e\n \u003cth\u003e\u003cbr\u003e\u003c/th\u003e\n \u003cth\u003e\u003cbr\u003e\u003c/th\u003e\n \u003cth\u003e\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLAB Strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean mucin/mucus thickness for HT-29 + Mucin (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean mucin/mucus thickness for HT-29 + Mucin + 20 mM Lactate (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean mucin/mucus thickness for HT-29 + Mucin + 50 mM Lactate (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NRRL-B 1910\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e71.2\u0026plusmn;19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22\u0026plusmn;28.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.64\u0026plusmn;7.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;DSM 1954\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.23\u0026plusmn;12.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.59\u0026plusmn;7.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e54.9\u0026plusmn;42.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36.08\u0026plusmn;5.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.63\u0026plusmn;13.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.6\u0026plusmn;57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eO. AK50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.81\u0026plusmn;8.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.68\u0026plusmn;5.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40.8\u0026plusmn;40.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eL. paracasei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.4\u0026plusmn;38.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.9\u0026plusmn;25.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70.4\u0026plusmn;44.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;UIN9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.2\u0026plusmn;21.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u0026plusmn;17.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e58.4\u0026plusmn;48.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e[1] *\u0026lt;10\u003csup\u003e5\u003c/sup\u003e for simulated gastric fluid and \u0026lt;10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003efor simulated intestinal fluid.\u003c/p\u003e\n\u003cdiv id=\"ftn2\"\u003e\n \u003cp\u003e[2] + (strong positive), + (\u0026plusmn;) (weak positive), - (\u0026plusmn;) (weak negative), - (strong negative).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"ftn3\"\u003e\n \u003cp\u003e[3] Statistical analyzed were applied by one way analysis of variance (ANOVA) and Tukey test (p \u0026gt; 0.05) for the differences between mean mucin amounts of 6 lactic acid bacteria strains. The data sets present normal distribution and results were expressed as mean \u0026plusmn; standard deviation in duplicates.\u003c/p\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactic acid bacteria, HT-29 cell culture, mucin/mucus layer, anti-obesity, probiotic, Akkermansia","lastPublishedDoi":"10.21203/rs.3.rs-4535093/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535093/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims: \u003c/strong\u003eThe primary goals of this work are to explore the potential of probiotic LAB's mucin/mucus layer thickening properties and to identify anti-obesity candidate strains that improve appropriate habitat for use with the \u003cem\u003eAkkermansia\u003c/em\u003e group population in the future.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e The HT-29 cell binding, antimicrobial properties, adhesion to the mucin/mucus layer, growth in the presence of mucin, stability during \u003cem\u003ein vitro\u003c/em\u003egastrointestinal (GI) conditions, biofilm formation, and mucin/mucus thickness increment abilities were all assessed for artisanal LAB strains. Sixteen LAB strains out of 40 were chosen for further analysis based on their ability to withstand GI conditions. Thirteen strains maintained their vitality in the simulated intestinal fluid, whereas the majority of the strains displayed high viability in the gastric juice simulation. Furthermore, 35.2-65.4% of those 16 bacteria adhered to the mucin layer. Besides, different lactate levels were produced, and \u003cem\u003eStreptococcus thermophilus\u003c/em\u003eUIN9 exhibited the highest biofilm development. Also, with a 50 mM lactate concentration and the presence of mucin that had been injected originally, \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e NRRL-B 441 had the maximum amount of mucin (321.6 µg/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Two isolates of olive bacteria from \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e were chosen as the anti-obesity prototype candidates; these strains did not consume mucin sugars.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignificance and Impact of the Study\u003c/strong\u003e: Probiotic LAB's attachment to the colonic mucosa and its ability to stimulate HT-29 cells to secrete mucus are critical mechanisms that may support the development of \u003cem\u003eAkkermansia.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Studies on the Probiotic, Adhesion and Induction Properties of Artisanal Lactic Acid Bacteria: To Customize a Gastrointestinal Niche to Trigger Anti-Obesity Functions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 13:19:55","doi":"10.21203/rs.3.rs-4535093/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-07T20:23:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T04:55:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-21T02:39:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267699934941564686836818526983712714696","date":"2024-06-14T20:34:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45467847146936046219599697515265793821","date":"2024-06-14T10:33:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-14T04:33:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-10T00:51:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T00:49:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2024-06-05T15:20:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5a73b9c6-1867-4ed6-a45d-b5a3dc7397b3","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:00:27+00:00","versionOfRecord":{"articleIdentity":"rs-4535093","link":"https://doi.org/10.1007/s12602-024-10357-6","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2024-10-09 15:57:16","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-06-24 13:19:55","video":"","vorDoi":"10.1007/s12602-024-10357-6","vorDoiUrl":"https://doi.org/10.1007/s12602-024-10357-6","workflowStages":[]},"version":"v1","identity":"rs-4535093","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535093","identity":"rs-4535093","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}