Screening of lactic acid bacteria with anti-adipogenic effect and potential probiotic properties from grains

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The bacterial strains were assigned as members of Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Lactococcus taiwanesis, Pediococcus pentosaceus, and Weissella paramesenteroides based on the closest similarity using 16S rRNA gene sequence analysis. The strains belonging to the same species were analyzed using RAPD-PCR, and one or two among strains showing the same band pattern were selected. Finally, 25 representative strains were selected for further functional study. Inhibitory effects of lipid accumulation were observed in the strains tested. Pediococcus pentosaceus K28, Levilactobacillus brevis RP21 and Lactiplantibacillus plantarum RP12 significantly reduced lipid accumulation and decreased expression of six adipogenic marker genes in C3H10T1/2 adipocytes. The three strains survived under strong acidity and bile salt conditions. The three strains showed adhesion to Caco-2 cells similar to a reference strain LGG. The resistance of the three strains to several antibiotics was also assessed. Strains RP12 and K28 were confirmed not to produce harmful enzymes based on API ZYM kit results. Based on these results, strains K28, RP21 and RP12 isolated from grains had the ability to inhibit adipogenesis in adipocytes and potentially be useful as probiotics. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Diet-induced obesity is a major factor in many chronic diseases such as cardiovascular disease, non-alcoholic fatty liver disease (NAFLD) and type2 diabetes, and is considered a serious health concern 1 , 2 . Currently, drug treatment and surgery with mechanisms that inhibit lipid absorption and suppress appetite are used worldwide to prevent and treat obesity. However, in the case of drugs, continuous administration is limited due to side effects, and a risk of complications in surgery is possible 3 , 4 . Therefore, non-drug therapies that are safer and improve the balance of metabolism are needed to treat and prevent obesity and probiotics have been proposed as an alternative 5 . Probiotics are defined as live microorganisms that provide the host with health benefits when administered in proper quantities 6 . The proven effects of probiotics include alleviation of intestinal inflammation, allergy prevention, reduced total serum cholesterol and inflammation and anti-cancer activity 7 – 11 . Among them, several probiotics are also known to have potential anti-obesity effects such as reduced body fat, blood sugar control and cholesterol improvement 12 . Lactic acid bacteria (LAB) is the main bacteria in probiotics 13 . According to the American Food and Drug Administration, LAB are generally regarded as safe (GRAS). LAB is widely used in the food industry, and studies of LAB functionality are growing in popularity. In several recent studies, LAB was shown to alleviate obesity by decreasing body weight, improving inflammatory state or glucose tolerance, and altering gut microbiota in diet-induced obese mice 14 – 16 . Grains are a major staple food in Asia. Grains mainly contain carbohydrates including fiber and oligosaccharides, which makes them an excellent source of prebiotics defined as “a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” 17 , 18 . A microbial community composed of LAB was shown to exist in grains 18 . In a previous study, LAB isolated from grains exerted antimicrobial properties 19 ; however, few studies about useful properties, including anti-obesity effects, have been performed. The objectives of the present study were to isolate LAB from various grains collected in the Republic of Korea and to screen lactic acid bacteria with inhibition of adipogenesis. The selected strains with anti-adipogenic effects were investigated to determine whether they have useful properties as probiotic candidates for development as functional food. Material And Methods Isolation of LAB strains and growth conditions. Four types of grains, rice, brown rice, black rice and hulled barley, were collected in the Republic of Korea. The grains were ground using blender and enriched for 7 days with distilled water under aerobic conditions. The grain samples were serially diluted with 0.85% (w/v) saline solution and spread on De Man-Rogosa-Sharpe (MRS; BD Difco, Sparks, MD, USA) agar. After incubation for 48 h at 30 ℃ or 37 ℃, LAB were isolated from the MRS agar plates and cultivated for 18 h at 30 ℃ or 37 °C in MRS agar. Lacticaseibacillus rhamnosus LGG (KCTC 5033), which was used as the experimental control strain for comparative analyses, was purchased from the Korean Collection for Type Cultures (Daejeon, Republic of Korea), and cultured at 37 ℃. All strains, including the experimental control strain, were stored at - 80 ℃ after suspension in 20% (w/v) glycerol solution (Georgiachem, GA, USA). 16S rRNA gene sequence analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. Genomic DNA was extracted using a G-spin genomic extraction kit (iNtRON, Seongnam, Republic of Korea), according to the manufacturer’s protocol. Polymerase chain reaction (PCR) amplification, purification, and sequencing of 16S rRNA gene were performed as described previously 20 . Identification of the closest phylogenetic species based on 16S rRNA gene sequence was performed using the EzBioCloud server (https://www.ezbiocloud.net/) 21 . Random amplified polymorphic DNA-PCR (RAPD-PCR), which was used to exclude replicates among LAB strains, was performed using two primers, ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) and ERIC1R (5’-ATGTAAGCTCCTGGGGATTCAC-3’) as described previously 22 . The PCR products were electrophoresed on 1.5% (w/v) agarose (LPS Solution, Daejeon, Republic of Korea) gel for 60 min, and after electrophoresis, the gel was stained with RedSafe (iNtRON, Seongnam, Republic of Korea). Preparation of cell extract from LAB. Cell extracts of LAB were prepared as described previously 23 with minor modifications. The cell mass was harvested using centrifugation and washed twice with phosphate-buffered saline (PBS, pH 7.2). The washed cells were resuspended in distilled water at a concentration of 100 mg/mL and sonicated using the method described previously 16 . The sonicated cell extracts were centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were filtered using a 0.45 µM syringe filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany) and lyophilized. The resulting powder was dissolved in sterile water to appropriate concentrations. Cell culture, adipocyte differentiation and intracellular triglyceride content. C3H10T1/2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured following the method described previously 24 . A subculture of C3H10T1/2 cells was performed with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (penicillin and streptomycin, Hyclone). After seeding in 12-well plates, C3H10T1/2 cells were cultured in DMEM containing 10% FBS and antibiotics until confluency. Confluent cells were induced into adipocytes in DMEM supplemented with 10% FBS, antibiotics, 20 nM GW1929 (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/mL insulin (Sigma-Aldrich). After 48 h, the differentiating cells were refreshed with media containing DMEM, 10% FBS, 20 nM GW1929, and 10 μg/mL insulin. Cell extracts of LAB were adjusted by suspending to a concentration of 25,50, and100 μg/mL with sterile distilled water to create the same conditions. LAB cell extracts were treated during adipocyte differentiation of C3H10T1/2 cells, and sterile distilled water was treated as control. Then, the differentiated C3H10T1/2 cells were fixed with 4% formaldehyde (Sigma-Aldrich) in PBS (Hyclone) at room temperature overnight and stained with Oil Red O (Sigma-Aldrich). To quantify intracellular triglyceride content, stained cells from at least two independent experiments were resolved in isopropanol (Sigma-Aldrich) and measured with a spectrophotometer at 520 nm. Cell viability assay. Cell viability was determined using methyl thiazolyl tetrazolium salt (MTS) colorimetric assays (ab197010, Abcam). C3H10T1/2 cells were seeded at 1.5 × 10 4 cells per well in 96-well plates, then treated with various strain doses (1, 12.5, 25, 50, 100, and 200 μg/mL) and sterile distilled water as control in triplicate. After 24 h, MTS (20 μL/100 μL in medium) was added into the media and cells incubated for 4 h at 37 ℃. The absorbance of formazan dye was measured at 490 nm using a microplate reader (BioTek, Winooski, VT, USA). Quantitative real-time polymerase chain reaction (RT PCR) analysis. Total RNA was extracted from C3H10T1/2 cells using QIAzol lysis reagent (QIAGEN, Germantown, MD, USA). First-strand complementary DNA was synthesized from 0.5 μg of total RNA using ReverTra Ace Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Quantitative RT PCR was performed in 25 μL final reaction volume containing Power SYBR Premix ExTaq (RP041A; Takara, Shiga, Japan), primers, and cDNA using thermal cycler machine (Takara). The primer sequences used for the PCR were described previously 24 . Tolerance assays against acid and bile salts. Tolerance to acid was measured as described previously 25 with minor modifications. LAB strains were cultured overnight (18 h) at 30 °C (for RP21 and K28) or 37 °C (for RP12 and LGG), harvested for 10 min at 7,000 rpm at 4 °C, and washed twice with PBS buffer (pH 7.2). Bacterial cells (approximately 10 9 CFU/mL) were resuspended in liquid MRS medium (pre-adjusted to pH 1.0, 2.0, 2.5, and 3.0) and incubated for 3 h at optimal temperatures. Viability was determined in triplicate in terms of viable colony counts using the plate count method. Tolerance to bile salts was measured as described previously 26 with minor modifications. LAB strains were suspended in liquid MRS medium containing 0.3, 0.5, 1.0, and 2.0% oxgall (Sigma-Aldrich) at a concentration of approximately 10 9 CFU/mL. After incubation for 6 h at 30 °C or 37 °C, suspension was poured into MRS agar plates and incubated at optimum growth temperatures for 48 h. Tolerance assays against acid and bile salts were performed in triplicate and LGG was used as the comparative strain. In vitro adhesion assays. The adherence assay was performed according to the method described previously 27 with minor modifications. Caco-2 cells used for the adherence assay were purchased from the Korean Cell Line Bank (Seoul, Korea). The Caco-2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) FBS (Hyclone) and 1% (v/v) penicillin-streptomycin at 37 °C in 5% CO 2 atmosphere. The Caco-2 cells were seeded at 2 × 10 5 cells/well in 6-well tissue culture plates. The adherence assay was performed at post-confluence. The monolayer was washed with sterile PBS (Hyclone) twice. LAB cells were diluted with DMEM to approximately 10 9 CFU/mL and added to the wells. Plates were incubated for 90 min at 37 °C in 5% CO 2 atmosphere. The Caco-2 monolayers were washed three times with sterile PBS (Hyclone) and treated with EDTA-trypsin solution for 3 min. The cell suspensions were serially diluted and spread on MRS agar plates. Cell viability was counted after incubation for 48 h. The adhesion ability of LAB was calculated as the percentage between remaining bacteria and initial bacteria per well. The same passage Caco-2 cells were used in adhesion assays and assays were repeated in triplicate. Antibiotic susceptibility. Susceptibility to antibiotics was examined using the disc-diffusion method with application of modified agar diffusion method described previously 28,29 . LAB inoculated in MRS agar were adjusted to approximately 10 8 CFU/mL and paper discs (Advantec, Tokyo, Japan) were dispensed. Each disc was treated with 10 μL of specific antibiotic. The concentrations of antibiotics tested are listed in Supplementary Table 1. The inhibition zone diameters were measured and evaluated in terms of sensitive, intermediate sensitive, and resistant according to the interpretative standard table (Supplementary Table 1). The 2013 Clinical and Laboratory Standards Institute criteria 30 were used for interpretation. Enzyme activity test. Enzyme activity of the LAB was investigated as described previously 16 using the API ZYM kit (BioMérieux, Marcy l’Etoile, France). Statistical analysis. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. Significance differences between groups in triglyceride content were determined using Duncan's multi-range test. Significance differences in gene expression and adhesion ability were determined by comparison with control using two-tailed unpaired Student’s t -test. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Inc. software (version 19.0). Results And Discussion Isolation and identification of LAB strains from grains . Bacterial strains were isolated from four types of grains collected in the Republic of Korea and a total of 187 LAB strains were obtained through 16S rRNA gene sequencing followed by identification. From the 16S rRNA gene sequence analyses of the LAB strains, 20 strains had the closest similarities to the type strain of Levilactobacillus (previously Lactobacillus ) brevis , 3 strains had the closest similarities to the type strain of Latilactobacillus (previously Lactobacillus ) curvatus , 22 strains had the closest similarities to the type strain of Lactiplantibacillus (previously Lactobacillus ) plantarum , 17 strains had the closest similarities to the type strain of Lactococcus taiwanesis , 82 strains had the closest similarities to the type strain of Pediococcus pentosaceus , and 43 strains had the closest similarities to the type strain of Weissella paramesenteroides . The genus Lactobacillus has been recently reclassified as 25 genera including Levilactobacillus , Latilactobacillus , and Lactiplantibacillus 31 . Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Pediococcus pentosaceus , and Weissella paramesenteroides have been shown to be isolated from grains 32,33 . In RAPD-PCR analysis, six different band patterns were assigned to 82 strains with the closest 16S rRNA gene sequence similarities to Pediococcus pentosaceus , and four different band patterns were assigned to 43 strains with the closest 16S rRNA gene sequence similarities to Weissella paramesenteroides (Supplementary Fig. 1). The strains assigned as Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum , and Lactococcus taiwanesis each showed only one type of band pattern (Supplementary Fig. 1). Finally, two representative strains from each group, except for the three groups having only one strain, were randomly selected, and used for further functional characterization (Supplementary Fig. 1; Table 1). Screening of strains with anti-adipogenic effects. Inhibitory effects of lipid accumulation were tested by treating the LAB cell extract on C3H10T1/2 cells. Because the strains assigned to Lactococcus taiwanensis and Weissella paramesenteroides caused cell damage during the treatment process, they were excluded from the test. A wide range of inhibitory effects of lipid accumulation was observed in the strains selected. Among the strains tested, five strains ( Pediococcus pentosaceus K28; Levilactobacillus brevis RP20 and RP21; Lactiplantibacillus plantarum RP11 and RP12) reduced lipid accumulation by more than 20% compared with the control, indicating that they have anti-adipogenic effects (Fig. 1). The two strains (RP20 and RP21) assigned to Levilactobacillus brevis and the two strains (RP11 and RP12) of Lactiplantibacillus plantarum showed similar results, respectively (Fig. 1). Thus, one strain from RP20 and RP21 and one strain from RP11 and RP12 were selected, and the three strains (K28, RP21 and RP12) were used for further experiments. The above results indicate that the components of LAB cell extract might influence the adipocyte differentiation process, thereby suppressing fat production. Significant diversity exists among LAB strains regarding functional characteristics that benefit health, such as antioxidant, antitumor, immunomodulatory, and hypocholesterolemic activities 34-40 . In several studies, the cellular components of LAB have shown beneficial effects on improving health 38,41,42 . It is not clear which substance(s) in LAB cell extract induce the anti-adipogenic effects. Exopolysaccharide (EPS) has been known to have anti-adipogenic effects 23 . The EPS, a cell wall component of LAB cells, is loosely associated with the cell envelope and easily released into the surrounding environment 43,44 . Effects of LAB strains on cell viability of C3H10T1/2. The cytotoxicity at various concentrations of strains LGG, K28, RP21, and RP12 on C3H10T1/2 cells was investigated by measuring cell viability using the MTS assay. C3H10T1/2 cells were found viable at all treatment concentrations of the four strains (Fig. 2). Inhibition of adipogenic gene expression by LAB extract during adipocyte differentiation. Inhibition of adipogenesis by strains K28, RP21 and RP12 was investigated by measuring expression of six adipogenic genes using quantitative RT PCR (Fig. 3). PPARγ and C/EBPα are transcription factors that regulate the process of adipocyte differentiation 45,46 . In addition, the activation of PPARγ promotes the expression of adipogenic genes, such as CD36 and LPL , which are important for the uptake and storage of triglycerides 47 . The down regulation of these adipogenic genes may affect decreased lipid accumulation in cells. Fatty acid synthase ( FAS ) gene is a downstream adipocyte gene that contributes to fatty acid synthesis 48 . Acetyl-coenzyme A carboxylase ( ACC ) is another key enzyme for fatty acid synthesis that catalyzes the synthesis of malonyl-CoA 49 . Cell extracts from strains K28, RP21, and RP12 decreased the expression of adipocyte-related genes in C3H10T1/2 cells (Fig. 3). The expression of the six genes decreased proportionally with increasing concentrations of the extracts of the three strains (Fig. 3). The three strains significantly reduced (p < 0.01 or 0.001) the expression of PPARγ and C/EBPα in all concentrations tested. In addition, expression of four other genes associated with adipogenesis, was significantly reduced (p < 0.05 or 0.01) in the three strains, except for LPL expression in 25 mg/mL treatment of strains K28 and RP12. Strain K28, which showed the lowest lipid accumulation, was analyzed to have the lowest values in expressions of the six genes after 100 mg/mL treatment (Fig. 3). These results indicate that the three strains may have anti-adipogenic effects by inhibiting the expression of adipogenesis-related genes. Tolerance against acid and bile salts. To have specific functionality, a probiotic must reach the intestines alive with resistance to acid and bile salts 50 . The acid tolerance of the selected strains and LGG as a reference strain was examined after incubation for 3 h in pH 3.0, 2.5, 2.0, and 1.0 (Table 2). The three strains and LGG maintained the values of more than 9 log CFU/mL at pH 3. Under pH 2.5 condition, the survival rates of strains K28 and RP21 decreased more than 2 log and approximately 1 log, respectively, whereas strains RP12 and LGG showed decreases of more than 3 log and 2 log, respectively (Table 2). Under pH 2 condition, the survival rate of strains K28 and RP21 decreased approximately 3 log and 2 log, respectively, and the survival rate of strains RP12 and LGG decreased approximately 6 log (Table 2). The three strains, except RP21 with approximately 3 log CFU/mL, showed low viability of less than 2 log CFU/mL at pH 1 (Table 2). Strain RP21 was also found to have higher acid resistance, as a strain of Levilactobacillus ( Lactobacillus ) brevis was shown highly acid-resistant in a previous study 51 . The pH of gastric fluid in the body is maintained at approximately 3.0, and probiotics are generally known to be highly acid-resistant if they are maintained at pH 3 for approximately 3 h 52 . Thus, the three strains were concluded to be highly tolerant to acid. Because food matrix can help the survival of LAB in the gastrointestinal tract due to its buffering capacity, the strains are expected to have stronger viability when used with carrier foods 53 . Bile salts are another factor that can reduce bacterial survival in the gastrointestinal tract by destroying cell membranes 50 . Strains K28, RP21 and RP12 were found to survive after 6 h exposure to 0.3, 0.5, 1.0, and 2.0% bile salts, similar to LGG which is known to be highly resistant to bile salts (Table 2). Although the in vitro assay cannot provide the same conditions as the gastrointestinal tract, it is recognized as an effective evaluation method to select potential strains when using proper criteria 27 . Adherence to Caco-2 cells. The adhesion ability of probiotics is a main factor that can increase the possibility of their survival and colonization in the gastrointestinal tract 54 . Adhesion is also required to prevent attachment of pathogenic bacteria through competition in intestinal epithelium 55 . Thus, the adherence ability has been considered an important biological property for the selection of useful probiotic strains 56 . In the present study, the adhesion ability of the three strains was evaluated using Caco-2 cells, which have morphological and physiological properties of human enterocytes, and their adhesion abilities were compared with that of the reference strain LGG (Fig. 4). Strain K28 had stronger adhesion ability than those of LGG and the two other strains (Fig. 4). The adhesion ability of strain K28 was highest at 1.95%, followed by LGG (1.79%), RP12 (1.67%), and RP21 (1.46%). Antibiotic susceptibility. Probiotics have been widely used in various fields including food and medical industries. Antimicrobial sensitivity for evaluation of probiotics is considered important for safety, because the resistant genes can be horizontally transferred to pathogenic bacteria, which can become a serious threat 57 . Sensitivity results of the strains for nine antibiotics used in this study are listed in Supplementary Table 1. For the nine antibiotics tested, strains K28, RP21 and RP12 showed sensitivity patterns similar to strain LGG. In this study, all four strains were equally sensitive to chloramphenicol and rifampicin, whereas strains K28 and RP21 were intermediate sensitive to tetracycline and strains RP12 and LGG were sensitive to tetracycline (Supplementary Table 1). Sensitivity or intermediate sensitivity of Lactobacillus species and Pediococcus species to chloramphenicol and tetracycline has been previously reported 58,59 . Strains K28, RP21, RP12 and LGG were resistant to gentamycin, kanamycin, and streptomycin, which are known to inhibit protein synthesis targeting Gram-negative bacteria. The resistance to aminoglycoside antibiotics is an intrinsic property among Lactobacillus species and Pediococcus species 60 . Therefore, the three strains are unlikely to cause safety problems based on antibiotic susceptibility profile tested. Enzyme production. For the safety of probiotic strains, it may be required to assess whether the strains produce harmful enzyme. β-glucuronidase is known as the carcinogen enzyme, which may increase the likelihood of tumor induction in the colon 61,62 . When the three strains were evaluated using API ZYM kit, strains RP12 and K28 did not produce any harmful enzymes such as β-glucuronidase, but strain RP21 was observed to produce β-glucuronidase (Supplementary Table 2). Conclusion In the present study, 187 LAB strains were isolated from four types of grains and identified using 16S rRNA gene sequence analysis. The 25 strains selected based on RAPD-PCR analysis were subjected to functional characterization. Among the strains tested, Pediococcus pentosaceus K28, Levilactobacillus brevis RP21, and Lactiplantibacillus plantarum RP12 had the potential to be useful probiotic candidates based on several characteristic analyses. The three strains exerted inhibitory effects on lipid accumulation and adipocyte differentiation by decreasing the expression of adipocyte-related genes. In addition, the three strains showed good tolerance against acid and bile salts, good intestinal cell adhesion, and were sensitive to chloramphenicol and rifampicin. In particular, strains RP12 and K28 did not produce β-glucuronidase. Therefore, Pediococcus pentosaceus K28, Levilactobacillus brevis RP21 and Lactiplantibacillus plantarum RP12 were concluded to have potential as probiotic candidates for use as functional neutraceutical foods. Declarations Acknowledgements This work was supported by "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ015247)" of Rural Development Administration, Republic of Korea and BK21 plus project of the Ministry of Education, Republic of Korea. Author contributions M.J.S.and J.H.Y. conceived and designed the study. S.M.W., M.J.K., J.H.S., E.B.L., and J.H.C. performed the experiments. M.J.S.and J.H.Y. analyzed the data and wrote the manuscript. K.W.P. supported and discussed the study. J.H.Y. reviewed and edited manuscript. K.W.P. and J.H.Y. supervised the study. All authors read and approved the manuscript. Data Availability 16S rRNA gene sequences of strains K28, RP21 and RP12 have been deposited in the National Centre for Biotechnology Information (NCBI) under GenBank accession numbers ON724233, ON724232 and ON724263, respectively. Competing interests The authors declare that there is no conflict of interest. References Ford, N. D,, Patel, S. A. & Narayan, K. M. Obesity in low- and middle-income countries: burden, drivers, and emerging challenges. Annu. Rev. Public Health 38, 145-164 (2017). Mazzotti, A. et al. Which treatment for type 2 diabetes associated with non-alcoholic fatty liver disease? Dig. Liver Dis. 49, 235-240 (2017). Bessesen, D. H. & Van Gaal, L. F. 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Lactobacillus rhamnosus GG improves insulin sensitivity and reduces adiposity in high-fat diet-fed mice through enhancement of adiponectin production. Biochem. Biophys. Res. Commun. 431, 258-263 (2013). Lee, J. W. et al . Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum . J. Vet. Sci. 5, 41-48 (2004). Liu, C. F. et al . Immunomodulatory and antioxidant potential of Lactobacillus exopolysaccharides. J. Sci. Food Agric. 91, 2284-2291 (2011). Miyoshi, M. et al . Anti-obesity effect of Lactobacillus gasseri SBT2055 accompanied by inhibition of pro-inflammatory gene expression in the visceral adipose tissue in diet-induced obese mice. Eur. J. Nutr. 53, 599-606 (2014). Jeung, W. H. et al . Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 cell extracts inhibit adipogenesis in 3T3-L1 and HepG2 cells. J. Med. Food 21, 876-886 (2018). Kim, J. Y. et al . Screening for antiproliferative effects of cellular components from lactic acid bacteria against human cancer cell lines. Biotechnol. Lett. 24, 1431-1436 (2002). Bhat, B. & Bajaj, B. K. Hypocholesterolemic potential and bioactivity spectrum of an exopolysaccharide from a probiotic isolate Lactobacillus paracasei M7. Bioact. Carbohydr. Diet. Fibre. 19, 100191 (2019). Kleerebezem, M. et al . The extracellular biology of the lactobacilli. FEMS Microbiol. Rev. 34, 199-230 (2010). Gregoire, F. M., Smas, C. M. & Sul, H. S. Understanding adipocyte differentiation. Physiol. Rev. 78, 783-809 (1998). Rosen, E. D. et al . PPARγ is required for the differentiation of adipose tissue in vivo and in vitro. Mol. Cell 4, 611-617 (1999). Tontonoz, P. & Spiegelman, B. M. Fat and beyond: the diverse biology of PPARg. Annu. Rev. Biochem. 77, 289-312 (2008). Liou, C. J. et al . Protective effects of licochalcone A ameliorates obesity and non-alcoholic fatty liver disease via promotion of the Sirt-1/AMPK pathway in mice fed a high-fat diet. Cells 8, 47 (2019). Li, K. K. et al . Cocoa tea (Camellia ptilophylla ) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes. Sci. Rep. 6, 20172 (2016). Succi, M. et al . Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol. Lett. 244, 129-137 (2005). Wu, C. H. et al . Characterization of a potential probiotic Lactobacillus brevis RK03 and efficient production of γ-aminobutyric acid in batch fermentation. Int. J. Mol. Sci. 19, 143 (2018). Guo, X. H. et al . Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties. Anaerobe 16, 321-326 (2010). Linares, D. M. et al . Lactic acid bacteria and bifidobacteria with potential to design natural biofunctional health-promoting dairy foods. Front. Microbiol. 8, 846 (2017). García-Cayuela, T. et al . Adhesion abilities of dairy Lactobacillus plantarum strains showing an aggregation phenotype. Food Res. Int. 57, 44-50 (2014). Monteagudo-Mera, A. et al . Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health. Appl. Microbiol. Biotechnol. 103, 6463-6472 (2019). Tuomola, E. M. & Salminen, S. J. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int. J. Food Microbiol. 41, 45-51 (1998). Gueimonde, M. et al . Antibiotic resistance in probiotic bacteria. Front. Microbiol. 4, 202 (2013). Hummel, A. S. et al . Antibiotic resistances of starter and probiotic strains of lactic acid bacteria. Appl. Environ. Microbiol. 73, 730-739 (2007). Maragkoudakis, P. A. et al . Probiotic potential of Lactobacillus strains isolated from dairy products. Int. Dairy J. 16, 189-199 (2006). Singla, V. et al . Antibiotic susceptibility profile of Pediococcus spp. from diverse sources. 3 Biotech. 8, 489 (2018). Hatakka, K. et al . The influence of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in human colon. Int. J. Food Microbiol. 128, 406-410 (2008). Monteagudo-Mera, A. et al . Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products. J. Food Prot. 74, 1379-1386 (2011). Tables Table 1 Lactic acid bacteria (n= 25) isolated from the four kinds of grains and a reference strain (LGG) used in this study. Selected strain Closest species by 16S rRNA gene sequence analysis Isolation source Number of strain selected RP20, RP21 Levilactobacillus brevis Rice 2 RP42, RP50 Latilactobacillus curvatus Rice 2 RP11, RP12 Lactiplantibacillus plantarum Rice 2 B5, B6 Lactococcus taiwanensis Black rice 2 H2, H6 Pediococcus pentosaceus Brown rice 2 K21, K22 Hulled barley 2 H13, H15 Brown rice 2 K28 Hulled barley 1 H11 Brown rice 1 K25 Hulled barley 1 B12, B13 Weissella paramesenteroides Black rice 2 B32, B45 Black rice 2 H8, H23 Brown rice 2 B40, H7 Brown rice, Black rice 2 LGG (KCTC 5033) Lacticaseibacillus rhamnosus Reference strain 1 Table 2 Acid and bile tolerance of strains K28, RP21 and RP12 and a reference strain (LGG) (log CFU/ml). Strain Acid condition Bile salts pH 3.0 pH 2.5 pH 2.0 pH 1.0 0.3% 0.5% 1.0% 2.0% Initial mean counts 3h Initial mean counts 3h Initial mean counts 3h Initial mean counts 3h K28 9.58±0.08 9.56±0.04 9.63±0.01 7.30±0.05 9.74±0.01 6.76±0.01 9.82±0.01 1.78±0.04 + a + + + RP21 9.71±0.07 9.59±0.03 9.87±0.01 8.66±0.01 9.73±0.01 7.65±0.01 9.96±0.04 3.41±0.06 + + + + RP12 9.20±0.04 9.18±0.05 9.24±0.01 5.38±0.09 9.24±0.01 3.15±0.10 9.12±0.04 1.65±0.09 + + + + LGG 9.55±0.03 9.59±0.09 9.34±0.04 6.94±0.03 9.48±0.07 3.56±0.09 9.58±0.09 1.76±0.03 + + + + a +, Survival. All values are Mean±SEM (n=3) Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2023 Read the published version in Scientific Reports → Version 2 posted Editorial decision: Major revision 16 Sep, 2022 Reviews received at journal 11 Aug, 2022 Reviewers agreed at journal 20 Jul, 2022 Reviewers invited by journal 20 Jul, 2022 Editor assigned by journal 14 Jul, 2022 Editor invited by journal 14 Jul, 2022 Submission checks completed at journal 14 Jul, 2022 First submitted to journal 27 May, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-1640993","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-05-18 16:17:44","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Article","associatedPublications":[],"authors":[{"id":125941270,"identity":"61eef707-0197-44aa-b3b1-9e67d191b6d8","order_by":0,"name":"Min Ju Seo","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"Ju","lastName":"Seo","suffix":""},{"id":125941271,"identity":"d7284acf-d028-40ea-821f-eb28a49aedf7","order_by":1,"name":"Sung-Min Won","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung-Min","middleName":"","lastName":"Won","suffix":""},{"id":125941272,"identity":"226dee44-870b-4e4c-a03e-2d80e8c0f5f7","order_by":2,"name":"Min Ju Kwon","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"Ju","lastName":"Kwon","suffix":""},{"id":125941273,"identity":"a8456561-2db4-4a74-9804-35ff731053c3","order_by":3,"name":"Ji Hyeon Song","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Hyeon","lastName":"Song","suffix":""},{"id":125941274,"identity":"ba040ce4-65d6-4203-aa53-f479c46ec51f","order_by":4,"name":"Eun Bee Lee","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eun","middleName":"Bee","lastName":"Lee","suffix":""},{"id":125941275,"identity":"e227f12e-ef0c-4199-8a79-da62f60d5a68","order_by":5,"name":"Jun Hyeong Cho","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"Hyeong","lastName":"Cho","suffix":""},{"id":125941276,"identity":"3434fb56-2a3c-41c4-9349-78c5149bcaf1","order_by":6,"name":"Kye Won Park","email":"","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kye","middleName":"Won","lastName":"Park","suffix":""},{"id":125941277,"identity":"d0b6c459-8ea2-48dc-8980-7467904b2ee4","order_by":7,"name":"Jung-Hoon Yoon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxhlgykaOn4GNGSzQQKSWNGPJBmK1MEiAyUOJGw4Qq4V5do/hg487DiRuPn8s2ZiHwUZ2wwFCDptzxthw5pk7xttupB1O5gG6kLCWGTlm0rxtz2S33WBvPszDcDiRWC2HGTf3Hwdp+U+8FsUNDGCHHSBGS1qx4cy2NGOJG2nJhnMMko1nEtJiOCN544OPbcCo7D9mLPGmwk62j6CWBg4DJK4BToUIIM/A/oAIZaNgFIyCUTCiAQBph0aNUIteLAAAAABJRU5ErkJggg==","orcid":"","institution":"Sungkyunkwan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jung-Hoon","middleName":"","lastName":"Yoon","suffix":""}],"badges":[],"createdAt":"2022-05-10 08:29:21","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1640993/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1640993/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-36961-0","type":"published","date":"2023-07-07T21:31:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":24732007,"identity":"cdc7d7f6-9be5-43f2-be9a-f88f2584756d","added_by":"auto","created_at":"2022-08-03 16:55:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27880,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect on lipid accumulation of LAB strains isolated from grains. Each strain was treated as cell extract at a concentration of 50 μg/mL. Values of each sample were determined relative to control. Values are presented as the mean ± SEM of three independent experiments.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/599579807df2071b02e4383e.png"},{"id":24732009,"identity":"8186fb8d-b893-4925-ba87-7367a9875740","added_by":"auto","created_at":"2022-08-03 16:55:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20423,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LAB treatment at different concentrations on viability of C3H10T1/2 cells. The C3H10T1/2 cells were treated with sterile distilled water (control) or LAB strains and their viability was determined using the MTS assay. Data are presented as the mean ± SEM from three independent experiments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/bcecf5a185a4a72d536fd7ab.png"},{"id":24732010,"identity":"a9f514e9-92f5-4b99-9828-23466ca9dd56","added_by":"auto","created_at":"2022-08-03 16:55:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20745,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of selected LAB treatment on expression of adipogenic genes in C3H10T1/2 cells. The C3H10T1/2 cells were treated with the indicated concentrations during differentiation for 6 days and expression of adipocyte markers was measured. The mRNA expression levels of peroxisome proliferator-activated receptor γ (\u003cem\u003ePPARγ\u003c/em\u003e), CCAAT-enhancer-binding protein-α (\u003cem\u003eC/EBPα\u003c/em\u003e), lipoprotein lipase (\u003cem\u003eLPL\u003c/em\u003e), fatty acid synthase (\u003cem\u003eFAS\u003c/em\u003e), cluster of differentiation 36 (\u003cem\u003eCD36\u003c/em\u003e), and acetyl-coenzyme A carboxylase (\u003cem\u003eACC\u003c/em\u003e) were measured using quantitative real-time polymerase chain reaction. Data are expressed as mean ± SEM of three independent experiments. Significant difference between selected strains (\u003cem\u003ePediococcus pentosaceus \u003c/em\u003eK28, \u003cem\u003eLevilactobacillus brevis \u003c/em\u003eRP21 and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e RP12) and control are indicated as *p \u0026lt; 0.05, ** p\u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/14e5f9b42b824b9f905ad131.png"},{"id":24732008,"identity":"43e3c40b-a1e0-4ef3-aa48-d474f3413779","added_by":"auto","created_at":"2022-08-03 16:55:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10802,"visible":true,"origin":"","legend":"\u003cp\u003eAdhesion ability of \u003cem\u003ePediococcus pentosaceus \u003c/em\u003eK28, \u003cem\u003eLevilactobacillus brevis \u003c/em\u003eRP21, and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e RP12 to Caco-2 epithelial cells compared with the reference strain LGG. Data are expressed as mean ± SEM of three independent experiments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/79d361bfe5d40f1d704726be.png"},{"id":44732647,"identity":"9acf9fda-746d-4bb9-a0b2-739ae00a67c9","added_by":"auto","created_at":"2023-10-16 21:57:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":582149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/f9e18e4b-dcd5-47a8-8eb3-eadfad2a1bdc.pdf"},{"id":24733170,"identity":"fb72e558-cf70-4d3c-91c8-d1a8166b9fab","added_by":"auto","created_at":"2022-08-03 17:00:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1230420,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1640993/v2/14d0487a727b1db5ed827202.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening of lactic acid bacteria with anti-adipogenic effect and potential probiotic properties from grains","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiet-induced obesity is a major factor in many chronic diseases such as cardiovascular disease, non-alcoholic fatty liver disease (NAFLD) and type2 diabetes, and is considered a serious health concern\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Currently, drug treatment and surgery with mechanisms that inhibit lipid absorption and suppress appetite are used worldwide to prevent and treat obesity. However, in the case of drugs, continuous administration is limited due to side effects, and a risk of complications in surgery is possible\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, non-drug therapies that are safer and improve the balance of metabolism are needed to treat and prevent obesity and probiotics have been proposed as an alternative\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Probiotics are defined as live microorganisms that provide the host with health benefits when administered in proper quantities\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The proven effects of probiotics include alleviation of intestinal inflammation, allergy prevention, reduced total serum cholesterol and inflammation and anti-cancer activity\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among them, several probiotics are also known to have potential anti-obesity effects such as reduced body fat, blood sugar control and cholesterol improvement\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Lactic acid bacteria (LAB) is the main bacteria in probiotics\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. According to the American Food and Drug Administration, LAB are generally regarded as safe (GRAS). LAB is widely used in the food industry, and studies of LAB functionality are growing in popularity. In several recent studies, LAB was shown to alleviate obesity by decreasing body weight, improving inflammatory state or glucose tolerance, and altering gut microbiota in diet-induced obese mice\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGrains are a major staple food in Asia. Grains mainly contain carbohydrates including fiber and oligosaccharides, which makes them an excellent source of prebiotics defined as \u0026ldquo;a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A microbial community composed of LAB was shown to exist in grains\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In a previous study, LAB isolated from grains exerted antimicrobial properties\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; however, few studies about useful properties, including anti-obesity effects, have been performed.\u003c/p\u003e \u003cp\u003eThe objectives of the present study were to isolate LAB from various grains collected in the Republic of Korea and to screen lactic acid bacteria with inhibition of adipogenesis. The selected strains with anti-adipogenic effects were investigated to determine whether they have useful properties as probiotic candidates for development as functional food.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eIsolation of LAB strains and growth conditions.\u003c/strong\u003e Four types of grains, rice, brown rice, black rice and hulled barley, were collected in the Republic of Korea. The grains were ground using blender and enriched for 7 days with distilled water under aerobic conditions. The grain samples were serially diluted with 0.85% (w/v) saline solution and spread on De Man-Rogosa-Sharpe (MRS; BD Difco, Sparks, MD, USA) agar. After incubation for 48 h at 30 ℃ or 37 ℃, LAB were isolated from the MRS agar plates and cultivated for 18 h at 30 ℃ or 37 \u0026deg;C in MRS agar. \u003cem\u003eLacticaseibacillus\u0026nbsp;\u003c/em\u003e\u003cem\u003erhamnosus\u003c/em\u003e LGG (KCTC 5033), which was used as the experimental control strain for comparative analyses, was purchased from the Korean Collection for Type Cultures (Daejeon, Republic of Korea), and cultured at 37 ℃. All strains, including the experimental control strain, were stored at - 80 ℃ after suspension in 20% (w/v) glycerol solution (Georgiachem, GA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16S rRNA gene sequence analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis.\u003c/strong\u003e Genomic DNA was extracted using a G-spin genomic extraction kit (iNtRON, Seongnam, Republic of Korea), according to the manufacturer\u0026rsquo;s protocol. Polymerase chain reaction (PCR) amplification, purification, and sequencing of 16S rRNA gene were performed as described previously\u003csup\u003e20\u003c/sup\u003e. Identification of the closest phylogenetic species based on 16S rRNA gene sequence was performed using the EzBioCloud server (https://www.ezbiocloud.net/)\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRandom amplified polymorphic DNA-PCR (RAPD-PCR), which was used to exclude replicates among LAB strains, was performed using two primers, ERIC2 (5\u0026prime;-AAGTAAGTGACTGGGGTGAGCG-3\u0026prime;) and ERIC1R (5\u0026rsquo;-ATGTAAGCTCCTGGGGATTCAC-3\u0026rsquo;) as described previously\u003csup\u003e22\u003c/sup\u003e. The PCR products were electrophoresed on 1.5% (w/v) agarose (LPS Solution, Daejeon, Republic of Korea) gel for 60 min, and after electrophoresis, the gel was stained with RedSafe (iNtRON, Seongnam, Republic of Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of cell extract from LAB.\u003c/strong\u003e Cell extracts of LAB were prepared as described previously\u003csup\u003e23\u003c/sup\u003e with minor modifications. The cell mass was harvested using centrifugation and washed twice with phosphate-buffered saline (PBS, pH 7.2). The washed cells were resuspended in distilled water at a concentration of 100 mg/mL and sonicated using the method described previously\u003csup\u003e16\u003c/sup\u003e. The sonicated cell extracts were centrifuged at 13,000 rpm for 15 min at 4 \u0026deg;C and the supernatants were filtered using a 0.45 \u0026micro;M syringe filter (Sartorius Stedim Biotech GmbH, G\u0026ouml;ttingen, Germany) and lyophilized. The resulting powder was dissolved in sterile water to appropriate concentrations.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture, adipocyte differentiation and intracellular triglyceride content.\u003c/strong\u003e C3H10T1/2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured following the method described previously\u003csup\u003e24\u003c/sup\u003e. A subculture of C3H10T1/2 cells was performed with Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (penicillin and streptomycin, Hyclone). After seeding in 12-well plates, C3H10T1/2 cells were cultured in DMEM containing 10% FBS and antibiotics until confluency. Confluent cells were induced into adipocytes in DMEM supplemented with 10% FBS, antibiotics, 20 nM GW1929 (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 1 \u0026mu;M dexamethasone (Sigma-Aldrich), and 10 \u0026mu;g/mL insulin (Sigma-Aldrich). After 48 h, the differentiating cells were refreshed with media containing DMEM, 10% FBS, 20 nM GW1929, and 10 \u0026mu;g/mL insulin.\u003c/p\u003e\n\u003cp\u003eCell extracts of LAB were adjusted by suspending to a concentration of 25,50, and100 \u0026mu;g/mL with sterile distilled water to create the same conditions. LAB cell extracts were treated during adipocyte differentiation of C3H10T1/2 cells, and sterile distilled water was treated as control. Then, the differentiated C3H10T1/2 cells were fixed with 4% formaldehyde (Sigma-Aldrich) in PBS (Hyclone) at room temperature overnight and stained with Oil Red O (Sigma-Aldrich). To quantify intracellular triglyceride content, stained cells from at least two independent experiments were resolved in isopropanol (Sigma-Aldrich) and measured with a spectrophotometer at 520 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay.\u003c/strong\u003e Cell viability was determined using methyl thiazolyl tetrazolium salt (MTS) colorimetric assays (ab197010, Abcam). C3H10T1/2 cells were seeded at 1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates, then treated with various strain doses (1, 12.5, 25, 50, 100, and 200 \u0026mu;g/mL) and sterile distilled water as control in triplicate. After 24 h, MTS (20 \u0026mu;L/100 \u0026mu;L in medium) was added into the media and cells incubated for 4 h at 37 ℃. The absorbance of formazan dye was measured at 490 nm using a microplate reader (BioTek, Winooski, VT, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time polymerase chain reaction (RT PCR) analysis.\u003c/strong\u003e Total RNA was extracted from C3H10T1/2 cells using QIAzol lysis reagent (QIAGEN, Germantown, MD, USA). First-strand complementary DNA was synthesized from 0.5 \u0026mu;g of total RNA using ReverTra Ace Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer\u0026rsquo;s instructions. Quantitative RT PCR was performed in 25 \u0026mu;L final reaction volume containing Power SYBR Premix ExTaq (RP041A; Takara, Shiga, Japan), primers, and cDNA using thermal cycler machine (Takara). The primer sequences used for the PCR were described previously\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTolerance assays against acid and bile salts.\u003c/strong\u003e Tolerance to acid was measured as described previously\u003csup\u003e25\u003c/sup\u003e with minor modifications. LAB strains were cultured overnight (18 h) at 30 \u0026deg;C (for RP21 and K28) or 37 \u0026deg;C (for RP12 and LGG), harvested for 10 min at 7,000 rpm at 4 \u0026deg;C, and washed twice with PBS buffer (pH 7.2). Bacterial cells (approximately 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) were resuspended in liquid MRS medium (pre-adjusted to pH 1.0, 2.0, 2.5, and 3.0) and incubated for 3 h at optimal temperatures. Viability was determined in triplicate in terms of viable colony counts using the plate count method. Tolerance to bile salts was measured as described previously\u003csup\u003e26\u003c/sup\u003e with minor modifications. LAB strains were suspended in liquid MRS medium containing 0.3, 0.5, 1.0, and 2.0% oxgall (Sigma-Aldrich) at a concentration of approximately 10\u003csup\u003e9\u003c/sup\u003e CFU/mL. After incubation for 6 h at 30 \u0026deg;C or 37 \u0026deg;C, suspension was poured into MRS agar plates and incubated at optimum growth temperatures for 48 h. Tolerance assays against acid and bile salts were performed in triplicate and LGG was used as the comparative strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro adhesion assays.\u003c/strong\u003e The adherence assay was performed according to the method described previously\u003csup\u003e27\u003c/sup\u003e with minor modifications. Caco-2 cells used for the adherence assay were purchased from the Korean Cell Line Bank (Seoul, Korea). The Caco-2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) FBS (Hyclone) and 1% (v/v) penicillin-streptomycin at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The Caco-2 cells were seeded at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well tissue culture plates. The adherence assay was performed at post-confluence. The monolayer was washed with sterile PBS (Hyclone) twice. LAB cells were diluted with DMEM to approximately 10\u003csup\u003e9\u003c/sup\u003e CFU/mL and added to the wells. Plates were incubated for 90 min at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The Caco-2 monolayers were washed three times with sterile PBS (Hyclone) and treated with EDTA-trypsin solution for 3 min. The cell suspensions were serially diluted and spread on MRS agar plates. Cell viability was counted after incubation for 48 h. The adhesion ability of LAB was calculated as the percentage between remaining bacteria and initial bacteria per well. The same passage Caco-2 cells were used in adhesion assays and assays were repeated in triplicate.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic susceptibility.\u003c/strong\u003e Susceptibility to antibiotics was examined using the disc-diffusion method with application of modified agar diffusion method described previously\u003csup\u003e28,29\u003c/sup\u003e. LAB inoculated in MRS agar were adjusted to approximately 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and paper discs (Advantec, Tokyo, Japan) were dispensed. Each disc was treated with 10 \u0026mu;L of specific antibiotic. The concentrations of antibiotics tested are listed in Supplementary Table 1. The inhibition zone diameters were measured and evaluated in terms of sensitive, intermediate sensitive, and resistant according to the interpretative standard table (Supplementary Table 1). The 2013 Clinical and Laboratory Standards Institute criteria\u003csup\u003e30\u003c/sup\u003e were used for interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme activity test.\u003c/strong\u003e Enzyme activity of the LAB was investigated as described previously\u003csup\u003e16\u003c/sup\u003e using the API ZYM kit (BioM\u0026eacute;rieux, Marcy l\u0026rsquo;Etoile, France).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e Results are presented as mean \u0026plusmn; standard error of the mean (SEM) of three independent experiments. Significance differences between groups in triglyceride content were determined using Duncan\u0026apos;s multi-range test. Significance differences in gene expression and adhesion ability were determined by comparison with control using two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. A p-value \u0026lt; 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Inc. software (version 19.0).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of LAB strains from grains\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Bacterial strains were isolated from four types of grains collected in the Republic of Korea and a total of 187 LAB strains were obtained through 16S rRNA gene sequencing followed by identification. From the 16S rRNA gene sequence analyses of the LAB strains, 20 strains had the closest similarities to the type strain of \u003cem\u003eLevilactobacillus \u003c/em\u003e(previously \u003cem\u003eLactobacillus\u003c/em\u003e) \u003cem\u003ebrevis\u003c/em\u003e, 3 strains had the closest similarities to\u003cem\u003e \u003c/em\u003ethe type strain of \u003cem\u003eLatilactobacillus \u003c/em\u003e(previously \u003cem\u003eLactobacillus\u003c/em\u003e) \u003cem\u003ecurvatus\u003c/em\u003e, 22 strains had the closest similarities to the type strain of \u003cem\u003eLactiplantibacillus \u003c/em\u003e(previously \u003cem\u003eLactobacillus\u003c/em\u003e) \u003cem\u003eplantarum\u003c/em\u003e, 17 strains had the closest similarities to\u003cem\u003e \u003c/em\u003ethe type strain of \u003cem\u003eLactococcus taiwanesis\u003c/em\u003e, 82 strains had the closest similarities to\u003cem\u003e \u003c/em\u003ethe type strain of \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, and 43 strains had the closest similarities to\u003cem\u003e \u003c/em\u003ethe type strain of \u003cem\u003eWeissella paramesenteroides\u003c/em\u003e. The genus \u003cem\u003eLactobacillus \u003c/em\u003ehas been recently reclassified as 25 genera including \u003cem\u003eLevilactobacillus\u003c/em\u003e, \u003cem\u003eLatilactobacillus\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eLactiplantibacillus\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e. \u003cem\u003eLevilactobacillus \u003c/em\u003e\u003cem\u003ebrevis, \u003c/em\u003e\u003cem\u003eLatilactobacillus \u003c/em\u003e\u003cem\u003ecurvatus,\u003c/em\u003e \u003cem\u003eLactiplantibacillus \u003c/em\u003e\u003cem\u003eplantarum,\u003c/em\u003e \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, and \u003cem\u003eWeissella paramesenteroides\u003c/em\u003e have been shown to be isolated from grains\u003csup\u003e32,33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn RAPD-PCR analysis, six different band patterns were assigned to 82 strains with the closest 16S rRNA gene sequence similarities to \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, and four different band patterns were assigned to 43 strains with the closest 16S rRNA gene sequence similarities to \u003cem\u003eWeissella paramesenteroides\u003c/em\u003e (Supplementary Fig. 1). The strains assigned as \u003cem\u003eLevilactobacillus \u003c/em\u003e\u003cem\u003ebrevis, \u003c/em\u003e\u003cem\u003eLatilactobacillus \u003c/em\u003e\u003cem\u003ecurvatus, \u003c/em\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, and \u003cem\u003eLactococcus taiwanesis \u003c/em\u003eeach showed only one type of band pattern (Supplementary Fig. 1). Finally, two representative strains from each group, except for the three groups having only one strain, were randomly selected, and used for further functional characterization (Supplementary Fig. 1; Table 1).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eScreening of strains with anti-adipogenic effects.\u003c/strong\u003e Inhibitory effects of lipid accumulation were tested by treating the LAB cell extract on C3H10T1/2 cells. Because the strains assigned to \u003cem\u003eLactococcus taiwanensis\u003c/em\u003e and \u003cem\u003eWeissella paramesenteroides \u003c/em\u003ecaused cell damage during the treatment process, they were excluded from the test. A wide range of inhibitory effects of lipid accumulation was observed in the strains selected. Among the strains tested, five strains (\u003cem\u003ePediococcus pentosaceus \u003c/em\u003eK28; \u003cem\u003eLevilactobacillus \u003c/em\u003e\u003cem\u003ebrevis \u003c/em\u003eRP20 and RP21; \u003cem\u003eLactiplantibacillus \u003c/em\u003e\u003cem\u003eplantarum\u003c/em\u003e RP11 and RP12) reduced lipid accumulation by more than 20% compared with the control, indicating that they have anti-adipogenic effects (Fig. 1). The two strains (RP20 and RP21) assigned to \u003cem\u003eLevilactobacillus \u003c/em\u003e\u003cem\u003ebrevis \u003c/em\u003eand the two strains (RP11 and RP12) of \u003cem\u003eLactiplantibacillus \u003c/em\u003e\u003cem\u003eplantarum\u003c/em\u003e showed similar results, respectively (Fig. 1). Thus, one strain from RP20 and RP21 and one strain from RP11 and RP12 were selected, and the three strains (K28, RP21 and RP12) were used for further experiments. The above results indicate that the components of LAB cell extract might influence the adipocyte differentiation process, thereby suppressing fat production.\u003c/p\u003e\n\u003cp\u003eSignificant diversity exists among LAB strains regarding functional characteristics that benefit health, such as antioxidant, antitumor, immunomodulatory, and hypocholesterolemic activities\u003csup\u003e34-40\u003c/sup\u003e. In several studies, the cellular components of LAB have shown beneficial effects on improving health\u003csup\u003e38,41,42\u003c/sup\u003e. It is not clear which substance(s) in LAB cell extract induce the anti-adipogenic effects. Exopolysaccharide (EPS) has been known to have anti-adipogenic effects\u003csup\u003e23\u003c/sup\u003e. The EPS, a cell wall component of LAB cells, is loosely associated with the cell envelope and easily released into the surrounding environment\u003csup\u003e43,44\u003c/sup\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEffects of LAB strains on cell viability of C3H10T1/2.\u003c/strong\u003e The cytotoxicity at various concentrations of strains LGG, K28, RP21, and RP12 on C3H10T1/2 cells was investigated by measuring cell viability using the MTS assay. C3H10T1/2 cells were found viable at all treatment concentrations of the four strains (Fig. 2).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eInhibition of adipogenic gene expression by LAB extract during adipocyte differentiation.\u003c/strong\u003e Inhibition of adipogenesis by strains K28, RP21 and RP12 was investigated by measuring expression of six adipogenic genes using quantitative RT PCR\u003cstrong\u003e \u003c/strong\u003e(Fig. 3). \u003cem\u003ePPAR\u0026gamma;\u003c/em\u003e and \u003cem\u003eC/EBP\u0026alpha;\u003c/em\u003e are transcription factors that regulate the process of adipocyte differentiation\u003csup\u003e45,46\u003c/sup\u003e. In addition, the activation of \u003cem\u003ePPAR\u0026gamma;\u003c/em\u003e promotes the expression of adipogenic genes, such as \u003cem\u003eCD36\u003c/em\u003e and \u003cem\u003eLPL\u003c/em\u003e, which are important for the uptake and storage of triglycerides\u003csup\u003e47\u003c/sup\u003e. The down regulation of these adipogenic genes may affect decreased lipid accumulation in cells. Fatty acid synthase (\u003cem\u003eFAS\u003c/em\u003e) gene is a downstream adipocyte gene that contributes to fatty acid synthesis\u003csup\u003e48\u003c/sup\u003e. Acetyl-coenzyme A carboxylase (\u003cem\u003eACC\u003c/em\u003e) is another key enzyme for fatty acid synthesis that catalyzes the synthesis of malonyl-CoA\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCell extracts from strains K28, RP21, and RP12 decreased the expression of adipocyte-related genes in C3H10T1/2 cells (Fig. 3). The expression of the six genes decreased proportionally with increasing concentrations of the extracts of the three strains (Fig. 3). The three strains significantly reduced (p \u0026lt; 0.01 or 0.001) the expression of \u003cem\u003ePPAR\u0026gamma;\u003c/em\u003e and \u003cem\u003eC/EBP\u0026alpha;\u003c/em\u003e in all concentrations tested. In addition, expression of four other genes associated with adipogenesis, was significantly reduced (p \u0026lt; 0.05 or 0.01) in the three strains, except for \u003cem\u003eLPL\u003c/em\u003e expression in 25 mg/mL treatment of strains K28 and RP12. Strain K28, which showed the lowest lipid accumulation, was analyzed to have the lowest values in expressions of the six genes after 100 mg/mL treatment (Fig. 3). These results indicate that the three strains may have anti-adipogenic effects by inhibiting the expression of adipogenesis-related genes.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTolerance \u003c/strong\u003e\u003cstrong\u003eagainst \u003c/strong\u003e\u003cstrong\u003eacid and bile salts.\u003c/strong\u003e To have specific functionality, a probiotic must reach the intestines alive with resistance to acid and bile salts\u003csup\u003e50\u003c/sup\u003e. The acid tolerance of the selected strains and LGG as a reference strain was examined after incubation for 3 h in pH 3.0, 2.5, 2.0, and 1.0 (Table 2). The three strains and LGG maintained the values of more than 9 log CFU/mL at pH 3. Under pH 2.5 condition, the survival rates of strains K28 and RP21 decreased more than 2 log and approximately 1 log, respectively, whereas strains RP12 and LGG showed decreases of more than 3 log and 2 log, respectively (Table 2). Under pH 2 condition, the survival rate of strains K28 and RP21 decreased approximately 3 log and 2 log, respectively, and the survival rate of strains RP12 and LGG decreased approximately 6 log (Table 2). The three strains, except RP21 with approximately 3 log CFU/mL, showed low viability of less than 2 log CFU/mL at pH 1 (Table 2). Strain RP21 was also found to have higher acid resistance, as a strain of\u003cem\u003e Levilactobacillus \u003c/em\u003e(\u003cem\u003eLactobacillus\u003c/em\u003e)\u003cem\u003e brevis \u003c/em\u003ewas shown highly acid-resistant in a previous study\u003csup\u003e51\u003c/sup\u003e.\u003cem\u003e \u003c/em\u003eThe pH of gastric fluid in the body is maintained at approximately 3.0, and probiotics are generally known to be highly acid-resistant if they are maintained at pH 3 for approximately 3 h\u003csup\u003e52\u003c/sup\u003e. Thus, the three strains were concluded to be highly tolerant to acid. Because food matrix can help the survival of LAB in the gastrointestinal tract due to its buffering capacity, the strains are expected to have stronger viability when used with carrier foods\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBile salts are another factor that can reduce bacterial survival in the gastrointestinal tract by destroying cell membranes\u003csup\u003e50\u003c/sup\u003e. Strains K28, RP21 and RP12 were found to survive after 6 h exposure to 0.3, 0.5, 1.0, and 2.0% bile salts, similar to LGG which is known to be highly resistant to bile salts (Table 2). Although the \u003cem\u003ein vitro \u003c/em\u003eassay\u003cem\u003e \u003c/em\u003ecannot provide the same conditions as the gastrointestinal tract, it is recognized as an effective evaluation method to select potential strains when using proper criteria\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAdherence to Caco-2 cells.\u003c/strong\u003e The adhesion ability of probiotics is a main factor that can increase the possibility of their survival and colonization in the gastrointestinal tract\u003csup\u003e54\u003c/sup\u003e. Adhesion is also required to prevent attachment of pathogenic bacteria through competition in intestinal epithelium\u003csup\u003e55\u003c/sup\u003e. Thus, the adherence ability has been considered an important biological property for the selection of useful probiotic strains\u003csup\u003e56\u003c/sup\u003e. In the present study, the adhesion ability of the three strains was evaluated using Caco-2 cells, which have morphological and physiological properties of human enterocytes, and their adhesion abilities were compared with that of the reference strain LGG (Fig. 4). Strain K28 had stronger adhesion ability than those of LGG and the two other strains (Fig. 4). The adhesion ability of strain K28 was highest at 1.95%, followed by LGG (1.79%), RP12 (1.67%), and RP21 (1.46%).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAntibiotic \u003c/strong\u003e\u003cstrong\u003esusceptibility.\u003c/strong\u003e Probiotics have been widely used in various fields including food and medical industries. Antimicrobial sensitivity for evaluation of probiotics is considered important for safety, because the resistant genes can be horizontally transferred to pathogenic bacteria, which can become a serious threat\u003csup\u003e57\u003c/sup\u003e. Sensitivity results of the strains for nine antibiotics used in this study are listed in Supplementary Table 1. For the nine antibiotics tested, strains K28, RP21 and RP12 showed sensitivity patterns similar to strain LGG. In this study, all four strains were equally sensitive to chloramphenicol and rifampicin, whereas strains K28 and RP21 were intermediate sensitive to tetracycline and strains RP12 and LGG were sensitive to tetracycline (Supplementary Table 1). Sensitivity or intermediate sensitivity of \u003cem\u003eLactobacillus\u003c/em\u003e species and \u003cem\u003ePediococcus\u003c/em\u003e species to chloramphenicol and tetracycline has been previously reported\u003csup\u003e58,59\u003c/sup\u003e. Strains K28, RP21, RP12 and LGG were resistant to gentamycin, kanamycin, and streptomycin, which are known to inhibit protein synthesis targeting Gram-negative bacteria. The resistance to aminoglycoside antibiotics is an intrinsic property among \u003cem\u003eLactobacillus\u003c/em\u003e species and \u003cem\u003ePediococcus\u003c/em\u003e species\u003csup\u003e60\u003c/sup\u003e. Therefore, the three strains are unlikely to cause safety problems based on antibiotic susceptibility profile tested.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEnzyme production.\u003c/strong\u003e For the safety of probiotic strains, it may be required to assess whether the strains produce harmful enzyme. \u0026beta;-glucuronidase is known as the carcinogen enzyme, which may increase the likelihood of tumor induction in the colon\u003csup\u003e61,62\u003c/sup\u003e. When the three strains were evaluated using API ZYM kit, strains RP12 and K28 did not produce any harmful enzymes such as \u0026beta;-glucuronidase, but strain RP21 was observed to produce \u0026beta;-glucuronidase (Supplementary Table 2).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, 187 LAB strains were isolated from four types of grains and identified using 16S rRNA gene sequence analysis. The 25 strains selected based on RAPD-PCR analysis were subjected to functional characterization. Among the strains tested, \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e K28, \u003cem\u003eLevilactobacillus brevis\u003c/em\u003e RP21, and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e RP12 had the potential to be useful probiotic candidates based on several characteristic analyses. The three strains exerted inhibitory effects on lipid accumulation and adipocyte differentiation by decreasing the expression of adipocyte-related genes. In addition, the three strains showed good tolerance against acid and bile salts, good intestinal cell adhesion, and were sensitive to chloramphenicol and rifampicin. In particular, strains RP12 and K28 did not produce β-glucuronidase. Therefore, \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e K28, \u003cem\u003eLevilactobacillus brevis\u003c/em\u003e RP21 and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e RP12 were concluded to have potential as probiotic candidates for use as functional neutraceutical foods.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by \u0026quot;Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ015247)\u0026quot; of Rural Development Administration, Republic of Korea and BK21 plus project of the Ministry of Education, Republic of Korea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.J.S.and\u0026nbsp;J.H.Y.\u0026nbsp;conceived and designed the study.\u0026nbsp;S.M.W., M.J.K., J.H.S., E.B.L., and J.H.C.\u0026nbsp;performed the experiments.\u0026nbsp;M.J.S.and\u0026nbsp;J.H.Y.\u0026nbsp;analyzed the data and wrote the manuscript.\u0026nbsp;K.W.P.\u0026nbsp;supported and discussed the study.\u0026nbsp;J.H.Y.\u0026nbsp;reviewed and edited manuscript.\u0026nbsp;K.W.P.\u0026nbsp;and\u0026nbsp;J.H.Y.\u0026nbsp;supervised the study. All authors read and approved the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e16S rRNA gene sequences of strains\u0026nbsp;K28,\u0026nbsp;RP21 and RP12\u0026nbsp;have been deposited in the National Centre for Biotechnology Information (NCBI) under GenBank accession numbers\u0026nbsp;ON724233, ON724232 and ON724263, respectively.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFord, N. D,, Patel, S. A. \u0026amp; Narayan, K. M. Obesity in low- and middle-income countries: burden, drivers, and emerging challenges. \u003cem\u003eAnnu. Rev. 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Food Microbiol.\u003c/em\u003e \u003cstrong\u003e41,\u003c/strong\u003e 45-51 (1998).\u003c/li\u003e\n\u003cli\u003eGueimonde, M. \u003cem\u003eet al\u003c/em\u003e. Antibiotic resistance in probiotic bacteria. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e4,\u003c/strong\u003e 202 (2013).\u003c/li\u003e\n\u003cli\u003eHummel, A. S. \u003cem\u003eet al\u003c/em\u003e. Antibiotic resistances of starter and probiotic strains of lactic acid bacteria. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cstrong\u003e73,\u003c/strong\u003e 730-739 (2007).\u003c/li\u003e\n\u003cli\u003eMaragkoudakis, P. A. \u003cem\u003eet al\u003c/em\u003e. Probiotic potential of \u003cem\u003eLactobacillus\u003c/em\u003e strains isolated from dairy products. \u003cem\u003eInt. Dairy J.\u003c/em\u003e \u003cstrong\u003e16,\u003c/strong\u003e 189-199 (2006).\u003c/li\u003e\n\u003cli\u003eSingla, V. \u003cem\u003eet al\u003c/em\u003e. Antibiotic susceptibility profile of \u003cem\u003ePediococcus\u003c/em\u003e spp. from diverse sources. \u003cem\u003e3 Biotech.\u003c/em\u003e \u003cstrong\u003e8,\u003c/strong\u003e 489 (2018).\u003c/li\u003e\n\u003cli\u003eHatakka, K. \u003cem\u003eet al\u003c/em\u003e. The influence of \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e LC705 together with \u003cem\u003ePropionibacterium\u003c/em\u003e \u003cem\u003efreudenreichii\u003c/em\u003e ssp. \u003cem\u003eshermanii\u003c/em\u003e JS on potentially carcinogenic bacterial activity in human colon. \u003cem\u003eInt. J. Food Microbiol.\u003c/em\u003e \u003cstrong\u003e128,\u003c/strong\u003e 406-410 (2008).\u003c/li\u003e\n\u003cli\u003eMonteagudo-Mera, A. \u003cem\u003eet al\u003c/em\u003e. Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products. \u003cem\u003eJ. Food Prot.\u003c/em\u003e \u003cstrong\u003e74,\u003c/strong\u003e 1379-1386 (2011).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLactic acid bacteria (n= 25) isolated from the four kinds of grains and a reference strain (LGG) used in this study.\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" style=\"border-collapse: collapse; margin: 0px auto;\" width=\"94%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSelected strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cstrong\u003eClosest species by 16S rRNA gene sequence analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolation source\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eof strain selected\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eRP20, RP21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eLevilactobacillus brevis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eRice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eRP42, RP50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eLatilactobacillus curvatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eRice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eRP11, RP12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eRice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eB5, B6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eLactococcus taiwanensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eBlack rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eH2, H6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eBrown rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eK21, K22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eHulled barley\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eH13, H15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eBrown rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eK28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eHulled barley\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eH11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eBrown rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eK25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eHulled barley\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eB12, B13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eWeissella paramesenteroides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eBlack rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eB32, B45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eBlack rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eH8, H23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eBrown rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.78688524590164%\"\u003e\n \u003cp\u003eB40, H7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.622950819672134%\"\u003e\n \u003cp\u003eBrown rice, Black rice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.59016393442623%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003eLGG (KCTC 5033)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.755102040816325%\"\u003e\n \u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.53061224489796%\"\u003e\n \u003cp\u003eReference strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003eAcid and bile tolerance of strains K28, RP21 and RP12 and a reference strain (LGG) (log CFU/ml).\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" width=\"5.1020408163265305%\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" width=\"65.3061224489796%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid condition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBile salts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" width=\"17.391304347826086%\"\u003e\n \u003cp\u003e\u003cstrong\u003epH 3.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"17.391304347826086%\"\u003e\n \u003cp\u003e\u003cstrong\u003epH 2.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"17.391304347826086%\"\u003e\n \u003cp\u003e\u003cstrong\u003epH 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"17.391304347826086%\"\u003e\n \u003cp\u003e\u003cstrong\u003epH 1.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.608695652173913%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.3%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.608695652173913%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.608695652173913%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.608695652173913%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.0%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003eInitial mean\u003c/p\u003e\n \u003cp\u003ecounts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003e3h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003eInitial mean\u003c/p\u003e\n \u003cp\u003ecounts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003e3h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003eInitial mean\u003c/p\u003e\n \u003cp\u003ecounts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003e3h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003eInitial mean\u003c/p\u003e\n \u003cp\u003ecounts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003e3h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.154639175257732%\"\u003e\n \u003cp\u003e\u003cstrong\u003eK28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e9.58\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n 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(n=3)\u003c/p\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1640993/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1640993/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA total of 187 lactic acid bacteria were isolated from four types of grains collected in South Korea. The bacterial strains were assigned as members of Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Lactococcus taiwanesis, Pediococcus pentosaceus, and Weissella paramesenteroides based on the closest similarity using 16S rRNA gene sequence analysis. The strains belonging to the same species were analyzed using RAPD-PCR, and one or two among strains showing the same band pattern were selected. Finally, 25 representative strains were selected for further functional study. Inhibitory effects of lipid accumulation were observed in the strains tested. Pediococcus pentosaceus K28, Levilactobacillus brevis RP21 and Lactiplantibacillus plantarum RP12 significantly reduced lipid accumulation and decreased expression of six adipogenic marker genes in C3H10T1/2 adipocytes. The three strains survived under strong acidity and bile salt conditions. The three strains showed adhesion to Caco-2 cells similar to a reference strain LGG. The resistance of the three strains to several antibiotics was also assessed. Strains RP12 and K28 were confirmed not to produce harmful enzymes based on API ZYM kit results. Based on these results, strains K28, RP21 and RP12 isolated from grains had the ability to inhibit adipogenesis in adipocytes and potentially be useful as probiotics.\u003c/p\u003e","manuscriptTitle":"Screening of lactic acid bacteria with anti-adipogenic effect and potential probiotic properties from grains","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-08-03 16:55:42","doi":"10.21203/rs.3.rs-1640993/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-16T19:20:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-11T10:21:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25b6b775-a097-4fd7-967a-a5fccf27c910","date":"2022-07-20T09:02:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-20T05:37:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-15T02:52:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-07-14T08:38:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-14T08:36:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-05-27T08:14:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0cf48bef-1d32-49d3-8d78-248311a82d01","owner":[],"postedDate":"August 3rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:43:00+00:00","versionOfRecord":{"articleIdentity":"rs-1640993","link":"https://doi.org/10.1038/s41598-023-36961-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-07-07 21:31:20","publishedOnDateReadable":"July 7th, 2023"},"versionCreatedAt":"2022-08-03 16:55:42","video":"","vorDoi":"10.1038/s41598-023-36961-0","vorDoiUrl":"https://doi.org/10.1038/s41598-023-36961-0","workflowStages":[]},"version":"v2","identity":"rs-1640993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1640993","identity":"rs-1640993","version":["v2"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}