Biological detoxification of Aflatoxin M₁ by lactic acid bacteria: experimental evidence from in vitro systems to fermented dairy products

Biological detoxification of Aflatoxin M₁ by lactic acid bacteria: experimental evidence from in vitro systems to fermented dairy products | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biological detoxification of Aflatoxin M₁ by lactic acid bacteria: experimental evidence from in vitro systems to fermented dairy products Mohammed Aladhadh, Mahmoud Al-Saman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8280315/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study evaluated the ability of various lactic acid bacteria (LAB) strains, in both viable and heat-inactivated forms, to reduce aflatoxin M₁ (AFM₁) levels in phosphate-buffered saline, MRS broth, and Karish cheese. Eight LAB strains were tested, including Lactobacillus acidophilus , L. salivarius , L. bulgaricus , Bifidobacterium lactis , and others. Viable cells exhibited strong detoxification efficiency, with L. acidophilus achieving the highest AFM₁ reduction (≈ 76%), followed by B. lactis and L. salivarius . Heat-killed counterparts showed minimal activity, confirming the essential role of metabolic processes in toxin removal. In MRS broth, a significant relationship was observed between pH decline and AFM₁ degradation; where up to 80% reduction occurred at pH 3.0 after 20 hrs, emphasizing the influence of acidic and enzymatic conditions. Application in Karish cheese demonstrated the practical relevance of LAB under real food conditions. Mixed cultures of S. thermophilus , L. bulgaricus , and L. acidophilus or L. salivarius reduced AFM₁ by 74–78% after 20 days at 4°C, accompanied by gradual acidification during ripening. These findings highlight the effectiveness of LAB, particularly multi-strain systems, as a natural and biotechnological approach for mitigating AFM₁ contamination and improving the safety of fermented dairy products. Aflatoxin M₁ Lactic acid bacteria Detoxification Karish cheese Food safety Figures Figure 1 INTRODUCTION Aflatoxin M₁ (AFM₁) is one of the most hazardous mycotoxins found in milk and dairy products, originating as a hydroxylated metabolite of aflatoxin B₁ produced by Aspergillus flavus and A. parasiticus . Due to its heat stability and resistance to conventional pasteurization or fermentation processes, AFM₁ poses a persistent risk to consumer health ( Ajma et al. 2022 ). Chronic exposure to AFM₁, even at trace concentrations, has been associated with hepatotoxicity, immuno-suppression, and carcinogenic effects ( Rushing and Selim, 2019 ) , which have prompted the establishment of strict regulatory limits worldwide. The attendance of this toxin in dairy products not only threatens food safety but also affects international trade and public confidence in dairy quality. AFM 1 has been confirmed to have cytotoxic and carcinogenic effects in many species, although being around ten times less dangerous than AFB1. According to the International Agency for Research on Cancer ( IARC, 2002 ) , AFM 1 is classified as category 1 carcinogenic to humans. In recent years, biological detoxification approaches have gained increasing attention as promising alternatives to chemical and physical methods, which often compromise the dietetic and sensory properties of food. Among these biological agents, lactic acid bacteria (LAB) have shown significant potential for binding, degrading, or sequestering AFM₁ through various mechanisms, including cell wall adsorption, enzymatic modification, and metabolic transformation. LAB is commonly recognized as safe (GRAS) and have long been used as starter cultures in fermented dairy products, making them ideal candidates for natural detoxification strategies ( Aween et al. 2012 ). Probiotic-based microbial food supplements have been shown to benefit both the body and the mind, including increased immunity, decreased lactose intolerance, a lower risk of cancer, improved intestinal tract health, pathogen antagonism, and increased nutrient synthesis and bioavailability ( Misra et al. 2021 ). A new technique for mycotoxin-free food is cold plasma, which produces reactive oxygen and nitrogen species. Physical methods such washing, de-hulling, aqueous extraction, cleaning, and milling have been shown to partially lower mycotoxins in grains ( Shetty and Bhat, 1999 ) . The amounts of AFs and fumonisin in tortillas were considerably decreased by alkaline cooking ( Voss et al. 2001 ). Fermentation has been used for centuries to preserve food, and research has demonstrated that LAB prevents the growth of mould and the creation of amyloid ( Mokoena et al. 2006 ). The present research was therefore designed to evaluate the capability of eight different LAB strains-both individually and in combination-to reduce AFM₁ levels under various experimental conditions , including incubation in phosphate-buffered saline, MRS broth, and real dairy systems such as Karish cheese. The study also aimed to compare the detoxification performance of viable and heat-killed cells and to monitor associated changes in pH during incubation and storage. This investigation is of great importance as it bridges the gap between laboratory findings and practical application in dairy processing. By identifying effective LAB strains capable of mitigating AFM₁ contamination without altering product quality, this study contributes to the development of safer, naturally fermented dairy products and supports sustainable strategies for mycotoxin control in the food industry. MATERIALS AND METHODS Chemicals The AFM 1 standard was supplied by Sigma (St. Louis, MO, USA). HPLC-pure solvents were provided by Merk (Darmstadt, Germany). The AFM 1 immune-affinity columns were provided by VICAM (Watertown, MA, USA). The water was cleaned using a MilliQWater System (Baunstead, Dubuque, USA). Bacterial strains and culture conditions Eight probiotic strains obtained from international culture collections ( Table 1 ) were selected for their industrial relevance and reported effects on food mutagens. Cultivation was performed on De Man, Rogosa, and Sharpe (MRS; Difco Laboratories) agar under optimal temperature and aeration conditions. Anaerobic strains were maintained in Oxoid anaerobic jars, while fully developed colonies were stored at 4°C and sub-cultured monthly. For long-term preservation, cells were cryostored at − 80°C in 90% glycerol. Lactobacilli were cultured in MRS broth and bifidobacteria in MRS supplemented with cysteine. After 24 hrs incubation at 37°C, cultures were centrifuged, and cells were washed with sterile phosphate-buffered saline (PBS, pH 7.3) for AFM₁ detoxification assays ( El-Nezami et al. 1998a ). Table 1 Sources of bacterial strains Strain Source Oxygen requirement Lactobacillus helveticus LH-BO2 1 Aerobic Bifidobactrium lactis Bb12 1 Anaerobic Lactobacillus salivarius TISTR 390 2 Anaerobic Lactobacillus paracasei TISTR 453 3 Aerobic Lactobacillus johnsonii ATCC 3320 4 Anaerobic Lactobacillus acidophilus ATCC4356 5 Aerobic Streptococcus thermophilus 2 Aerobic Lactobacillus bulgaricus 2 Anaerobic 1 , Chr. Hansen-Denmark; 2 , Egyptian Microbial Culture Collection (EMCC) at Cairo Microbiological Resources Centre (Cairo, MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt; 3 , Thailand Institute of Scientific and Technological Research, Bangkok, Thailand; 4 , German Centre for Culture Collection (DSMZ-Germany); 5 , The American Type Culture Collection (ATCC) Milk powder 100 mL of demonized water was added to a flask holding 10 g of milk powder. After 10 min of stirring, the mixture was centrifuged for an additional 10 min at 20°C and 3500 × g. 100 mL of skim milk was utilized in the following HPLC analysis to detect AFM 1 after the top fatty layer was eliminated by centrifugation. Preparation of Karish cheese Karish cheese was prepared following Ahmed et al. ( 2005 ) by reconstituting 1 kg skim milk powder in 9 L water. Milk was heated to 90°C for 15 min, cooled to 47°C, and inoculated with three different starter cultures: Starter #1 ( S. thermophilus + L. bulgaricus , 1:1), Starter #2 (+ L. acidophilus ATCC4356, 1:1:1), and Starter #3 (+ L. salivarius TISTR 390, 1:1:1). After coagulation (pH 4.5–4.7), curd was drained, salted (0.5%), cut into cubes, wrapped, and stored at 4°C for 20 days. Samples were collected at days 5, 10, 15, and 20 for AFM₁ analysis, with some stored at − 20°C for further tests. Inoculum preparation The microorganisms listed in Table 1 were grown in 100 mL of MRS broth (Oxoid CM 359) at 37 ° C daily. S. thermophilus was grown in 25 mL of M17 broth (Oxoid CM817) at 37 ° C. The Neubauer hemocytometer counting was used to assess the bacterial growth status 2 hrs until a consistent growth pattern appeared. Then the concentration of each bacterial inoculum was maintained at 1x10 6 cells/mL after adjustment. Assessment of AFM 1 removal by LAB strains in contaminated PBS In order to vertify the ability of the strains under investigation to bind AFM 1 -contaminated PBS buffer, 2 mL of each pure culture (made as previously described) was suspended in 98 mL of 50 µg/L AFM 1 -contaminated PBS in a Falcon tube, and the tube was then incubated at 37 ° C for 24 hrs. Regarding the cultures, 0.5 mL of each inoculum was collected and added to the volume of PBS contaminated with AFM 1 . Every 2 hrs throughout the incubation period, 2 mL of each bacterial suspension and pure cultures were obtained, mixed, and centrifuged for 10 min at 3500 × g. Using HPLC, the amount of unbound AFM 1 in the supernatant was confirmed. The samples were prepared according AOAC (2005) for the HPLC analysis. As a positive control, cell-free PBS tainted with AFM 1 was employed. During the experiment period, pure species of bacteria were used for bacterial growth, and as a negative control, they were suspended in non-contaminated PBS. Impact of acidity and heat on AFM removal To test the stability of the bacteria and AFM 1 , 2 mL of each pure culture were suspended in a bottle containing 98 mL of PBS contaminated with AFM 1 at a concentration of 50 µg/L. The bottle was then incubated at 37°C for 24 hrs. Regarding the cultures, 0.5 mL of each inoculum sample was added and mixed with a volume of PBS tainted with AFM 1 . 10 6 cells of cultured bacteria were used for the binding study. Bacteria were either heat-treated (boiled in 4 mL of PBS for 1 hrs) or cultivated as viable (in 4 mL of PBS for 1 hrs) in order to examine the impact of pH on the capacity of LAB and bifidobacteria to eliminate AFM 1 from purposefully contaminated MRS broth. Samples of the infected MRS broth were taken following 1, 5, 10, 15, and 20 days of media storage at 4°C. All bacterial samples were centrifuged at 3500 × g for 10 min before to conducting AFM 1 binding or degradation studies. After then, the supernatant was discarded. The AOAC (2005) method was used to quantify the concentration of AFM 1 . Forty mL of dichloromethane was mixed with 2 g of the sample for 15 min, and then removed. After filtering the suspension, 10 mL of the filtrate was heated to 60 ° C and then evaporated. A new solution of oily residue was prepared with 0.5 mL of methanol, 0.5 mL of PBS buffer, and 1 mL of hexane. The mixture was centrifuged for 15 min at 3500 x g and 15 ° C. After removing the top layer of hexane, 400 mL of deionized water (Fluka, Germany) was used to dilute 100 mL of the aliquot. Diluted samples were used for determine AFM 1 . Determination of AFM₁ by high-performance liquid chromatography (HPLC) AFM₁ levels were analyzed following the VICAM method using an affinity immunoassay column. Samples (20 g) were extracted with 80 mL dichloromethane, centrifuged to remove fat, and 50 mL of the extract was eluted from the column with methanol and a methanol–acetonitrile mixture (3:2, v/v). Eluates were diluted 1:3 with deionized water, filtered, and 10 µL injected into an HPLC system (Agilent 1100) equipped with a fluorescence detector (excitation 360 nm, emission 430 nm). Separation was performed on a C 18 column using an isocratic mobile phase of acetonitrile, methanol, and water (16:22:62, v/v/v). A calibration curve was generated from AFM₁ standards in acetonitrile. Statistical and cluster analysis All experimental data were expressed as the mean ± standard deviation (SD) of triplicate determinations. Statistical significance among treatments was analyzed using one-way ANOVA followed by Duncan’s multiple range test at a significance level of p < 0.05. To explore the relationship among the tested LAB strains based on their ability to remove AFM₁ in MRS broth, a hierarchical cluster analysis (HCA) was performed using Ward’s linkage method and Euclidean distance as a measure of similarity. The dendrogram was constructed to visualize the clustering patterns and to identify groups of LAB strains exhibiting similar AFM₁ detoxification capacities. All analyses were conducted using standard statistical software packages. RESULTS Characterization of lactic acid bacteria strains utilized for aflatoxin M₁ detoxification Table 1 presents the eight LAB strains employed in the current study, highlighting their sources and oxygen requirements. The selected strains represent a diverse taxonomic and physiological spectrum of LAB, encompassing both Lactobacillus and Streptococcus genera, in addition to Bifidobacterium . This diversity was intentionally included to provide a comprehensive evaluation of their detoxification potential against aflatoxin M₁ (AFM₁) under varying environmental and metabolic conditions. The strains originated from internationally recognized microbial repositories, including Chr. Hansen (Denmark), the Egyptian Microbial Culture Collection (EMCC, Ain Shams University), the Thailand Institute of Scientific and Technological Research (TISTR), the German Centre for Culture Collection (DSMZ), and the American Type Culture Collection (ATCC). Such a broad range of sources ensured the inclusion of strains adapted to different ecological niches and fermentation practices, thereby increasing the representativeness of the study. Regarding oxygen requirements, the collection included both aerobic and anaerobic species, which is particularly relevant for assessing AFM₁ detoxification mechanisms. Aerobic strains such as L. helveticus , L. paracasei , L. acidophilus , and S. thermophilus are typically associated with surface fermentation and rapid acidification, potentially enhancing AFM₁ adsorption through cell wall charge modification. In contrast, anaerobic strains like B. lactis , L. salivarius , L. johnsonii , and L. bulgaricus may rely more on metabolic or enzymatic degradation pathways that function optimally under low-oxygen environments. The inclusion of both homofermentative and heterofermentative LAB species allows for comparative analysis of AFM₁ removal through distinct physiological routes - adsorption to cell wall components such as peptidoglycan and teichoic acids in viable or heat-treated cells, or possible enzymatic biotransformation in metabolically active strains. Therefore, the composition of this bacterial panel provides a strong foundation for interpreting the subsequent results on AFM₁ detoxification efficiency across different culture systems and storage conditions. Detection of aflatoxin M₁ in dairy matrix by high-performance liquid chromatography (HPLC) S 1 illustrates the HPLC chromatograms used for the detection and quantification of AFM₁. Chromatogram (A) represents the AFM₁ standard solution at a concentration of 3.6 ng mL⁻¹, showing a distinct and sharp peak at a retention time of approximately 12.87 min, which confirms the elution position of pure AFM₁ under the applied chromatographic conditions. In contrast, chromatogram (B) corresponds to a Karish cheese sample, in which the same characteristic AFM₁ peak appeared at the same retention time (12.87 min), indicating the presence of the toxin in the analyzed dairy matrix. However, additional smaller peaks observed at 2.154 min and 5.798 min correspond to other naturally occurring compounds or matrix interferences commonly found in fermented dairy products. The similarity in retention time between the standard and the sample confirms the identity of AFM₁ in the tested cheese. The relatively lower peak area in the sample chromatogram compared with the standard demonstrates a reduced AFM₁ concentration, likely due to the binding or degradation effect of LAB used during fermentation. This finding highlights the effectiveness of LAB strains in mitigating AFM₁ contamination through adsorption or biotransformation mechanisms during cheese processing and storage. Comparison of AFM₁ detoxification efficiency among viable LAB strains The data presented in Table 2 evaluate the efficiency of various viable LAB strains in reducing AFM 1 concentration from PBS. The initial concentration of AFM 1 was 50 µg/L, and changes in its residual level were recorded after 4, 8, 16, and 24 hrs of incubation at 37°C. Across all LAB strains tested, a progressive reduction in AFM 1 concentration was observed with increasing incubation time. This indicates that viable LAB cells possess the ability to interact with and remove AFM 1 , most likely through adsorption to cell wall components or by partial metabolic transformation. Table 2 Effect of viable lactic acid bacteria strains on aflatoxin M1 (AFM 1 ) reduction in PBS Viable strain a Concentration of AFM 1 (µg/L) 4 hrs b 8 hrs 16 hrs 24 hrs % AFM 1 removal after 24 hrs L. helveticus LH-BO2 35.2 ± 2.2 25.2 ± 2.3 19.9 ± 3.1 15.8 ± 1.5 68.4 ± 1.43 bc B. lactis Bb12 35.5 ± 2.7 37.9 ± 2.7 38.9 ± 2.9 14.96 ± 0.5 70.1 ± 0.89 b L. salivarius TISTR 390 36.8 ± 2.1 24.3 ± 2.3 19.8 ± 4.1 15.4 ± 1.3 69.2 ± 2.23 b L. paracasei TISTR 453 37.4 ± 3.1 34.8 ± 3.7 31.1 ± 1.3 16.1 ± 0.1 67.8 ± 1.54 c L. johnsonii ATCC 3320 38.8 ± 2.2 28.8 ± 2.3 18.7 ± 2.1 16.2 ± 3.1 67.6 ± 1.6 7c L. acidophilus ATCC4356 31.5 ± 3.1 21.1 ± 3.6 18.5 ± 2.3 11.8 ± 0.46 76.4 ± 2.04 a S. thermophilus 36.6 ± 0.55 28.5 ± 1.89 22.9 ± 0.78 17.6 ± 0.57 64.8 ± 0.61 d L. bulgaricus 35.1 ± 0.69 29.1 ± 1.32 21.8 ± 0.35 15.4 ± 0.89 69.2 ± 0.85 b a Viable bacteria were incubated in PBS (4 mL) at 37°C for 1 hrs b Incubation period The initial AFM 1 concentration was 50 µg/L Mean ± standard deviation (SD) of triplicate samples Different superscript letters in the same column indicate significant difference at p < 0.05 After 24 hrs, all strains achieved substantial AFM 1 reduction, ranging from 64.8 to 76.4%. Among the tested LAB, L. acidophilus ATCC 4356 exhibited the highest detoxification efficiency, reducing the AFM 1 concentration to 11.8 µg/L, equivalent to 76.4% removal. B. lactis Bb12 and L. salivarius TISTR 390 also demonstrated strong performance with 70.1 and 69.2% removal, respectively. In contrast, S. thermophilus showed the lowest reduction (only 64.8%) after 24 hrs, suggesting comparatively weaker binding affinity or fewer active cell wall sites for AFM 1 interaction. At the early incubation stage (4 hrs), the AFM 1 concentration remained relatively high for all strains, with values between 31.5 and 38.8 µg/L, corresponding to 22–37% removal. After 8 hrs, a marked destabilization was observed, especially for B. lactis (37.9 ± 2.7 µg/L removed). However, between 16 and 24 hrs, the reduction rate tended to stabilize, suggesting that the binding capacity of bacterial cells reached equilibrium-possibly due to saturation of adsorption sites or reversible toxin release at prolonged exposure. The trend indicates that most of the toxin removal occurred within the first 8–16 hrs, followed by a slower rate of reduction up to 24 hrs. For example, L. acidophilus reduced AFM 1 from 31.5 µg/L at 4 hrs to 18.5 µg/L at 16 hrs, and finally to 11.8 µg/L at 24 hrs, representing an incremental improvement of ~ 25% between 16 and 24 hrs. This plateau effect implies that extending incubation beyond 24 hrs may not yield significant additional detoxification. The detoxification mechanism likely involves non-covalent adsorption of AFM 1 molecules onto bacterial surface components such as peptidoglycans, polysaccharides, and teichoic acids. The greater efficiency of L. acidophilus could be attributed to its high surface area-to-volume ratio and hydrophobic cell surface, which enhance toxin binding. Strains like S. thermophilus and L. paracasei , with smoother or less dense cell wall structures, showed lower binding potential. Moreover, since viable cells were used, partial metabolic degradation of AFM 1 cannot be completely ruled out, particularly for species known to exhibit active enzymatic systems during incubation. All LAB strains tested are capable of reducing AFM 1 levels significantly, confirming their potential as biological adsorbents for mycotoxin decontamination. L. acidophilus ATCC 4356 is the most promising candidate, achieving over three-quarters removal under the tested conditions. The use of LAB in detoxification systems offers a safe, natural, and cost-effective alternative to chemical decontamination methods. Residual AFM₁ concentrations after incubation with HK-LAB Table 3 shows residual AFM₁ concentrations (µg/L) after incubation of heat-killed LAB strains with an initial AFM₁ level of 50 µg/L, measured at 4, 8, 16 and 24 hrs. Bacteria boiled in PBS for 1 hrs (heat treated). Triplicate means ± SD are presented. Heat-killed cells produced only modest AFM₁ reductions over 24 hrs. Residual AFM₁ at 24 hrs ranged from 41.3 to 46.7 µg/L, corresponding to 6.6–17.4% removal of the initial 50 µg/L. These removal levels are substantially lower than those achieved with viable LAB in the previous table (where removals were ~ 65–76% after 24 hrs). This indicates that viability (or the cell state prior to heat treatment) strongly affects AFM₁ removal under the tested conditions. Table 3 Effect of heat-killed lactic acid bacteria (HK-LAB) strains on AFM 1 removal from PBS Heat-killed strain a concentration of AFM 1 (µg/L) 4 hrs b 8 hrs 16 hrs 24 hrs % AFM 1 removal after 24 hrs L. helveticus LH-BO2 44.8 ± 0.55 43.9 ± 0.67 42.9 ± 0.28 41.9 ± 0.39 16.2 ± 1.46 a B. lactis Bb12 47.9 ± 2.8 46.7 ± 0.11 46.5 ± 0.27 46.7 ± 0.16 6.6 ± 1.89 c L. salivarius TISTR 390 45.9 ± 2.3 44.4 ± 0.34 44.2 ± 0.24 43.0 ± 0.21 14.0 ± 3.21 b L. paracasei TISTR 453 47.9 ± 1.7 46.9 ± 0.45 46.7 ± 0.34 46.2 ± 0.56 7.6 ± 0.89 c L. johnsonii ATCC 3320 47.9 ± 2.8 46.9 ± 4.6 46.7 ± 0.14 46.4 ± 0.23 7.2 ± 0.91 c L. acidophilus ATCC4356 42.4 ± 1.7 41.0 ± 0.67 41.2 ± 0.11 41.3 ± 0.46 17.4 ± 1.46 a S. thermophilus 44.1 ± 0.33 43.8 ± 0.32 43.8 ± 0.15 43.2 ± 0.15 13.6 ± 2.62 b L. bulgaricus 44.6 ± 0.55 42.9 ± 067 42.3 ± 0.28 41.9 ± 0.29 16.2 ± 1.09 a a Bacteria were boiled in PBS (4 mL) for 1 hrs (heat treated) b Incubation period The initial AFM 1 concentration was 50 µg/L Mean ± standard deviation of triplicate samples Different superscript letters in the same column indicate significant difference at p < 0.05 L. acidophilus , L. helveticus and L. bulgaricus performed best among heat-killed strains (~ 16–17% removal), while B. lactis , L. paracasei and L. johnsonii showed minimal effect (~ 6–8%). The change from 4 → 24 hrs is small for all strains (typical reductions of ~ 2–6 µg/L over 20 hrs). For example, L. helveticus decreased from 44.8 µg/L (4 hrs) to 41.9 µg/L (24 hrs) (a net removal of 2.9 µg/L, ~ 5.8% absolute). The near-plateau behavior indicates rapid attainment of equilibrium between AFM₁ in solution and binding sites on heated cells, with little progressive removal after early contact. Heat killing likely alters cell wall structure (protein denaturation, polysaccharide rearrangement) and abolishes metabolic activity. Two non-exclusive effects are plausible: Loss of active metabolic degradation pathways (so enzymatic breakdown is minimal). Alteration of adsorption sites - heating may expose some hydrophobic sites in some strains (slightly improving passive adsorption), or conversely destroy/lower affinity of ligand sites, reducing binding. The substantially lower removal compared with viable cells suggests that active physiological factors (cell surface properties maintained in viable cells, or metabolism-linked processes) play a major role in AFM₁ removal observed with live cultures. Heat-killed LAB are poor standalone solutions for AFM₁ removal under the tested conditions (max ~ 17% removal). For meaningful decontamination, viable cultures or other interventions are required. Effect of incubation period and pH variation on the reduction of AFM₁ by LAB strains in MRS broth The data in Table 4 summarize the relationship between pH variation and AFM₁ degradation by different lactic acid bacteria (LAB) strains during incubation in MRS broth for 1, 5, 10, 15, and 20 days. The initial AFM₁ concentration was 50 µg/L. Both AFM₁ reduction and acidification of the medium (pH decline) were monitored as indicators of bacterial metabolic activity and detoxification potential. All LAB strains exhibited a progressive decrease in AFM₁ concentration with increasing incubation time, accompanied by a steady decline in pH values from approximately 4.5 on day 1 to around 3.0 by day 20. This simultaneous decline suggests that acid production and active metabolism play crucial roles in enhancing AFM₁ removal. Table 4 Effect of LAB strains and pH changes during incubation periods on the reduction of AFM 1 in MRS broth Strain Incubation time % AFM 1 removal 1 D 5 D 10 D 15 D 20 D pH# AFM 1 @ pH AFM 1 pH AFM 1 pH AFM 1 pH AFM 1 L. helveticus LH-BO2 4.48 40.12 ± 1.2 4.27 34.12 ± 1.8 3.84 25.11 ± 1.5 3.55 18.38 ± 1.2 3.01 13.2 ± 4.5 73.6 ± 2.04 c B. lactis Bb12 4.66 39.81 ± 2.3 4.22 31.99 ± 2.8 3.88 24.45 ± 2.8 3.45 17.99.2.3 3.00 11.91 ± 1.9 76.2 ± 1.90 b L. salivarius TISTR 390 4.38 39.94 ± 2.7 4.01 25.88 ± 1.1 3.87 17.56 ± 4.1 3.41 14.21 ± 3.4 3.01 10.15 ± 5.2 79.8 ± 1.04 a L. paracasei TISTR 453 4.27 38.61 ± 3.1 4..00 34.89 ± 2.3 3.80 28.65 ± 1.9 3.41 19.98 ± 3.3 3.02 12.14 ± 1.8 75.8 ± 0.94 b L. johnsonii ATCC 3320 4.57 38.89 ± 3.2 4.17 29.97 ± 1.8 3.78 25.22 ± 1.5 3.44 18.56 ± 4.1 3.11 13.23 ± 2.5 73.6 ± 1.09 c L. acidophilus ATCC4356 4.52 31.59 ± 3.5 4.02 23.89 ± 2.66 3.76 15.99 ± 1.89 3.45 13.12 ± 2.2 3.00 9.98 ± ± 2.5 80.0 ± 2.10 a S. thermophilus 4.34 36.58 ± 0.97 3.99 28.33 ± 1.3 3.68 22.11 ± 3.3 3.51 18.99 ± 4.2 2.99 14.87 ± 2.4 70.4 ± 1.50 de L. bulgaricus 4.47 35.97 ± 1.4 4.00 26.11 ± 3.4 3.61 21.89 ± 2.7 3.43 19.01 ± 1.2 2.98 13.89 ± 2.8 72.2 ± 0.71 cd # measured pH value @ Concentration of AFM 1 (µg/L) The initial AFM 1 concentration was 50 µg/L Mean ± standard deviation of triplicate samples Different superscript letters in the same column indicate significant difference at p < 0.05 After 20 days, the most efficient strain was L. acidophilus ATCC 4356, which reduced AFM₁ concentration from 50 µg/L to 9.98 µg/L, corresponding to an 80.0% removal rate. L. salivarius followed closely with 79.8% removal, while B. lactis and L. paracasei achieved approximately 75–76% reduction. The least effective strain was S. thermophilus with 70.4% removal, though still significant compared to the initial concentration. A clear correlation was observed between pH decline and AFM₁ reduction. For instance, in L. acidophilus , pH dropped from 4.52 (day 1) to 3.00 (day 20), while AFM₁ fell from 31.5 µg/L to 9.98 µg/L. Similarly, L. salivarius showed a pH drop from 4.38 → 3.41 → 3.00, with AFM₁ decreasing from 39.9 µg/L → 14.2 µg/L → 10.1 µg/L. This pattern indicates that the acidic environment produced by LAB metabolism enhances AFM₁ binding or degradation. Lower pH values may promote electrostatic interactions between AFM₁ molecules and bacterial cell wall components, facilitating adsorption. In early stage (day 1–5): only partial removal observed (typically 20–35%); metabolic activity just beginning. Intermediate phase (day 10–15): marked acceleration in detoxification - AFM₁ reduced to ~ 35–40% of initial levels, coinciding with rapid acidification (pH ≈ 3.4–3.6). Late phase (day 20): near-maximum detoxification achieved (70–80% removal) as bacterial growth reached stationary phase, and pH stabilized near 3.0. These results suggest that extended incubation enhances AFM₁ removal due to cumulative adsorption and possible enzymatic modification over time. LAB strains, especially L. acidophilus , L. salivarius , and B. lactis , demonstrate strong potential for biological detoxification of AFM₁ in liquid matrices. The correlation between pH reduction and AFM₁ removal supports the use of controlled fermentation as a safe, natural method to reduce mycotoxin contamination in dairy environments. However, long incubation times (≥ 15 days) may be impractical industrially; optimization of cell density, medium composition, and initial pH could accelerate detoxification. Hierarchical cluster analysis (HCA) was performed to classify the eight LAB strains according to their efficiency in removing AFM₁ after 20 days of incubation in MRS broth. The dendrogram revealed two major clusters. The first cluster grouped L. acidophilus ATCC4356 and L. salivarius TISTR 390, which exhibited the highest AFM₁ removal rates of 80.0 and 79.8%, respectively, indicating their strong detoxification capacity. The second cluster included the remaining strains, with S. thermophilus (70.4%) and L. bulgaricus (72.2%) showing the lowest reduction levels ( Fig. 1 ) . These results indicate that L. acidophilus and L. salivarius are the most potent AFM₁-reducing strains, showing a distinct clustering pattern that reflects their superior detoxification potential. This clustering pattern demonstrates clear variability among the tested LAB strains, suggesting that the ability to remove AFM₁ is strain-dependent. The close association between L. acidophilus and L. salivarius emphasizes their potential use as effective bio-detoxifying cultures for reducing AFM₁ contamination in dairy systems. Effect of mixed LAB starter cultures on AFM₁ reduction and pH changes in Karish cheese during cold storage Table 5 illustrates the effect of three combinations of LAB starter cultures on the removal of AFM₁ from Karish cheese during processing and cold storage at 4°C for 20 days. The data clearly show that all bacterial combinations were effective in progressively reducing AFM₁ concentrations over time, accompanied by a consistent decrease in pH values, indicating enhanced acidification and metabolic activity of the cultures. Table 5 Removal of AFM 1 from Karish cheese using three different LAB strain combinations as starter cultures during processing and storage at 4°C Starter culture pH/storage period % AFM 1 removal 1 D 5 D 10 D 15 D 20 D pH # AFM 1 @ pH AFM 1 pH AFM 1 pH AFM 1 pH AFM 1 group No. 1 S. thermophilus L. bulgaricus 4.70 36.89 ± 1.61 4.33 28..78 ± 2.01 4.11 20.23 ± 0.49 3.88 17.54 ± 2.01 3.55 12.89 ± 1.51 74.2 ± 0.67 c group No. 2 S. thermophilus L. bulgaricus L. acidophilus ATCC4356 4.55 34.83 ± 1.79 4.23 27.16 ± 1.03 4.00 20.12 ± 1.30 3.67 16.88 ± 0.34 3.36 10.99 ± 1.34 78.0 ± 1.03 a group No. 3 S. thermophilus L. bulgaricus L. salivarius TISTR 390 4.67 33.39 ± 1.02 4.20 23.88 ± 1.80 3.99 19.37 ± 1.20 3..46 15.65 ± 1.71 3.30 11.82 ± 1.28 76.4 ± 1.39 b # measured pH value @ Concentration of AFM 1 (µg/L) The initial AFM 1 concentration was 50 µg/L Mean ± standard deviation of triplicate samples Different superscript letters in the same column indicate significant difference at p < 0.05 At the initial stage (1 day), the AFM₁ content ranged from 36.89 µg/L in Group No. 1 ( S. thermophilus + L. bulgaricus ) to 33.39 µg/L in Group No. 3 ( S. thermophilus , L. bulgaricus and L. salivarius ). As the incubation and storage progressed, the reduction in AFM₁ became more pronounced. After 5 days, AFM₁ concentrations declined to 28.78 µg/L, 27.16 µg/L, and 23.88 µg/L for Groups 1, 2, and 3, respectively, representing removal percentages of approximately 21.9%, 22.1%, and 28.5% compared to the initial 50 µg/L concentration. By 10 days, the reduction trend continued, with AFM₁ levels decreasing to 20.23 µg/L, 20.12 µg/L, and 19.37 µg/L for Groups 1, 2, and 3, respectively. The greatest detoxification efficiency was observed in Group No. 3, which achieved a 61.3% reduction at this stage. After 15 days, AFM₁ concentrations dropped further to 17.54 µg/L, 16.88 µg/L, and 15.65 µg/L, while the pH values reached 3.88, 3.67, and 3.46, respectively. The lowest AFM₁ value recorded at the final stage (20 days) was 10.99 µg/L for Group No. 2, followed closely by 11.82 µg/L for Group No. 3 and 12.89 µg/L for Group No. 1, corresponding to removal efficiencies of 78.0%, 76.4%, and 74.2%, respectively. The gradual decline in AFM₁ concentration with the decrease in pH suggests a strong correlation between bacterial metabolic activity and toxin binding or degradation. The inclusion of L. acidophilus (Group No. 2) and L. salivarius (Group No. 3) appeared to enhance AFM₁ removal compared with the traditional yogurt starter ( S. thermophilus + L. bulgaricus ). This improvement may be attributed to differences in cell wall composition and surface charge, which facilitate stronger adsorption of AFM₁ molecules. Group No. 2 achieved the highest total reduction (78%) after 20 days, indicating the synergistic detoxifying activity of L. acidophilus when combined with the traditional starter bacteria. DISCUSSION Similar results were obtained Elsanhoty et al. ( 2014 ), indicate that the initial concentrations of AFB 1 in yogurt influence the relative amounts of AFB 1 removed by viable bacteria and heat and acid treatment. Assaf et al. ( 2019 ) also confirmed these results discovering that different dairy strains of LAB and bifidobacteria with varying binding capabilities can reduce bioavailability of AFs. In Addition, heat-killed bacteria have been reported to be associated with a specific strain AFB 1 . It has been reported that the AFB 1 binds to the surface elements of LAB ( Haskard et al. 2001 ). The results also support the findings of Khadivi et al. ( 2020 ) who found that a group of dairy strains, species, and genus LAB are uniquely associated with AFB 1 . The results obtained were consistent with those published by Conte et al. ( 2020 ), who reported that the amount of AFB 1 removed by viable bacteria or heat and acid treatment is determined by the initial concentration of AFB 1 . With similar results Ondiek et al. ( 2022 ) reported, they discovered that toxin clearance usually increases with increased toxin concentration. It has been shown that AFB 1 and T-2 can be eliminated by both living and denatured cells. Given their ability to bind mycotoxins and reduce their absorption into the bloodstream through the gastrointestinal tract, it can be inferred from the data that these LAB species could act as biological agents to reduce AFB 1 and T-2. Studies have demonstrated that Lactobacillus strains can remove mycotoxins from contaminated liquid media ( Freire et al. 2021 ). Liu et al. ( 2020 ) showed that AFs molecules exhibited non-covalent reversible connectivity regardless of cellular activity through their ability to bind to the L. plantarum cell wall. However, several variables, including the growth media, the bacterial state (alive or dead), the incubation temperature, the incubation duration, and the pH level of medium, influence the degree to which AFs bind to the L. plantarum cell wall ( Sadiq et al. 2019 ). Heat and acid treatments severely affect the structural stability of polysaccharides and peptidoglycans in bacterial cell wall. According to Piotrowsika (2014) , many strains of Lactobacillus bacteria undergo a change in cell surface polarity from hydrophilic to hydrophobic upon heat treatment; As a result, heat-treated L. plantarum was able to adhere to AFs more easily. Yao et al. ( 2024 ) found that AFB 1 binds to L. brucei and L. plantarum most strongly at pH 2.5 and less strongly at pH 8.5. As a result of denaturing the cell surface proteins and exposing additional binding area to AFB1, the results showed that acid treatment enhanced hydrophobic interactions. The results shown in Table 4 showed that the type of starter had an effect on the variability of the AFB 1 degradation rate. Furthermore, a proportional relationship was found between the decrease in pH values and the corresponding decrease in AFM 1 content, where the greater the decrease in pH-values, the greater the decrease in AFM 1 content. The researchers hypothesized that suppression of AFs might be caused by lactic acid and/or LAB metabolites. These metabolites are low-molecular-weight compounds that are thermally-stable. Differences in extraction techniques, toxin concentration, and time before analysis, storage temperature, milk contamination method, milk composition, or the behavior of the starter cultures used in making the yogurt are likely responsible for these discrepancies in the results. The reduction of AFM₁ in yogurt during storage may be attributed to glucose oxidase activity, which catalyzes glucose oxidation to produce gluconolactone and hydrogen peroxide. The latter can generate reactive oxygen species capable of attacking the double bond in the AFM₁ dihydrofuran ring ( Yousef and Marth, 1989 ) . Hydrolysis of gluconolactone yields gluconic acid, lowering the pH (~ 3.9) and further promoting AFM₁ degradation. Several studies have reported the ability of Lactobacillus strains to remove mycotoxins from liquid media ( Freire et al., 2021 ). L. plantarum was shown to reduce ochratoxin A by 32–58% ( Mohammad and Hashemi, 2019 ) and aflatoxin B₁ by 69.1% ( Damayanti et al., 2017 ). According to literature reviews ( Dawlal et al. 2019 ), mycotoxins are associated with the LAB cell wall and are therefore extracted from the contaminated medium. According to reports Zhang et al. ( 2016 ), the main components of the LAB cell wall, associated with the mycotoxin binding mechanism, are teichoic acids, polysaccharides, and peptidoglycans. Thermal inactivation altered these components, leading to the formation of pores and protein denaturation in the bacterial cell wall, resulting in the emergence of new binding sites ( Haskard et al. 2001 ). This could explain the binding obtained in our study with inactivated LAB cells. The ability of LAB to bind mycotoxin has been the subject of numerous studies, which have shown that it depends on a variety of factors, including the type of LAB strains, the density of LAB cells, the viability of LAB, the concentration of mycotoxin, pH medium, temperature and the incubation time ( Zhao et al. 2016 ). The binding sites on the cell walls of microorganisms are essential for LAB to eliminate mycotoxins. In discussing this dependence, Badji et al. ( 2023 ) offered two explanations for the observed results: first, the binding sites for OTA and AFB 1 were different from each other, and second, the concentration of mycotoxin was insufficient to saturate all binding sites on the cell walls of the microorganisms. Probiotic LAB strains from various origins can reduce or eliminate AFM₁ in foods, exhibiting antimutagenic and anticancer properties. AFM₁ removal in Karish cheese lowered toxin levels to safe limits. This approach should be applied to improve food and feed safety. Further studies are needed to elucidate the detoxification mechanisms in LAB and Bifidobacteria, including the genetic traits enabling mycotoxin removal, to facilitate practical applications in the dairy industry. CONCLUSION This study demonstrates the effective biological detoxification of aflatoxin M₁ by eight lactic acid bacteria (LAB) strains. Viable cells showed superior AFM₁ removal (up to 76%) compared to heat-killed ones (6.6–17.4%), emphasizing the importance of cell viability and surface integrity. L. acidophilus ATCC 4356 exhibited the highest efficiency, followed by B. lactis and L. salivarius . In MRS broth and Karish cheese, toxin reduction (70–80%) correlated with acidification during fermentation. The mixed culture of S. thermophilus , L. bulgaricus , and L. acidophilus achieved the greatest detoxification, highlighting LAB’s potential as natural bio-detoxifying agents for safer dairy products. Declarations Authors’ contribution MA carried out the experiments and investigated the literature; MAA-S investigated, wrote, reviewed and edited the manuscript; all authors have read and agreed to the published version of the manuscript. Funding The authors received no financial support for the research, authorship, and/or publication of this article. Data availability The data presented in this study are available in this manuscript. Code availability Not applicable. Conflict of interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Ethical approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. References Ahmed, N.H., El Soda, M., Hassan, A.N., Frank, J. (2005). Improving the textural properties of an acid-coagulated (Karish) cheese using exopolysaccharide producing cultures. LWT-Food science and technology, 38(8), 843-847 Ajma, l M., Bedale, W., Akram, A., Yu, J.H. (2022). Comprehensive Review of Aflatoxin Contamination, Impact on Health and Food Security, and Management Strategies in Pakistan. Toxins (Basel). 2;14(12):845 AOAC Official Method 2000.08 (2005). Aflatoxin M 1 in liquid milk, immunoaffinity column by liquid chromatography. Natural Toxins-chapter 49 (pp. 45-47). Official Methods of Analysis of AOAC International, 18th edition, AOAC International. Gaithersburg, Maryland 20877-2417, USA Assaf, J.C., Nahle, S., Chokr, A., Louka, N., Atoui, A., El Khoury, A. (2019). Assorted Methods for Decontamination of Aflatoxin M1 in Milk Using Microbial Adsorbents. Toxins (Basel). 29;11(6):304. doi: 10.3390/toxins11060304 Aween, M.M., Hassan, Z., Muhialdin, B.J., et al. (2012 ). Antibacterial activity of Lactobacillus acidophilus strains isolated from honey marketed in Malaysia against selected multiple antibiotic resistant (MAR) Gram-positive bacteria. J Food Sci.;77(7):364-371 Badji, T., Durand , N., Bendali , F., PiroMetayer , I., Zinedine , A., SalahAbbès ,J.B., Abbès, S., Montet, D., Riba , A., Brabet , C. (2023) . In vitro detoxification of aflatoxin B1 and ochratoxin A by lactic acid bacteria isolated from Algerian fermented foods. Biological Control, 179, 105-181 Conte, G., Fontanelli, M., Galli, F., Cotrozzi, L., Pagni, L., Pellegrini, E. (2020). Mycotoxins in feed and food and the role of ozone in their detoxification and degradation: an update. Toxins 12(8): 486 Damayanti, E., Istiqomah, L., Saragih, J.E., Purwoko, T., Sardjono, (2017). Characterization of lactic acid bacteria as poultry probiotic candidates with aflatoxin B1 binding activities. Earth Environ. Sci. 101,https://doi.org/10.1088/1755-1315 Dawlal, P., Brabet, C., Thantsha, M.S., Buys, E.M. (2019) . Visualisation and quantification of fumonisins bound by lactic acid bacteria isolates from traditional African maize-based fermented cereals, ogi and mahewu. Food Additives Contaminants - Part A Chem., Anal., Control, Exposure Risk Assess. 36 (2), 296-307. El-Nezami, H., Kankaanpaa, P., Salminen, S., & Ahokas, J. (1998a). Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1. Food and Chemical Toxicology, 36, 321-326 Elsanhoty, R.M.,Salam S. A., Ramadan M.F., Badr F. H. (2014). Detoxification of aflatoxin M1 in yoghurt using probiotics and lactic acid bacteria. Food Control, 43: 129-134 Freire, L., Souza, A.D., Ana, S., Corassin, C.H., Rattray, F.P., Augusto, C., Oliveira, F.D. (2021). Effect of lactic acid bacteria strains on the growth and aflatoxin production potential of Aspergillus parasiticus , and their ability to bind aflatoxin B1, ochratoxin A, and zearalenone in vitro . Front. Microbiol. 12 Haskard, C.A., El-Nezami, H.S., Kankaanpaa, P.E., Salminen, S., Ahokas, J.T., (2001). Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria. Appl. Environ. Microbiol. 67 (7), 3086-3091. IARC. (2002). IARC monograph on the evaluation of carcinogenic risk to humans (Vol. 82) (p. 171). Lyon: International Agency for Research on Cancer-World Health Organization Khadivi, R., Razavilar, V., Anvar, A., Akbari, Adreghani, B. (2020). Aflatoxin M1-Binding Ability of Selected Lactic Acid Bacteria Strains and Saccharomyces boulardii in the Experimentally Contaminated Milk Treated with Some Biophysical Factors. Arch Razi Inst. 2020 Mar;75(1):63-73 Liu, A.,Zheng, Y., Liu, L., Chen, S., He, L., Ao, X., Yang, Y.,Liu, S. (2020). Decontamination of Aflatoxins by Lactic Acid Bacteria. Current Microbiology. 77:3821-3830 Misra, S.; Pandey, P.; Mishra, H.N . ( 2021). Novel approaches for co-encapsulation of probiotic bacteria with bioactive compounds, their health benefits and functional food product development: A review. Trends Food Sci. Technol. 109, 340-351 Mohammad, S., Hashemi, B. (2019). Fermentation of table cream by Lactobacillus plantarum strains: effect on fungal growth, aflatoxin M 1 and ochratoxin A. Int. J. Food Sci. Technol. 54, 347-353 Mokoena,M.P., Chelule, P.K Gqaleni, N. (2006). The toxicity and decreased concentration of aflatoxin B 1 in natural lactic acid fermented maize meal. Journal of Applied Microbiology 100: 773-777 Ondiek, W., Wang, Y., Sun, L., Zhou, L., On, S.L., Zheng, H., Ravi, G. (2022 ). Removal of aflatoxin B1 and t-2 toxin by bacteria isolated from commercially available probiotic dairy foods. Food Sci Technol Int. 28(1):15-25. Piotrowska, M. (2014). The adsorption of ochratoxin a by Lactobacillus species. Toxins 6 (9), 2826-2839. Rushing, B. and Selim , R. (2019). Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food Chem. Toxicol. 124, 81-100. Sadiq, F. A., Yan, B., Tian, F., Zhao, J., Zhang, H., & Chen, W. (2019). LAB as Antifungal and Anti‐Mycotoxigenic Agents: A Comprehensive Review. Comprehensive Reviews in Food Science and Food Safety, 18(5), 1403-1436. Shetty, P. H., & Bhat, R. V. (1999). A physical method for segregation of umonisin contaminated maize. Food Chemistry, 66, 371-374. Voss, K. A., Poling, S. M., Meredith, F. I., Bacon, C.W., & Saunders, D. S. (2001). Fate of fumonisins during the production of fried tortilla chips. Journal of Agricultural and Food Chemistry, 49, 3120-3126. Yao, Y.; Luo, J.; Zhang, P.; Wang, Y.; Lu, B.; Wu, G.; Zhang, J.; Luo, X.; Wang, L. (2024). Screening, Identification and Application of Lactic Acid Bacteria for Degrading Mycotoxin Isolated from the Rumen of Yaks. Microorganisms 2024, 12, 2260. Yousef, A. E., & Marth, E. H. (1989). Stability and degradation of aflatoxin M1. In H. P. Van Egmond (Ed.), Mycotoxins in dairy products (pp. 127-161). London: Elsevier Applied Science. Zhang, J., Zhang, J., Zhang, B. (2016). The mechanism of Lactobacillus strains for their ability to remove fumonisins B1 and B2. Food Chem. Toxicol. 97, 40-46. Zhao, H., Wang, X., Zhang, J., Zhang, J., Zhang, B. (2016). The mechanism of Lactobacillus strains for their ability to remove fumonisins B1 and B2. Food Chem. Toxicol. 97, 40-46. https://doi.org/10.1016/j.fct.2016.08.028. Additional Declarations No competing interests reported. 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16:10:22","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155214,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8280315/v1/ee45ba62cb997a1145caf60b.html"},{"id":98898352,"identity":"f865c21f-cabc-4e88-878f-12f036303042","added_by":"auto","created_at":"2025-12-23 18:31:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27987,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical cluster analysis (HCA) of LAB strains based on their ability to remove AFM₁ after 20 days of incubation in MRS broth. The dendrogram illustrates two major clusters: Cluster \u003cstrong\u003eI\u003c/strong\u003e includes \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e as the most effective strains, while Cluster \u003cstrong\u003eII\u003c/strong\u003e comprises the remaining strains showing moderate to lower AFM₁removal efficiency.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8280315/v1/6218b724ba2e53ccf5ce0910.png"},{"id":104404209,"identity":"a3f0e82d-3dd2-4dee-8b83-d80c903cc4cb","added_by":"auto","created_at":"2026-03-11 12:19:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2474864,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8280315/v1/0c9cfd54-8019-4e56-b5fb-4c3cf3c5f6e5.pdf"},{"id":99309515,"identity":"7ce8d5f6-f486-45d1-bbe2-894eb3150982","added_by":"auto","created_at":"2025-12-31 16:10:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":116439,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8280315/v1/a8cf005657c97090c96d756a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological detoxification of Aflatoxin M₁ by lactic acid bacteria: experimental evidence from in vitro systems to fermented dairy products","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAflatoxin M₁ (AFM₁) is one of the most hazardous mycotoxins found in milk and dairy products, originating as a hydroxylated metabolite of aflatoxin B₁ produced by \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eA. parasiticus\u003c/em\u003e. Due to its heat stability and resistance to conventional pasteurization or fermentation processes, AFM₁ poses a persistent risk to consumer health \u003cb\u003e(\u003c/b\u003eAjma et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Chronic exposure to AFM₁, even at trace concentrations, has been associated with hepatotoxicity, immuno-suppression, and carcinogenic effects \u003cb\u003e(\u003c/b\u003eRushing and Selim, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, which have prompted the establishment of strict regulatory limits worldwide. The attendance of this toxin in dairy products not only threatens food safety but also affects international trade and public confidence in dairy quality. AFM\u003csub\u003e1\u003c/sub\u003e has been confirmed to have cytotoxic and carcinogenic effects in many species, although being around ten times less dangerous than AFB1. According to the International Agency for Research on Cancer \u003cb\u003e(\u003c/b\u003eIARC, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, AFM\u003csub\u003e1\u003c/sub\u003e is classified as category \u003cb\u003e1\u003c/b\u003e carcinogenic to humans. In recent years, biological detoxification approaches have gained increasing attention as promising alternatives to chemical and physical methods, which often compromise the dietetic and sensory properties of food. Among these biological agents, \u003cb\u003elactic acid bacteria (LAB)\u003c/b\u003e have shown significant potential for binding, degrading, or sequestering AFM₁ through various mechanisms, including cell wall adsorption, enzymatic modification, and metabolic transformation. LAB is commonly recognized as safe (GRAS) and have long been used as starter cultures in fermented dairy products, making them ideal candidates for natural detoxification strategies \u003cb\u003e(\u003c/b\u003eAween et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProbiotic-based microbial food supplements have been shown to benefit both the body and the mind, including increased immunity, decreased lactose intolerance, a lower risk of cancer, improved intestinal tract health, pathogen antagonism, and increased nutrient synthesis and bioavailability \u003cb\u003e(\u003c/b\u003eMisra et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A new technique for mycotoxin-free food is cold plasma, which produces reactive oxygen and nitrogen species. Physical methods such washing, de-hulling, aqueous extraction, cleaning, and milling have been shown to partially lower mycotoxins in grains \u003cb\u003e(\u003c/b\u003eShetty and Bhat, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1999\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The amounts of AFs and fumonisin in tortillas were considerably decreased by alkaline cooking \u003cb\u003e(\u003c/b\u003eVoss et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Fermentation has been used for centuries to preserve food, and research has demonstrated that LAB prevents the growth of mould and the creation of amyloid \u003cb\u003e(\u003c/b\u003eMokoena et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present research was therefore designed to \u003cb\u003eevaluate the capability of eight different LAB strains-both individually and in combination-to reduce AFM₁ levels under various experimental conditions\u003c/b\u003e, including incubation in phosphate-buffered saline, MRS broth, and real dairy systems such as Karish cheese. The study also aimed to compare the detoxification performance of viable and heat-killed cells and to monitor associated changes in pH during incubation and storage. \u003cb\u003eThis investigation is of great importance as it bridges the gap between laboratory findings and practical application in dairy processing.\u003c/b\u003e By identifying effective LAB strains capable of mitigating AFM₁ contamination without altering product quality, this study contributes to the development of safer, naturally fermented dairy products and supports sustainable strategies for mycotoxin control in the food industry.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eThe AFM\u003csub\u003e1\u003c/sub\u003e standard was supplied by Sigma (St. Louis, MO, USA). HPLC-pure solvents were provided by Merk (Darmstadt, Germany). The AFM\u003csub\u003e1\u003c/sub\u003e immune-affinity columns were provided by VICAM (Watertown, MA, USA). The water was cleaned using a MilliQWater System (Baunstead, Dubuque, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial strains and culture conditions\u003c/h3\u003e\n\u003cp\u003eEight probiotic strains obtained from international culture collections \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e were selected for their industrial relevance and reported effects on food mutagens. Cultivation was performed on De Man, Rogosa, and Sharpe (MRS; Difco Laboratories) agar under optimal temperature and aeration conditions. Anaerobic strains were maintained in Oxoid anaerobic jars, while fully developed colonies were stored at 4\u0026deg;C and sub-cultured monthly. For long-term preservation, cells were cryostored at \u0026minus;\u0026thinsp;80\u0026deg;C in 90% glycerol. Lactobacilli were cultured in MRS broth and bifidobacteria in MRS supplemented with cysteine. After 24 hrs incubation at 37\u0026deg;C, cultures were centrifuged, and cells were washed with sterile phosphate-buffered saline (PBS, pH 7.3) for AFM₁ detoxification assays \u003cb\u003e(\u003c/b\u003eEl-Nezami et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSources of bacterial strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOxygen requirement\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus helveticus\u003c/em\u003e LH-BO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBifidobactrium lactis\u003c/em\u003e Bb12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus salivarius\u003c/em\u003e TISTR 390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus paracasei\u003c/em\u003e TISTR 453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus johnsonii\u003c/em\u003e ATCC 3320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e ATCC4356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus thermophilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cb\u003e1\u003c/b\u003e, Chr. Hansen-Denmark; \u003cb\u003e2\u003c/b\u003e, Egyptian Microbial Culture Collection (EMCC) at Cairo Microbiological Resources Centre (Cairo, MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt; \u003cb\u003e3\u003c/b\u003e, Thailand Institute of Scientific and Technological Research, Bangkok, Thailand; \u003cb\u003e4\u003c/b\u003e, German Centre for Culture Collection (DSMZ-Germany); \u003cb\u003e5\u003c/b\u003e, The American Type Culture Collection (ATCC)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMilk powder\u003c/h3\u003e\n\u003cp\u003e100 mL of demonized water was added to a flask holding 10 g of milk powder. After 10 min of stirring, the mixture was centrifuged for an additional 10 min at 20\u0026deg;C and 3500 \u0026times; g. 100 mL of skim milk was utilized in the following HPLC analysis to detect AFM\u003csub\u003e1\u003c/sub\u003e after the top fatty layer was eliminated by centrifugation.\u003c/p\u003e\n\u003ch3\u003ePreparation of Karish cheese\u003c/h3\u003e\n\u003cp\u003eKarish cheese was prepared following Ahmed et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) by reconstituting 1 kg skim milk powder in 9 L water. Milk was heated to 90\u0026deg;C for 15 min, cooled to 47\u0026deg;C, and inoculated with three different starter cultures: Starter #1 (\u003cem\u003eS. thermophilus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. bulgaricus\u003c/em\u003e, 1:1), Starter #2 (+\u0026thinsp;\u003cem\u003eL. acidophilus\u003c/em\u003e ATCC4356, 1:1:1), and Starter #3 (+\u0026thinsp;\u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390, 1:1:1). After coagulation (pH 4.5\u0026ndash;4.7), curd was drained, salted (0.5%), cut into cubes, wrapped, and stored at 4\u0026deg;C for 20 days. Samples were collected at days 5, 10, 15, and 20 for AFM₁ analysis, with some stored at \u0026minus;\u0026thinsp;20\u0026deg;C for further tests.\u003c/p\u003e\n\u003ch3\u003eInoculum preparation\u003c/h3\u003e\n\u003cp\u003eThe microorganisms listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were grown in 100 mL of MRS broth (Oxoid CM 359) at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC daily. \u003cem\u003eS. thermophilus\u003c/em\u003e was grown in 25 mL of M17 broth (Oxoid CM817) at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC. The Neubauer hemocytometer counting was used to assess the bacterial growth status 2 hrs until a consistent growth pattern appeared. Then the concentration of each bacterial inoculum was maintained at 1x10\u003csup\u003e6\u003c/sup\u003e cells/mL after adjustment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of AFM\u003csub\u003e1\u003c/sub\u003e removal by LAB strains in contaminated PBS\u003c/h2\u003e \u003cp\u003eIn order to vertify the ability of the strains under investigation to bind AFM\u003csub\u003e1\u003c/sub\u003e-contaminated PBS buffer, 2 mL of each pure culture (made as previously described) was suspended in 98 mL of 50 \u0026micro;g/L AFM\u003csub\u003e1\u003c/sub\u003e-contaminated PBS in a Falcon tube, and the tube was then incubated at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 24 hrs. Regarding the cultures, 0.5 mL of each inoculum was collected and added to the volume of PBS contaminated with AFM\u003csub\u003e1\u003c/sub\u003e. Every 2 hrs throughout the incubation period, 2 mL of each bacterial suspension and pure cultures were obtained, mixed, and centrifuged for 10 min at 3500 \u0026times; g. Using HPLC, the amount of unbound AFM\u003csub\u003e1\u003c/sub\u003e in the supernatant was confirmed. The samples were prepared according \u003cb\u003eAOAC (2005)\u003c/b\u003e for the HPLC analysis. As a positive control, cell-free PBS tainted with AFM\u003csub\u003e1\u003c/sub\u003e was employed. During the experiment period, pure species of bacteria were used for bacterial growth, and as a negative control, they were suspended in non-contaminated PBS.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImpact of acidity and heat on AFM removal\u003c/h3\u003e\n\u003cp\u003eTo test the stability of the bacteria and AFM\u003csub\u003e1\u003c/sub\u003e, 2 mL of each pure culture were suspended in a bottle containing 98 mL of PBS contaminated with AFM\u003csub\u003e1\u003c/sub\u003e at a concentration of 50 \u0026micro;g/L. The bottle was then incubated at 37\u0026deg;C for 24 hrs. Regarding the cultures, 0.5 mL of each inoculum sample was added and mixed with a volume of PBS tainted with AFM\u003csub\u003e1\u003c/sub\u003e. 10\u003csup\u003e6\u003c/sup\u003e cells of cultured bacteria were used for the binding study. Bacteria were either heat-treated (boiled in 4 mL of PBS for 1 hrs) or cultivated as viable (in 4 mL of PBS for 1 hrs) in order to examine the impact of pH on the capacity of LAB and bifidobacteria to eliminate AFM\u003csub\u003e1\u003c/sub\u003e from purposefully contaminated MRS broth. Samples of the infected MRS broth were taken following 1, 5, 10, 15, and 20 days of media storage at 4\u0026deg;C. All bacterial samples were centrifuged at 3500 \u0026times; g for 10 min before to conducting AFM\u003csub\u003e1\u003c/sub\u003e binding or degradation studies. After then, the supernatant was discarded. The \u003cb\u003eAOAC (2005)\u003c/b\u003e method was used to quantify the concentration of AFM\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eForty mL of dichloromethane was mixed with 2 g of the sample for 15 min, and then removed. After filtering the suspension, 10 mL of the filtrate was heated to 60\u003csup\u003e\u0026deg;\u003c/sup\u003eC and then evaporated. A new solution of oily residue was prepared with 0.5 mL of methanol, 0.5 mL of PBS buffer, and 1 mL of hexane. The mixture was centrifuged for 15 min at 3500 x g and 15\u003csup\u003e\u0026deg;\u003c/sup\u003eC. After removing the top layer of hexane, 400 mL of deionized water (Fluka, Germany) was used to dilute 100 mL of the aliquot. Diluted samples were used for determine AFM\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eDetermination of AFM₁ by high-performance liquid chromatography (HPLC)\u003c/h3\u003e\n\u003cp\u003eAFM₁ levels were analyzed following the VICAM method using an affinity immunoassay column. Samples (20 g) were extracted with 80 mL dichloromethane, centrifuged to remove fat, and 50 mL of the extract was eluted from the column with methanol and a methanol\u0026ndash;acetonitrile mixture (3:2, v/v). Eluates were diluted 1:3 with deionized water, filtered, and 10 \u0026micro;L injected into an HPLC system (Agilent 1100) equipped with a fluorescence detector (excitation 360 nm, emission 430 nm). Separation was performed on a C\u003csub\u003e18\u003c/sub\u003e column using an isocratic mobile phase of acetonitrile, methanol, and water (16:22:62, v/v/v). A calibration curve was generated from AFM₁ standards in acetonitrile.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical and cluster analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of triplicate determinations. Statistical significance among treatments was analyzed using one-way ANOVA followed by Duncan\u0026rsquo;s multiple range test at a significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To explore the relationship among the tested LAB strains based on their ability to remove AFM₁ in MRS broth, a hierarchical cluster analysis (HCA) was performed using Ward\u0026rsquo;s linkage method and Euclidean distance as a measure of similarity. The dendrogram was constructed to visualize the clustering patterns and to identify groups of LAB strains exhibiting similar AFM₁ detoxification capacities. All analyses were conducted using standard statistical software packages.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of lactic acid bacteria strains utilized for aflatoxin M₁ detoxification\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the eight LAB strains employed in the current study, highlighting their sources and oxygen requirements. The selected strains represent a diverse taxonomic and physiological spectrum of LAB, encompassing both \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eStreptococcus\u003c/em\u003e genera, in addition to \u003cem\u003eBifidobacterium\u003c/em\u003e. This diversity was intentionally included to provide a comprehensive evaluation of their detoxification potential against aflatoxin M₁ (AFM₁) under varying environmental and metabolic conditions. The strains originated from internationally recognized microbial repositories, including Chr. Hansen (Denmark), the Egyptian Microbial Culture Collection (EMCC, Ain Shams University), the Thailand Institute of Scientific and Technological Research (TISTR), the German Centre for Culture Collection (DSMZ), and the American Type Culture Collection (ATCC). Such a broad range of sources ensured the inclusion of strains adapted to different ecological niches and fermentation practices, thereby increasing the representativeness of the study.\u003c/p\u003e \u003cp\u003eRegarding oxygen requirements, the collection included both aerobic and anaerobic species, which is particularly relevant for assessing AFM₁ detoxification mechanisms. Aerobic strains such as \u003cem\u003eL. helveticus\u003c/em\u003e, \u003cem\u003eL. paracasei\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, and \u003cem\u003eS. thermophilus\u003c/em\u003e are typically associated with surface fermentation and rapid acidification, potentially enhancing AFM₁ adsorption through cell wall charge modification. In contrast, anaerobic strains like \u003cem\u003eB. lactis\u003c/em\u003e, \u003cem\u003eL. salivarius\u003c/em\u003e, \u003cem\u003eL. johnsonii\u003c/em\u003e, and \u003cem\u003eL. bulgaricus\u003c/em\u003e may rely more on metabolic or enzymatic degradation pathways that function optimally under low-oxygen environments.\u003c/p\u003e \u003cp\u003eThe inclusion of both \u003cem\u003ehomofermentative\u003c/em\u003e and \u003cem\u003eheterofermentative\u003c/em\u003e LAB species allows for comparative analysis of AFM₁ removal through distinct physiological routes - adsorption to cell wall components such as peptidoglycan and teichoic acids in viable or heat-treated cells, or possible enzymatic biotransformation in metabolically active strains. Therefore, the composition of this bacterial panel provides a strong foundation for interpreting the subsequent results on AFM₁ detoxification efficiency across different culture systems and storage conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetection of aflatoxin M₁ in dairy matrix by high-performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003e \u003cb\u003eS 1\u003c/b\u003e illustrates the HPLC chromatograms used for the detection and quantification of AFM₁. Chromatogram (A) represents the AFM₁ standard solution at a concentration of 3.6 ng mL⁻\u0026sup1;, showing a distinct and sharp peak at a retention time of approximately 12.87 min, which confirms the elution position of pure AFM₁ under the applied chromatographic conditions. In contrast, chromatogram (B) corresponds to a Karish cheese sample, in which the same characteristic AFM₁ peak appeared at the same retention time (12.87 min), indicating the presence of the toxin in the analyzed dairy matrix. However, additional smaller peaks observed at 2.154 min and 5.798 min correspond to other naturally occurring compounds or matrix interferences commonly found in fermented dairy products.\u003c/p\u003e \u003cp\u003eThe similarity in retention time between the standard and the sample confirms the identity of AFM₁ in the tested cheese. The relatively lower peak area in the sample chromatogram compared with the standard demonstrates a reduced AFM₁ concentration, likely due to the binding or degradation effect of LAB used during fermentation. This finding highlights the effectiveness of LAB strains in mitigating AFM₁ contamination through adsorption or biotransformation mechanisms during cheese processing and storage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eComparison of AFM₁ detoxification efficiency among viable LAB strains\u003c/h2\u003e \u003cp\u003eThe data presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e evaluate the efficiency of various viable LAB strains in reducing AFM\u003csub\u003e1\u003c/sub\u003e concentration from PBS. The initial concentration of AFM\u003csub\u003e1\u003c/sub\u003e was 50 \u0026micro;g/L, and changes in its residual level were recorded after 4, 8, 16, and 24 hrs of incubation at 37\u0026deg;C. Across all LAB strains tested, a progressive reduction in AFM\u003csub\u003e1\u003c/sub\u003e concentration was observed with increasing incubation time. This indicates that viable LAB cells possess the ability to interact with and remove AFM\u003csub\u003e1\u003c/sub\u003e, most likely through adsorption to cell wall components or by partial metabolic transformation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of viable lactic acid bacteria strains on aflatoxin M1 (AFM\u003csub\u003e1\u003c/sub\u003e) reduction in PBS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eViable strain \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eConcentration of AFM\u003csub\u003e1\u003c/sub\u003e (\u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hrs \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e% AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eremoval after 24 hrs\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. helveticus\u003c/em\u003e LH-BO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e68.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/b\u003e\u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. lactis\u003c/em\u003e Bb12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e37.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e70.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. paracasei\u003c/em\u003e TISTR 453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e34.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e67.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. johnsonii\u003c/em\u003e ATCC 3320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/b\u003e\u003csup\u003e\u003cb\u003e7c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e ATCC4356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e31.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e76.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e36.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e64.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/b\u003e\u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e35.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eViable bacteria were incubated in PBS (4 mL) at 37\u0026deg;C for 1 hrs\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eIncubation period\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eThe initial AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003econcentration was 50 \u0026micro;g/L\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of triplicate samples\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eDifferent superscript letters in the same column indicate significant difference at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter 24 hrs, all strains achieved substantial AFM\u003csub\u003e1\u003c/sub\u003e reduction, ranging from 64.8 to 76.4%. Among the tested LAB, \u003cem\u003eL. acidophilus\u003c/em\u003e ATCC 4356 exhibited the highest detoxification efficiency, reducing the AFM\u003csub\u003e1\u003c/sub\u003e concentration to 11.8 \u0026micro;g/L, equivalent to 76.4% removal.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. lactis\u003c/em\u003e Bb12 and \u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390 also demonstrated strong performance with 70.1 and 69.2% removal, respectively. In contrast, \u003cem\u003eS. thermophilus\u003c/em\u003e showed the lowest reduction (only 64.8%) after 24 hrs, suggesting comparatively weaker binding affinity or fewer active cell wall sites for AFM\u003csub\u003e1\u003c/sub\u003e interaction.\u003c/p\u003e \u003cp\u003eAt the early incubation stage (4 hrs), the AFM\u003csub\u003e1\u003c/sub\u003e concentration remained relatively high for all strains, with values between 31.5 and 38.8 \u0026micro;g/L, corresponding to 22\u0026ndash;37% removal. After 8 hrs, a marked destabilization was observed, especially for \u003cem\u003eB. lactis\u003c/em\u003e (37.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u0026micro;g/L removed). However, between 16 and 24 hrs, the reduction rate tended to stabilize, suggesting that the binding capacity of bacterial cells reached equilibrium-possibly due to saturation of adsorption sites or reversible toxin release at prolonged exposure.\u003c/p\u003e \u003cp\u003eThe trend indicates that most of the toxin removal occurred within the first 8\u0026ndash;16 hrs, followed by a slower rate of reduction up to 24 hrs. For example, \u003cem\u003eL. acidophilus\u003c/em\u003e reduced AFM\u003csub\u003e1\u003c/sub\u003e from 31.5 \u0026micro;g/L at 4 hrs to 18.5 \u0026micro;g/L at 16 hrs, and finally to 11.8 \u0026micro;g/L at 24 hrs, representing an incremental improvement of ~\u0026thinsp;25% between 16 and 24 hrs. This plateau effect implies that extending incubation beyond 24 hrs may not yield significant additional detoxification.\u003c/p\u003e \u003cp\u003eThe detoxification mechanism likely involves non-covalent adsorption of AFM\u003csub\u003e1\u003c/sub\u003e molecules onto bacterial surface components such as peptidoglycans, polysaccharides, and teichoic acids. The greater efficiency of \u003cem\u003eL. acidophilus\u003c/em\u003e could be attributed to its high surface area-to-volume ratio and hydrophobic cell surface, which enhance toxin binding. Strains like \u003cem\u003eS. thermophilus\u003c/em\u003e and \u003cem\u003eL. paracasei\u003c/em\u003e, with smoother or less dense cell wall structures, showed lower binding potential. Moreover, since viable cells were used, partial metabolic degradation of AFM\u003csub\u003e1\u003c/sub\u003e cannot be completely ruled out, particularly for species known to exhibit active enzymatic systems during incubation.\u003c/p\u003e \u003cp\u003eAll LAB strains tested are capable of reducing AFM\u003csub\u003e1\u003c/sub\u003e levels significantly, confirming their potential as biological adsorbents for mycotoxin decontamination. \u003cem\u003eL. acidophilus\u003c/em\u003e ATCC 4356 is the most promising candidate, achieving over three-quarters removal under the tested conditions. The use of LAB in detoxification systems offers a safe, natural, and cost-effective alternative to chemical decontamination methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eResidual AFM₁ concentrations after incubation with HK-LAB\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows residual AFM₁ concentrations (\u0026micro;g/L) after incubation of heat-killed LAB strains with an initial AFM₁ level of 50 \u0026micro;g/L, measured at 4, 8, 16 and 24 hrs. Bacteria boiled in PBS for 1 hrs (heat treated). Triplicate means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD are presented. Heat-killed cells produced only modest AFM₁ reductions over 24 hrs. Residual AFM₁ at 24 hrs ranged from 41.3 to 46.7 \u0026micro;g/L, corresponding to 6.6\u0026ndash;17.4% removal of the initial 50 \u0026micro;g/L. These removal levels are substantially lower than those achieved with viable LAB in the previous table (where removals were ~\u0026thinsp;65\u0026ndash;76% after 24 hrs). This indicates that viability (or the cell state prior to heat treatment) strongly affects AFM₁ removal under the tested conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of heat-killed lactic acid bacteria (HK-LAB) strains on AFM\u003csub\u003e1\u003c/sub\u003e removal from PBS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHeat-killed\u003c/p\u003e \u003cp\u003estrain \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003econcentration of AFM\u003csub\u003e1\u003c/sub\u003e (\u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hrs \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e% AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eremoval after 24 hrs\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. helveticus\u003c/em\u003e LH-BO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. lactis\u003c/em\u003e Bb12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e45.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e44.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e43.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. paracasei\u003c/em\u003e TISTR 453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e46.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. johnsonii\u003c/em\u003e ATCC 3320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e ATCC4356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e42.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e44.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e43.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e43.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e44.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e42.9\u0026thinsp;\u0026plusmn;\u0026thinsp;067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eBacteria were boiled in PBS (4 mL) for 1 hrs (heat treated)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eIncubation period\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eThe initial AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003econcentration was 50 \u0026micro;g/L\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate samples\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eDifferent superscript letters in the same column indicate significant difference at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. helveticus\u003c/em\u003e and \u003cem\u003eL. bulgaricus\u003c/em\u003e performed best among heat-killed strains (~\u0026thinsp;16\u0026ndash;17% removal), while \u003cem\u003eB. lactis\u003c/em\u003e, \u003cem\u003eL. paracasei\u003c/em\u003e and \u003cem\u003eL. johnsonii\u003c/em\u003e showed minimal effect (~\u0026thinsp;6\u0026ndash;8%).\u003c/p\u003e \u003cp\u003eThe change from 4 \u0026rarr; 24 hrs is small for all strains (typical reductions of ~\u0026thinsp;2\u0026ndash;6 \u0026micro;g/L over 20 hrs). For example, \u003cem\u003eL. helveticus\u003c/em\u003e decreased from 44.8 \u0026micro;g/L (4 hrs) to 41.9 \u0026micro;g/L (24 hrs) (a net removal of 2.9 \u0026micro;g/L, ~\u0026thinsp;5.8% absolute). The near-plateau behavior indicates rapid attainment of equilibrium between AFM₁ in solution and binding sites on heated cells, with little progressive removal after early contact.\u003c/p\u003e \u003cp\u003eHeat killing likely alters cell wall structure (protein denaturation, polysaccharide rearrangement) and abolishes metabolic activity. Two non-exclusive effects are plausible:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eLoss of active metabolic degradation pathways (so enzymatic breakdown is minimal).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eAlteration of adsorption sites - heating may expose some hydrophobic sites in some strains (slightly improving passive adsorption), or conversely destroy/lower affinity of ligand sites, reducing binding.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe substantially lower removal compared with viable cells suggests that active physiological factors (cell surface properties maintained in viable cells, or metabolism-linked processes) play a major role in AFM₁ removal observed with live cultures.\u003c/p\u003e \u003cp\u003eHeat-killed LAB are poor standalone solutions for AFM₁ removal under the tested conditions (max\u0026thinsp;~\u0026thinsp;17% removal). For meaningful decontamination, viable cultures or other interventions are required.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of incubation period and pH variation on the reduction of AFM₁ by LAB strains in MRS broth\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e summarize the relationship between pH variation and AFM₁ degradation by different lactic acid bacteria (LAB) strains during incubation in MRS broth for 1, 5, 10, 15, and 20 days. The initial AFM₁ concentration was 50 \u0026micro;g/L. Both AFM₁ reduction and acidification of the medium (pH decline) were monitored as indicators of bacterial metabolic activity and detoxification potential. All LAB strains exhibited a progressive decrease in AFM₁ concentration with increasing incubation time, accompanied by a steady decline in pH values from approximately 4.5 on day 1 to around 3.0 by day 20. This simultaneous decline suggests that acid production and active metabolism play crucial roles in enhancing AFM₁ removal.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of LAB strains and pH changes during incubation periods on the reduction of AFM\u003csub\u003e1\u003c/sub\u003e in MRS broth\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eIncubation time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003e% AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eremoval\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e5 D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e10 D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e15 D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e20 D\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAFM\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e@\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAFM\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAFM\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAFM\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAFM\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. helveticus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eLH-BO2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e40.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e34.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e25.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e18.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e73.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB. lactis\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eBb12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e39.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e31.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.88\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e24.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e17.99.2.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e11.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e76.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. salivarius\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTISTR 390\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e39.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e25.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.87\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e17.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.41\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e14.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e10.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e79.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. paracasei\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTISTR 453\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e38.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4..00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e34.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.80\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e28.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.41\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e19.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e12.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e75.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. johnsonii\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eATCC 3320\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e38.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e29.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e25.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.44\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e18.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e13.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e73.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. acidophilus\u003c/b\u003e \u003cb\u003eATCC4356\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.52\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e31.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e23.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e15.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e13.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e9.98\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e80.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. thermophilus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e36.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e28.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.68\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e22.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.51\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e18.99\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e2.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e14.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e70.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/b\u003e\u003csup\u003e\u003cb\u003ede\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL. bulgaricus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.47\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e35.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e26.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e21.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.43\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e19.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e2.98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e13.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e72.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/b\u003e\u003csup\u003e\u003cb\u003ecd\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e\u003cb\u003e#\u003c/b\u003e\u003c/sup\u003e \u003cem\u003emeasured pH value\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e\u003cb\u003e@\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eConcentration of AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(\u0026micro;g/L)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eThe initial AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003econcentration was 50 \u0026micro;g/L\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate samples\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eDifferent superscript letters in the same column indicate significant difference at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter 20 days, the most efficient strain was \u003cem\u003eL. acidophilus\u003c/em\u003e ATCC 4356, which reduced AFM₁ concentration from 50 \u0026micro;g/L to 9.98 \u0026micro;g/L, corresponding to an 80.0% removal rate. \u003cem\u003eL. salivarius\u003c/em\u003e followed closely with 79.8% removal, while \u003cem\u003eB. lactis\u003c/em\u003e and \u003cem\u003eL. paracasei\u003c/em\u003e achieved approximately 75\u0026ndash;76% reduction. The least effective strain was \u003cem\u003eS. thermophilus\u003c/em\u003e with 70.4% removal, though still significant compared to the initial concentration.\u003c/p\u003e \u003cp\u003eA clear correlation was observed between pH decline and AFM₁ reduction. For instance, in \u003cem\u003eL. acidophilus\u003c/em\u003e, pH dropped from 4.52 (day 1) to 3.00 (day 20), while AFM₁ fell from 31.5 \u0026micro;g/L to 9.98 \u0026micro;g/L. Similarly, \u003cem\u003eL. salivarius\u003c/em\u003e showed a pH drop from 4.38 \u0026rarr; 3.41 \u0026rarr; 3.00, with AFM₁ decreasing from 39.9 \u0026micro;g/L \u0026rarr; 14.2 \u0026micro;g/L \u0026rarr; 10.1 \u0026micro;g/L. This pattern indicates that the acidic environment produced by LAB metabolism enhances AFM₁ binding or degradation. Lower pH values may promote electrostatic interactions between AFM₁ molecules and bacterial cell wall components, facilitating adsorption.\u003c/p\u003e \u003cp\u003eIn early stage (day 1\u0026ndash;5): only partial removal observed (typically 20\u0026ndash;35%); metabolic activity just beginning. Intermediate phase (day 10\u0026ndash;15): marked acceleration in detoxification - AFM₁ reduced to ~\u0026thinsp;35\u0026ndash;40% of initial levels, coinciding with rapid acidification (pH\u0026thinsp;\u0026asymp;\u0026thinsp;3.4\u0026ndash;3.6). Late phase (day 20): near-maximum detoxification achieved (70\u0026ndash;80% removal) as bacterial growth reached stationary phase, and pH stabilized near 3.0. These results suggest that extended incubation enhances AFM₁ removal due to cumulative adsorption and possible enzymatic modification over time.\u003c/p\u003e \u003cp\u003eLAB strains, especially \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. salivarius\u003c/em\u003e, and \u003cem\u003eB. lactis\u003c/em\u003e, demonstrate strong potential for biological detoxification of AFM₁ in liquid matrices. The correlation between pH reduction and AFM₁ removal supports the use of controlled fermentation as a safe, natural method to reduce mycotoxin contamination in dairy environments. However, long incubation times (\u0026ge;\u0026thinsp;15 days) may be impractical industrially; optimization of cell density, medium composition, and initial pH could accelerate detoxification.\u003c/p\u003e \u003cp\u003eHierarchical cluster analysis (HCA) was performed to classify the eight LAB strains according to their efficiency in removing AFM₁ after 20 days of incubation in MRS broth. The dendrogram revealed two major clusters. The first cluster grouped \u003cem\u003eL. acidophilus\u003c/em\u003e ATCC4356 and \u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390, which exhibited the highest AFM₁ removal rates of 80.0 and 79.8%, respectively, indicating their strong detoxification capacity. The second cluster included the remaining strains, with \u003cem\u003eS. thermophilus\u003c/em\u003e (70.4%) and \u003cem\u003eL. bulgaricus\u003c/em\u003e (72.2%) showing the lowest reduction levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results indicate that \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e are the most potent AFM₁-reducing strains, showing a distinct clustering pattern that reflects their superior detoxification potential. This clustering pattern demonstrates clear variability among the tested LAB strains, suggesting that the ability to remove AFM₁ is strain-dependent. The close association between \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e emphasizes their potential use as effective bio-detoxifying cultures for reducing AFM₁ contamination in dairy systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of mixed LAB starter cultures on AFM₁ reduction and pH changes in Karish cheese during cold storage\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the effect of three combinations of LAB starter cultures on the removal of AFM₁ from Karish cheese during processing and cold storage at 4\u0026deg;C for 20 days. The data clearly show that all bacterial combinations were effective in progressively reducing AFM₁ concentrations over time, accompanied by a consistent decrease in pH values, indicating enhanced acidification and metabolic activity of the cultures.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRemoval of AFM\u003csub\u003e1\u003c/sub\u003e from Karish cheese using three different LAB strain combinations as starter cultures during processing and storage at 4\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eStarter culture\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003epH/storage period\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003e% AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eremoval\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1 D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5 D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10 D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e15 D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u003cb\u003e20 D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003csup\u003e\u003cb\u003e#\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAFM\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003csup\u003e\u003cb\u003e@\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAFM\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eAFM\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eAFM\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eAFM\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003egroup No. 1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.70\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e36.89\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e28..78\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;2.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e20.23\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;0.49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.88\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e17.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e12.89\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.51\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e74.2\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;0.67\u003c/b\u003e \u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003egroup No. 2\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e ATCC4356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e34.83\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.79\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e27.16\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e20.12\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e10.99\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e78.0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.03\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003egroup No. 3\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. thermophilus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. salivarius\u003c/em\u003e TISTR 390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e33.39\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e23.88\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.80\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e19.37\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3..46\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e15.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e3.30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e11.82\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e76.4\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026plusmn;\u0026thinsp;1.39\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e\u003cb\u003e#\u003c/b\u003e\u003c/sup\u003e \u003cem\u003emeasured pH value\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e\u003cb\u003e@\u003c/b\u003e\u003c/sup\u003e \u003cem\u003eConcentration of AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(\u0026micro;g/L)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eThe initial AFM\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003econcentration was 50 \u0026micro;g/L\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate samples\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cem\u003eDifferent superscript letters in the same column indicate significant difference at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAt the initial stage (1 day), the AFM₁ content ranged from 36.89 \u0026micro;g/L in Group No. 1 (\u003cem\u003eS. thermophilus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. bulgaricus\u003c/em\u003e) to 33.39 \u0026micro;g/L in Group No. 3 (\u003cem\u003eS. thermophilus\u003c/em\u003e, \u003cem\u003eL. bulgaricus\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e). As the incubation and storage progressed, the reduction in AFM₁ became more pronounced. After 5 days, AFM₁ concentrations declined to 28.78 \u0026micro;g/L, 27.16 \u0026micro;g/L, and 23.88 \u0026micro;g/L for Groups 1, 2, and 3, respectively, representing removal percentages of approximately 21.9%, 22.1%, and 28.5% compared to the initial 50 \u0026micro;g/L concentration. By 10 days, the reduction trend continued, with AFM₁ levels decreasing to 20.23 \u0026micro;g/L, 20.12 \u0026micro;g/L, and 19.37 \u0026micro;g/L for Groups 1, 2, and 3, respectively. The greatest detoxification efficiency was observed in Group No. 3, which achieved a 61.3% reduction at this stage. After 15 days, AFM₁ concentrations dropped further to 17.54 \u0026micro;g/L, 16.88 \u0026micro;g/L, and 15.65 \u0026micro;g/L, while the pH values reached 3.88, 3.67, and 3.46, respectively. The lowest AFM₁ value recorded at the final stage (20 days) was 10.99 \u0026micro;g/L for Group No. 2, followed closely by 11.82 \u0026micro;g/L for Group No. 3 and 12.89 \u0026micro;g/L for Group No. 1, corresponding to removal efficiencies of 78.0%, 76.4%, and 74.2%, respectively. The gradual decline in AFM₁ concentration with the decrease in pH suggests a strong correlation between bacterial metabolic activity and toxin binding or degradation.\u003c/p\u003e \u003cp\u003eThe inclusion of \u003cem\u003eL. acidophilus\u003c/em\u003e (Group No. 2) and \u003cem\u003eL. salivarius\u003c/em\u003e (Group No. 3) appeared to enhance AFM₁ removal compared with the traditional yogurt starter (\u003cem\u003eS. thermophilus\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eL. bulgaricus\u003c/em\u003e). This improvement may be attributed to differences in cell wall composition and surface charge, which facilitate stronger adsorption of AFM₁ molecules. Group No. 2 achieved the highest total reduction (78%) after 20 days, indicating the synergistic detoxifying activity of \u003cem\u003eL. acidophilus\u003c/em\u003e when combined with the traditional starter bacteria.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSimilar results were obtained Elsanhoty et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), indicate that the initial concentrations of AFB\u003csub\u003e1\u003c/sub\u003e in yogurt influence the relative amounts of AFB\u003csub\u003e1\u003c/sub\u003e removed by viable bacteria and heat and acid treatment. Assaf et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) also confirmed these results discovering that different dairy strains of LAB and bifidobacteria with varying binding capabilities can reduce bioavailability of AFs. In Addition, heat-killed bacteria have been reported to be associated with a specific strain AFB\u003csub\u003e1\u003c/sub\u003e. It has been reported that the AFB\u003csub\u003e1\u003c/sub\u003e binds to the surface elements of LAB \u003cb\u003e(\u003c/b\u003eHaskard et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results also support the findings of Khadivi et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) who found that a group of dairy strains, species, and genus LAB are uniquely associated with AFB\u003csub\u003e1\u003c/sub\u003e. The results obtained were consistent with those published by Conte et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported that the amount of AFB\u003csub\u003e1\u003c/sub\u003e removed by viable bacteria or heat and acid treatment is determined by the initial concentration of AFB\u003csub\u003e1\u003c/sub\u003e. With similar results Ondiek et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported, they discovered that toxin clearance usually increases with increased toxin concentration. It has been shown that AFB\u003csub\u003e1\u003c/sub\u003e and T-2 can be eliminated by both living and denatured cells. Given their ability to bind mycotoxins and reduce their absorption into the bloodstream through the gastrointestinal tract, it can be inferred from the data that these LAB species could act as biological agents to reduce AFB\u003csup\u003e1\u003c/sup\u003e and T-2. Studies have demonstrated that \u003cem\u003eLactobacillus\u003c/em\u003e strains can remove mycotoxins from contaminated liquid media \u003cb\u003e(\u003c/b\u003eFreire et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLiu et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed that AFs molecules exhibited non-covalent reversible connectivity regardless of cellular activity through their ability to bind to the \u003cem\u003eL. plantarum\u003c/em\u003e cell wall. However, several variables, including the growth media, the bacterial state (alive or dead), the incubation temperature, the incubation duration, and the pH level of medium, influence the degree to which AFs bind to the \u003cem\u003eL. plantarum\u003c/em\u003e cell wall \u003cb\u003e(\u003c/b\u003eSadiq et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHeat and acid treatments severely affect the structural stability of polysaccharides and peptidoglycans in bacterial cell wall. According to \u003cb\u003ePiotrowsika (2014)\u003c/b\u003e, many strains of \u003cem\u003eLactobacillus\u003c/em\u003e bacteria undergo a change in cell surface polarity from hydrophilic to hydrophobic upon heat treatment; As a result, heat-treated \u003cem\u003eL. plantarum\u003c/em\u003e was able to adhere to AFs more easily. Yao et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that AFB\u003csub\u003e1\u003c/sub\u003e binds to \u003cem\u003eL. brucei\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e most strongly at pH 2.5 and less strongly at pH 8.5. As a result of denaturing the cell surface proteins and exposing additional binding area to AFB1, the results showed that acid treatment enhanced hydrophobic interactions.\u003c/p\u003e \u003cp\u003eThe results shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed that the type of starter had an effect on the variability of the AFB\u003csub\u003e1\u003c/sub\u003e degradation rate. Furthermore, a proportional relationship was found between the decrease in pH values and the corresponding decrease in AFM\u003csub\u003e1\u003c/sub\u003e content, where the greater the decrease in pH-values, the greater the decrease in AFM\u003csub\u003e1\u003c/sub\u003e content.\u003c/p\u003e \u003cp\u003eThe researchers hypothesized that suppression of AFs might be caused by lactic acid and/or LAB metabolites. These metabolites are low-molecular-weight compounds that are thermally-stable. Differences in extraction techniques, toxin concentration, and time before analysis, storage temperature, milk contamination method, milk composition, or the behavior of the starter cultures used in making the yogurt are likely responsible for these discrepancies in the results.\u003c/p\u003e \u003cp\u003eThe reduction of AFM₁ in yogurt during storage may be attributed to glucose oxidase activity, which catalyzes glucose oxidation to produce gluconolactone and hydrogen peroxide. The latter can generate reactive oxygen species capable of attacking the double bond in the AFM₁ dihydrofuran ring \u003cb\u003e(\u003c/b\u003eYousef and Marth, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1989\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Hydrolysis of gluconolactone yields gluconic acid, lowering the pH (~\u0026thinsp;3.9) and further promoting AFM₁ degradation. Several studies have reported the ability of \u003cem\u003eLactobacillus\u003c/em\u003e strains to remove mycotoxins from liquid media \u003cb\u003e(\u003c/b\u003eFreire et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eL. plantarum\u003c/em\u003e was shown to reduce ochratoxin A by 32\u0026ndash;58% \u003cb\u003e(\u003c/b\u003eMohammad and Hashemi, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e and aflatoxin B₁ by 69.1% \u003cb\u003e(\u003c/b\u003eDamayanti et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to literature reviews \u003cb\u003e(\u003c/b\u003eDawlal et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), mycotoxins are associated with the LAB cell wall and are therefore extracted from the contaminated medium. According to reports Zhang et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the main components of the LAB cell wall, associated with the mycotoxin binding mechanism, are teichoic acids, polysaccharides, and peptidoglycans. Thermal inactivation altered these components, leading to the formation of pores and protein denaturation in the bacterial cell wall, resulting in the emergence of new binding sites \u003cb\u003e(\u003c/b\u003eHaskard et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This could explain the binding obtained in our study with inactivated LAB cells. The ability of LAB to bind mycotoxin has been the subject of numerous studies, which have shown that it depends on a variety of factors, including the type of LAB strains, the density of LAB cells, the viability of LAB, the concentration of mycotoxin, pH medium, temperature and the incubation time \u003cb\u003e(\u003c/b\u003eZhao et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe binding sites on the cell walls of microorganisms are essential for LAB to eliminate mycotoxins. In discussing this dependence, Badji et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) offered two explanations for the observed results: first, the binding sites for OTA and AFB\u003csub\u003e1\u003c/sub\u003e were different from each other, and second, the concentration of mycotoxin was insufficient to saturate all binding sites on the cell walls of the microorganisms.\u003c/p\u003e \u003cp\u003eProbiotic LAB strains from various origins can reduce or eliminate AFM₁ in foods, exhibiting antimutagenic and anticancer properties. AFM₁ removal in Karish cheese lowered toxin levels to safe limits. This approach should be applied to improve food and feed safety. Further studies are needed to elucidate the detoxification mechanisms in LAB and Bifidobacteria, including the genetic traits enabling mycotoxin removal, to facilitate practical applications in the dairy industry.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrates the effective biological detoxification of aflatoxin M₁ by eight lactic acid bacteria (LAB) strains. Viable cells showed superior AFM₁ removal (up to 76%) compared to heat-killed ones (6.6\u0026ndash;17.4%), emphasizing the importance of cell viability and surface integrity. \u003cem\u003eL. acidophilus\u003c/em\u003e ATCC 4356 exhibited the highest efficiency, followed by \u003cem\u003eB. lactis\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e. In MRS broth and Karish cheese, toxin reduction (70\u0026ndash;80%) correlated with acidification during fermentation. The mixed culture of \u003cem\u003eS. thermophilus\u003c/em\u003e, \u003cem\u003eL. bulgaricus\u003c/em\u003e, and \u003cem\u003eL. acidophilus\u003c/em\u003e achieved the greatest detoxification, highlighting LAB\u0026rsquo;s potential as natural bio-detoxifying agents for safer dairy products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; contribution\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMA carried out the experiments and investigated the literature; MAA-S investigated, wrote, reviewed and edited the manuscript; all authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support for the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCode availability\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of interest\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eAhmed, N.H., El Soda, M., Hassan, A.N., Frank, J. 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(2001).\u003c/strong\u003e Fate of fumonisins during the production of fried tortilla chips. Journal of Agricultural and Food Chemistry, 49, 3120-3126.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eYao, Y.; Luo, J.; Zhang, P.; Wang, Y.; Lu, B.; Wu, G.; Zhang, J.; Luo, X.; Wang, L. \u003c/strong\u003e\u003cstrong\u003e(2024).\u003c/strong\u003e Screening, Identification and Application of Lactic Acid Bacteria for Degrading Mycotoxin Isolated from the Rumen of Yaks. Microorganisms 2024, 12, 2260.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eYousef, A. E., \u0026amp; Marth, E. H. (1989).\u003c/strong\u003e Stability and degradation of aflatoxin M1. In H. P. Van Egmond (Ed.), Mycotoxins in dairy products (pp. 127-161). London: Elsevier Applied Science.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eZhang, J., Zhang, J., Zhang, B. (2016).\u003c/strong\u003e The mechanism of \u003cem\u003eLactobacillus \u003c/em\u003estrains for their ability to remove fumonisins B1 and B2. 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Toxicol. 97, 40-46. https://doi.org/10.1016/j.fct.2016.08.028.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Aflatoxin M₁, Lactic acid bacteria, Detoxification, Karish cheese, Food safety","lastPublishedDoi":"10.21203/rs.3.rs-8280315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8280315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the ability of various lactic acid bacteria (LAB) strains, in both viable and heat-inactivated forms, to reduce aflatoxin M₁ (AFM₁) levels in phosphate-buffered saline, MRS broth, and Karish cheese. Eight LAB strains were tested, including \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, \u003cem\u003eL. salivarius\u003c/em\u003e, \u003cem\u003eL. bulgaricus\u003c/em\u003e, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e, and others. Viable cells exhibited strong detoxification efficiency, with \u003cem\u003eL. acidophilus\u003c/em\u003e achieving the highest AFM₁ reduction (\u0026asymp;\u0026thinsp;76%), followed by \u003cem\u003eB. lactis\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e. Heat-killed counterparts showed minimal activity, confirming the essential role of metabolic processes in toxin removal. In MRS broth, a significant relationship was observed between pH decline and AFM₁ degradation; where up to 80% reduction occurred at pH 3.0 after 20 hrs, emphasizing the influence of acidic and enzymatic conditions. Application in Karish cheese demonstrated the practical relevance of LAB under real food conditions. Mixed cultures of \u003cem\u003eS. thermophilus\u003c/em\u003e, \u003cem\u003eL. bulgaricus\u003c/em\u003e, and \u003cem\u003eL. acidophilus\u003c/em\u003e or \u003cem\u003eL. salivarius\u003c/em\u003e reduced AFM₁ by 74\u0026ndash;78% after 20 days at 4\u0026deg;C, accompanied by gradual acidification during ripening. These findings highlight the effectiveness of LAB, particularly multi-strain systems, as a natural and biotechnological approach for mitigating AFM₁ contamination and improving the safety of fermented dairy products.\u003c/p\u003e","manuscriptTitle":"Biological detoxification of Aflatoxin M₁ by lactic acid bacteria: experimental evidence from in vitro systems to fermented dairy products","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-23 18:31:26","doi":"10.21203/rs.3.rs-8280315/v1","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}}],"origin":"","ownerIdentity":"b795a22e-627d-47d4-a74f-677b500f96e5","owner":[],"postedDate":"December 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-08T21:09:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-23 18:31:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8280315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8280315","identity":"rs-8280315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}