Genotypic characterization of non starter lactic acid bacteria involved in the ripening of artisanal Bitto PDO cheese

Bitto of Valchiavenna, an artisanal Italian cheese produced without the addition of any starter cultures, has been attributed a protected designation origin (PDO) cheese, but the strain composition of the natural microbial population colonizing this traditional dairy product is still unknown. To obtain preliminary information on the non starter lactic acid bacteria involved in its ripening, a total of 136 NSLAB isolates, randomly selected from MRS and M17 agar plates, were collected from three different cheese samples after 120 days of ripening. The new isolates were identified by combining PCR 16S–23S rDNA spacer analyses, partial 16S rRNA gene sequencing, species‐specific probes and colony hybridization. Eighty‐two isolates, representing 60% of the total strains selected, were homofermentative cocci: 83% of them were enterococci, with Enterococcus durans being the predominant species found. Pediococcus spp. were also isolated, together with strains of Streptococcus thermophilus. Within lactobacilli, 57% of the isolates were identified as Lactobacillus paracasei; Lact. curvatus, Lact. plantarum, Lact. fermentum, were present in a lower amount. The isolates were differentiated at strain‐level by Rep‐PCR analysis. This is the first effort to microbiological characterization of Valchiavenna's Bitto; the results suggest the possibility of preserving the wild bacterial population in order to protect the typical organoleptic characteristics of this traditional raw milk cheese and to select new strains for the dairy industry. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular adaptation of sourdough Lactobacillus plantarum DC400 under co-cultivation with other lactobacilli

This work was aimed at investigating the molecular mechanisms of Quorum Sensing (QS) in Lactobacillus plantarum DC400 when co-cultured with other sourdough lactobacilli. The growth and survival of L. plantarum DC400 was not affected when co-cultivated with Lactobacillus sanfranciscensis DPPMA174 or Lactobacillus rossiae A7. Nevertheless, 2-DE analysis showed that the level of protein expression of L. plantarum DC400 increased under co-culture conditions. Although several proteins were commonly induced in both co-cultures, the highest induction was found in co-culture with L. rossiae A7. Overexpressed proteins, related to QS and stress response mechanisms, were identified: DnaK, GroEL, 30S ribosomal protein S1 and S6, ATP synthase subunit beta, adenosylmethionine synthetase (MetK), phosphopyruvate hydratase, phosphoglycerate kinase, elongation factor Tu, putative manganese-dependent inorganic pyrophosphatase, d-lactate dehydrogenase, triosephosphate isomerase, fructose-bisphosphate aldolase and nucleoside-diphosphate kinase. As shown by real-time PCR, expression of the luxS gene of L. plantarum DC400 was also affected during co-cultivation. According to overexpression of MetK and luxS during co-cultivation, synthesis of AI-2-like substances was also influenced by the type of microbial co-cultures.This study showed that expression of some genes/proteins, also QS-related, in L. plantarum was influenced by co-cultivation of other sourdough lactobacilli.     

Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Intestinal epithelial cells are confronted with many noxious stimuli which play an important role in the mucosal immune response. In the present study, Caco-2 cells treated with hydrogen peroxide were used as a model system for studying inflammatory responses and induction of cell death. Live lactobacilli and their concentrated spent culture supernatant (SCS) were applied for studying possible short- and long-term protective effects against hydrogen peroxide-mediated oxidative stress in Caco-2 cells. The secretion of pro-inflammatory cytokine interleukin-8 (IL-8) was investigated in non-filter grown Caco-2 cells, while transepithelial electrical resistance and cell death was studied in filter grown Caco-2 cells. Pre-incubation of Caco-2 cells with Lactobacillus plantarum 2142 did not decrease IL-8 levels induced by 1 mM hydrogen peroxide, nor did lactobacilli suppress IL-8 levels induced by hydrogen peroxide when Caco-2 cells were treated simultaneously with 1 mM hydrogen peroxide and L. plantarum 2142. Thus, lactobacilli did not exert a long-term protective effect against hydrogen peroxide in non-filter grown Caco-2 cells. However, the concentrated SCS of lactobacilli was able to reduce IL-8 levels by more than 6-fold, as determined 24 h after treatment. A short-term effect of lactobacilli was observed in filter grown Caco-2 cells, as they inhibited cell death induced by hydrogen peroxide in the concentration range 10-40 mM. Pre-incubation of epithelial cells with L. plantarum 2142 or simultaneous exposure to hydrogen peroxide and lactobacilli protected Caco-2 cells against cell death. In spite of the presence of lactobacilli, the permeability of membrane increased (transepithelial electrical resistance decreased), and exhibited a similar characteristic pattern as under treatment with hydrogen peroxide alone.     

Production of α-galactosidase by Lactobacillus plantarum isolated from diverse sources

α-Galactosidase activity was observed in six strains of Lactobacillus plantarum isolated from fermented cereal products, human intestinal flora and fermented tea. The cultural conditions under which the enzyme activity was detected suggest that the enzyme is inducible. Development of mutants in four out of the six strains was observed and the mutants recorded high enzyme activity than the parent strains. Effect of different carbohydrates on enzyme activity showed glucose and raffinose being repressive in the parent strains. Although all the carbohydrate sources supported growth, highest amount of enzyme activity was recorded on lactose and glucose. The enzymatic potential of L plantarum in reducing flatulence properties of the raw material base of some West African fermented foods is suggested.     

Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables

Ten Lactobacillus strains originally isolated from Thai fruits and vegetables fermentation were characterized by various phenotypic and genotypic methods. The phenotypic analysis using the method of carbohydrate fermentation patterns (API50CHL) revealed that the isolates belonged to the L. plantarum species. This was further confirmed by 16S rRNA gene sequencing. Multilocus sequence typing (MLST) revealed a strongly clonal population structure and a low genotypic diversity in this collection. However, the analyzed L. plantarum population demonstrated a higher level of diversification after API50CHL that reflects the role of available carbohydrate sources in bacterial evolution. Our results support the postulate that a combination of conventional biochemical and genotyping methods allows a thorough characterization and identification of isolates. We propose that genotypic characterization could be complemented by biochemical characterization to discriminate L. plantarum strains. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Influence of feeding fermented milk and non-fermented milk containing Lactobacillus casei on immune response in mice

The effect of feeding Lactobacillus casei in the form of fermented and non-fermented milk on non-specific as well as humoral immune response in mice was investigated. Feeding of L. casei fermented milk (LcF), its cell free supernatant (LcS), and L. casei non-fermented milk (LcNF) to mice for period of two, five and eight days resulted in increased activity of β-galactosidase and β-glucuronidase in peritoneal macrophages in comparison to skim milk (control). The phagocytic activity of peritoneal macrophages also increased significantly (p<0.01) in mice fed on LcF, LcS and LcNF compared to skim milk-fed mice. The maximum rise in β-galactosidase, β-glucuronidase and phagocytic activity was on day 5 post-feeding. In challenge studies with Shigella dysenteriae, it was observed that colonization of S. dysenteriae in the intestine, liver and spleen was significantly less in mice fed on LcNF and LcF in comparison to mice fed on skim milk for a period of seven days before challenge. Levels of secretory antibodies were higher in groups fed LcNF and LcF. The results suggest the immunomodulatory potential of L. casei.     

Characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from Irish pork and beef abattoirs

Lactic acid bacteria isolated from Irish pork and beef abattoirs were analysed for their susceptibility to antimicrobials. Thirty-seven isolates (12 enterococci, 10 lactobacilli, 8 streptococci, 3 lactococci, 2 Leuconostoc, and 2 pediococci) were examined for phenotypic resistance using the E-test and their minimum inhibitory concentration to a panel of six antibiotics (ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, and vancomycin) was recorded. The corresponding genetic determinants responsible were characterised by PCR. Also, the transferability of these resistance markers was assessed in filter mating assays. Of the 37 isolates, 33 were found to be resistant to one or more antibiotics. All strains were susceptible to ampicillin and chloramphenicol. The erm(B) and msrA/B genes were detected among the 11 erythromycin-resistant strains of enterococci, lactobacilli, and streptococci. Two tetracycline-resistant strains, Lactobacillus plantarum and Leuconostoc mesenteroides spp., contained tet(M) and tet(S) genes respectively. Intrinsic streptomycin resistance was observed in lactobacilli, streptococci, lactococci and Leuconostoc species; none of the common genetic determinants (strA, strB, aadA, aadE) were identified. Four of 10 strains of Enterococcus faecium were resistant to vancomycin; however, no corresponding genetic determinants for this phenotype were identified. Enterococcus faecalis strains were susceptible to vancomycin. L. plantarum, L. mesenteroides and Pediococcus pentosaceus were intrinsically resistant to vancomycin. Transfer of antibiotic resistance determinants was demonstrated in one strain, wherein the tet(M) gene of L. plantarum (23) isolated from a pork abattoir was transferred to Lactococcus lactis BU-2-60 and to E. faecalis JH2-2. This study identified the presence of antibiotic resistance markers in Irish meat isolates and, in one example, resistance was conjugally transferred to other LAB strains.     

The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection

In this study, we report that administration of Lactobacillus casei confers protection to mice against the intracellular protozoan Babesia microti. Mice treated with L. casei orally or intraperitoneally were inoculated 7 days later with an infectious dose of B. microti. Mice treated with lactobacilli showed significant reduction in the percentage of parasitized erythrocytes (PPE) compared to untreated mice. When mice were inoculated intraperitoneally with L. casei 3 or 0 days before challenge with B. microti, the PPE was significantly lower compared to untreated mice and there were no differences between treated mice and mice immune to B. microti infection. When mice treated with live or dead L. casei were compared to mice inoculated with Freund Complete Adjuvant before a B. microti infection, a significant reduction of PPE was observed. These results show the protective effect of L. casei administered to mice against a B. microti infection and suggest that it might act by stimulating the innate immune system.     

Lactic acid bacteria differ in their ability to induce functional regulatory T cells in humans

Background Trials with probiotic lactic acid bacteria have yielded different results, which may be due to the strains used. Lactobacilli and bifidobacteria are known to be potent modulators of the immune system. The capacity of these bacteria used as probiotics to influence both T helper type 1 (Th1)‐ and Th2‐mediated diseases has been shown before. However, the ability of strains to induce forkhead box P3 (FOXP3⁺) expressing regulatory T cells has not yet been investigated. Objective Test the inherent differences between strains in their capacity to induce functional regulatory T cells in human peripheral blood mononuclear cells (PBMC). Methods Human PBMC were co‐cultured in vitro with either Bifidobacterium lactis W51, Lactobacillus acidophilus W55 or Lactobacillus plantarum W62 or an Escherichia coli control strain. The percentage of FOXP3⁺ cells, the origin of the induced cells and the functionality of these cells were assessed. Results Probiotic strains differ in their capacity to induce regulatory T cells. FOXP3⁺ cells were induced from CD25^? cells and were able to suppress effector T cells. Naturally occurring regulatory T cells were not affected by co‐culture with lactobacilli. IL‐10 concentrations found in the supernatant showed a trend towards the same differences between strains. Blockade of IL‐10 did not influence the up‐regulation of FOXP3. No differences between lactic acid bacteria were found in IL‐17, IFN‐γ or IL‐13. Conclusions Some probiotic strains are potent inducers of regulatory cells, while others are not. The clear differences between strains imply that an in vitro characterization of probiotic strains before application is recommended. Cite this as: S. de Roock, M. van Elk, M. E. A. van Dijk, H. M. Timmerman, G. T. Rijkers, B. J. Prakken, M. O. Hoekstra and I. M. de Kleer, Clinical & Experimental Allergy, 2010 (40) 103–110.     

Monoclonal antibodies for the detection of Rhizomonas Suberifaciens causal agent of corky root of lettuce,with enzyme immunoassays

Monoclonal antibodies (MAbs) were produced to Rhizomonas suberifaciens strain CA1. Initial screening of MAbs was performed with strains CA1 and FL1 of R. suberifaciens and 82 strains of other genera in antibody capture indirect enzyme‐linked immunosorbent assay (ELISA) tests. One MAb (MAb‐Rs 1) reacted with both strains of R. suberifaciens and another (MAb‐Rs 2) only with R. suberifaciens strain CA1. Both MAbs were further screened for cross‐reactivity with 28 strains of R. suberifaciens, four strains of Rhizomonas sp., and 56 strains of unknown bacteria isolated from lettuce, prickly sowthistle and melon roots with symptoms of corky root. All these strains were tested for pathogenicity on lettuce, DNA homology with DNA of R. suberifaciens strain CA1, and cross‐reactivity with both MAbs in indirect antibody capture and indirect double antibody sandwich (DAS) ELISA tests. All strains of R. suberifaciens reacted with MAb‐Rs1 in both indirect ELISA tests, while none of the other strains reacted. In antibody capture indirect ELISA tests, MAb‐Rs2 had affinity to strains of R. suberifaciens from California and New York but not to those from Florida, Wisconsin and one from New York. In indirect DAS ELISA tests, MAb‐Rs2 reacted with all strains of R. suberifaciens. The indirect DAS ELISA tests were more sensitive than the indirect antibody capture tests.     

Bactericidal activity of culture fluid components of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> strain 90 TS-4 (21) clone 3, and their capacity to modulate adhesion of <Emphasis Type="Italic">Candida Albicans</Emphasis> yeast-like fungi to vaginal epithelial cells

Antagonistic activities of L. fermentum strain 90 TS-4 (21), L. casei ATCC 27216, and L. acidophilus ATCC 4356 and bactericidal activity of lactobacillus culture fluid towards E. coli strain K12, S. aureus, and S. epidermidis test cultures were studied. The bactericidal effect of L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation (pH 6.0) on the test cultures was dose-dependent. Adhesion of C. albicans yeast-like fungi to vaginal epitheliocytes was more pronounced for strains isolated from women with asymptomatic infection than for strains isolated from women with manifest forms. L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation modulated adhesion of yeast-like fungi only if the fungal strain was initially highly adherent. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 330–334, March, 2007     

Lack of correlation between in vitro antibiosis and in vivo protection against enteropathogenic bacteria by probiotic lactobacilli

Increased resistance to infection is one of the beneficial effects attributed to probiotic microorganisms. This effect may be due to several mechanisms: production of inhibitory substances, blocking of adhesion sites on the intestinal surface, competition for nutrients and stimulation of mucosal and systemic immunity. The present study aimed to investigate the correlation between in vitro and in vivo antimicrobial activity of probiotic lactobacilli. The agar spot test was used to show that twenty Lactobacillus strains were able to inhibit the enteropathogenic bacterium Yersinia enterocolitica. This inhibition was mainly attributable to a decrease in pH resulting from dextrose fermentation by lactobacilli. The inhibition of Y. enterocolitica, Salmonella enterica serovar Typhimurium and Listeria monocytogenes by two probiotic strains, Lactobacillus casei C1 and Lactobacillus plantarum C4, was also associated with the pH decrease. However, both strains lacked protective effects in mouse experimental infection models, with the exception of long-lasting pre-treatment with L. plantarum C4, which exerted a partial protective effect against S. Typhimurium that was attributable to an immunostimulatory mechanism. Our results show that in vitro antibiosis tests do not provide useful information on the probiotic potential of Lactobacillus strains.     

Use of 16S rRNA gene sequencing to identifyLactobacillus casei in septicaemia secondary to a paraprosthetic enteric fistula

Human infections caused byLactobacillus spp. are rarely reported in the literature. Underlying conditions are frequently reported, and identification of lactobacilli to the species level remains rare. A case ofLactobacillus casei septicaemia secondary to a vascular graft infection is reported. The 16S rRNA sequencing technique was used to definitively identifyLactobacillus casei.     

Immunomodulatory effects of dead Lactobacillus on murine splenocytes and macrophages

To elucidate the immunomodulation effects of dead lactobacilli, whole cells and gastrointestinal enzymatic hydrolysates of supernatants and precipitates from Lactobacillus paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 on RAW264.7 macrophages and splenocytes were investigated. Increased NO, COX-2 expression, IL-10 and IL-12 were observed in high-dose precipitates and whole cells of both strains after 24-h stimulation. All of the hydrolysates and whole cells from both strains induced lower pro-inflammatory cytokines (IL-1β and IL-6) than LPS. The supernatants activated cell division to the S phase or promoted advance to the G2/M phase. Regardless of the Lactobacillus strains, higher levels of TNF-α, IL-6, IL-10 and IL-12 in splenocytes were induced by the precipitates. Supernatant of NTU 101 increased the amounts of IFN-γ than precipitate in splenocytes. It shows that hydrolysates of NTU 101 induce the proliferations of macrophage and splenocyte and the release of IL-10 and IL-12 cytokines to modulate the innate and adaptive immune systems and inflammatory response.     

Role of arginine deiminase in thymic atrophy during experimental Streptococcus pyogenes infection

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L‐arginine deficiency in the host cells’ microenvironment. Bioavailability of L‐arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46‐16, we obtained a strain with inactivated arcA/sagp gene (M49‐16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L‐arginine concentration in the plasma of mice infected with S pyogenes M49‐16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49‐16 demonstrated gradual diminution of L‐arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49‐16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46‐16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49‐16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49‐16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49‐16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.     

Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2^−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Assessment of Safety of Lactobacillus Strains Based on Resistance to Host Innate Defense Mechanisms

Seven Lactobacillus strains belonging to four species were evaluated for pathogenicity as well as for in vitro sensitivity to the bactericidal mechanisms of macrophages in a rabbit infective endocarditis (IE) model. Two bacteremia-associated strains, L. rhamnosus PHLS A103/70 and L. casei PHLS A357/84, as well as the L. rhamnosus type strain and the probiotic L. rhamnosus strain ATCC 53103, showed moderate infectivity, and the virulence of the probiotic L. casei strain Shirota and type strains such as L. acidophilus ATCC 4356^T and L. gasseri DSM 20243^T in the model was negligible. The strains that showed pathogenic potential in the rabbit IE model (PHLS A357/84, PHLS A103/70, and ATCC 53103) were more resistant than strain Shirota to intracellular killing activity by mouse macrophages in vitro and also to bactericidal nitrogen intermediates, such as nitric oxide and NO₂⁻ ions. These results suggest that resistance to host innate defense systems, which would function at inflammatory lesions, should be considered in the safety assessment of Lactobacillus strains.     

Enhanced Antigen-Specific Delayed-Type Hypersensitivity and Immunoglobulin G2b Responses after Oral Administration of Viable Lactobacillus casei YIT9029 in Wistar and Brown Norway Rats

In this study, the effects of orally administered viable Lactobacillus casei Shirota strain YIT9029 on the immunity parameters of Wistar and Brown Norway rats were examined. For this purpose, we used the Trichinella spiralis host resistance model. Two weeks before and during T. spiralis infection, rats were fed 10⁹ viable L. casei bacteria 5 days per week. The T. spiralis-specific delayed-type hypersensitivity (DTH) response was significantly enhanced in both Wistar and Brown Norway rats given L. casei. In both rat strains fed L. casei, serum T. spiralis-specific immunoglobulin G2b (IgG2b) concentrations were also significantly increased. In the model, no significant effects of L. casei on larval counts or inflammatory reactions in the tongue musculature, body weights, or lymphoid organ weights were observed. Serum specific antibody responses, other than IgG2b, were not changed by feeding of L. casei. In contrast to L. casei, it was shown that orally administered Bifidobacterium breve or Bifidobacterium bifidum had no influence on the measured infection and immunity indices in the rat infection model. Since the rat DTH response is considered to be a manifestation of Th1 cell-mediated immunity and the IgG2b isotype has been associated with Th1 activity, it was concluded that Th1 cells could play an active role in the immunomodulatory effects of orally administered L. casei. Furthermore, our data do not indicate that the effect of oral supplementation with L. casei is dependent on the genetic background of the host.     

Ability of Lactobacillus rhamnosus GAF01 to remove AFM1 in vitro and to counteract AFM1 immunotoxicity in vivo

Aflatoxin M₁ (AFM₁) has been detected in many parts of the world both in raw milk and many dairy products, causing great economic losses and human disease. Unfortunately, there are few studies dealing with AFM₁ immunotoxicity/interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM₁ in vitro and evaluate whether the same organism(s) could impart a protective role against AFM₁-induced immunotoxicity in exposed Balb/c mice. Bacteria (Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM₁ from phosphate-buffered saline (PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM₁/ml) after 0, 6, and 24?h at 37°C. Results showed that the selected bacteria could ‘remove’ AFM₁ both in PBS and skimmed milk. The binding abilities of AFM₁ by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 10⁸ CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1–78.6% and 15.3–95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. ‘Removal’ appeared to be by simple binding; the bacteria/AFM₁ complex was stable and only a very small proportion of mycotoxin was released back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM₁-induced effects on total white and red blood cells, and lymphocyte subtypes, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM₁ in vitro and—by mechanisms that might also be related to a binding effect—counteract AFM₁-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an additive in dairy products and in biotechnology for mycotoxin detoxification.