Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

D. FIRA, M. KOJIC, A. BANINA, I. SPASOJEVIC, I. STRAHINIC AND L. TOPISIROVIC. 2001 . The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6·5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both α_S1-casein and β-casein, showing very low activity towards κ-casein. The BGPF1 proteinase completely hydrolysed only β-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA–DNA hybridization and PCR analysis showed that BGPF1 contains the prtB -like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Variable response to exogenous Lactobacillus acidophilus NCFM consumed in different delivery vehicles

AIMS: To study the effects of the delivery vehicle for Lactobacillus acidophilus on the human faecal microbiota. Our hypotheses were that (i) the delivery vehicle would influence faecal lactobacilli numbers and (ii) consumption of Lact. acidophilus would influence the populations of Bifidobacterium and hydrogen sulphide-producing bacteria. METHODS AND RESULTS: Ten subjects each received Lact. acidophilus with skim milk or water. Lactobacillus, Bifidobacterium and hydrogen sulphide-producing bacterial populations were analysed before, during and after each treatment. Regardless of the vehicle, faecal lactobacilli populations changed during treatment. Bifidobacteria and the hydrogen sulphide-producing bacteria underwent no statistically significant population changes. Intra- and intersubject variability was observed. CONCLUSIONS: The vehicle in which Lact. acidophilus was delivered did not influence faecal lactobacilli numbers. Consumption of Lact. acidophilus did not influence the populations of Bifidobacterium and hydrogen sulphide-producing bacteria. The lactobacilli populations of subjects were variable. The fed lactobacilli did not appear to colonize the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide evidence that (i) there was no collective advantage to using skim milk as a delivery vehicle vs water; (ii) exogenous Lact. acidophilus did not affect endogenous bifidobacteria or hydrogen sulphide-producing bacteria; (iii) data should be carefully examined before pooling for analysis and (iv) continuous feeding was required to maintain an elevated lactobacilli population.     

Manufacturing of fermented goat milk with a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus in a controlled bioreactor

AIMS: This work was undertaken to study the feasibility and the characteristics of a fermented product made of goat milk, using a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus under controlled conditions, and to determine their survival in the fermented milk during refrigerated storage. METHODS AND RESULTS: Goat milk was inoculated with Lact. acidophilus and Bif. animalis mixed starter, fermented in a glass bioreactor with controlled temperature (37 degrees C) and anaerobiosis, and monitored for growth and acidification. The fermented milk was then stored for 10 days under refrigeration, and monitored daily for starter microflora survival and pH changes. Lact. acidophilus viable counts reached a maximum of 7.1 x 10(8) colony-forming units (CFU) ml(-1), and Bif. animalis a maximum of 6.3 x 10(7) CFU ml(-1) by 20 h of fermentation. During refrigerated storage, both strains exhibited a good survival, with viable numbers remaining essentially constant throughout the experiment, whereas the pH of the fermented milk dropped slightly. CONCLUSIONS: Mixed cultures of Bif. animalis and Lact. acidophilus may be used to produce fermented goat milk with high counts of both probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Goat milk fermented with Bif. animalis and Lact. acidophilus can be manufactured as an alternative probiotic dairy product.     

16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM

AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.     

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80°C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2·4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.     

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2–3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.     

Amylase production by three Lactobacillus strains isolated from chicken crop

Three amylolytic Lactobacillus strains designated LEM 220, LEM 207 and LEM 202 were isolated from the chicken crop. They belonged to the subgenus Thermobacterium. Strain LEM 220 resembled Lact. acidophilus. Amylase production was more abundant in cells grown in media containing amylopectin or starch than in media containing glucose or maltose. Optimum pH and temperature of the amylase were 5.5 and 55°C respectively. Hydrolysis of amylopectin gave maltose, maltotriose and small amounts of glucose. Strain LEM 207 also resembled Lact. acidophilus , but differed from strain 220. It had a lower amylase activity. Optimum pH and temperature of the amylase were 6.4 and 40°C, respectively, and hydrolysis of amylopectin gave maltose, maltotriose and carbohydrates higher than maltopentaose. Strain LEM 202 was similar to Lact. vitelinus. It had the lowest amylase activity which was increased only in presence of maltose. Amylase properties were similar to those of LEM 220.     

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.     

Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 in milk medium

The effect of growth parameters and the molecular basis for antibacterial activity by a natural isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 was studied. The inhibition was tested against a toxigenic strain of Bacillus cereus F 4810. When grown in milk medium, the activity was highest at an incubation temperature of 37 °C in 48 h. The antibacterial activity appeared to be produced between late logarithmic and early stationary phases. The active principle was proteinaceous in nature (bacteriocin) and stable to low pH (3·8–5·0) and heat (75 °C for 30 min). There was also the possible role of hydrogen peroxide in bringing about inhibition. The strain of Lact. delbruecki ssp. bulgaricus CFR 2028 revealed the presence of plasmid DNA bands of 9·4 and 6·5 kbp, respectively, in agarose gel electrophoresis. The above strain has the potential to be used as a biopreservative in popular Indian fermented foods.     

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Influence of the incubation temperature on the autolytic activity of Lactobacillus acidophilus

M.L. FERNANDEZ MURGA, A. PESCE DE RUIZ HOLGADO AND G.F. DE VALDEZ, 1995. The effect of temperature and growth phase on the autolysis of Lactobacillus acidophilus CRL 640 was studied. The maximal rate of autolytic activity ( ca 48% cell lysis) was found at 45°C. At this temperature, two peaks were detected: the first one at the early exponential phase of growth and the second lysis peak during the transition stage from the exponential to the stationary phase. The release of intracellular compounds absorbing at 260 and 280 nm was also detected at 45°C. The microscopic observations revealed morphological changes and the presence of ghost cells. At 37°C, the low autolytic activity obtained would be related to the normal cell cycle of growth.     

Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 from ready-to-eat salad

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 °C. The bacteriocin was inactivated by α-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2·0 to 10·0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.     

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.     

β-Galactosidase and 6-phospho-β-galactosidase activities in strains of the Lactobacillus acidophilus complex

β-Galactosidase (β-gal) and 6-phospho-β-galactosidase (P-β-gal) activities were measured in a total of 34 strains from Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gasseri and Lactobacillus johnsonii. The Lact. gasseri strains have P-β-gal but little or no β-gal activities. The strains from other species have β-gal but only very little P-β-gal activities.     

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria

In this Study the effects of both pH and organic acids on Helicobacter pylori NCTC 11637 were tested. Lactobacillus acidophilus, Lact. casei, Lact. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were assayed for their lactic acid production, pH and inhibition of H. pylori growth. A standard antimicrobial plate well diffusion assay was employed to examine inhibitory effects. Lactic, acetic and hydrochloric acids demonstrated inhibition of H. pylori growth in a concentration-dependent manner with the lactic acid demonstrating the greatest inhibition. This inhibition was due both to the pH of the solution and its concentration. Six strains of Lact. acidophilus and one strain of Lact. casei subsp. rhamnosus inhibited H. pylori growth where as Bifidobacterium bifidus, Ped. pentosaceus and Lact. bulgaricus did not. Concentrations of lactic acid produced by these strains ranged from 50 to 156 mmol 1⁻¹ and correlated with H. pylori inhibition. The role of probiotic organisms and their metabolic by-products in the eradication of H. pylori in vivo remains to be determined.     

Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response

AIMS: The ability of two different Lactobacillus strains (Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716), isolated from human breast milk, to modulate the immune response was examined. METHODS AND RESULTS: In rodent bone-marrow-derived macrophages (BMDM), the presence of Lact. fermentum CECT5716 induced pro-inflammatory cytokines, in contrast to the activation of IL-10 induced by Lact. salivarius CECT5713. Although both strains reduced the lipopolysaccharide (LPS)-induced inflammatory response in BMDM, the effect of Lact. salivarius CECT5713 was more efficient, probably because of the production of higher amounts of IL-10 cytokine. In vivo assays in mice showed similar results; the consumption of Lact. fermentum CECT5716 enhanced the production of Th1 cytokines by spleen cells and increased the IgA concentration in faeces. However, the consumption of Lact. salivarius CECT5713 induced IL-10 production by spleen cells. CONCLUSION: Therefore, in general, the effect of Lact. fermentum CECT5716 is immunostimulatory in contrast to the anti-inflammatory effect of Lact. salivarius CECT5713. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that two Lactobacillus strains isolated from breast milk can exert different and even opposing effects on immune response demonstrating the specificity of each strain.