Survival of Lactobacillus rhamnosus strains in the upper gastrointestinal tract

In the present study six probiotic Lactobacillus rhamnosus strains were investigated for their ability to survive in the human upper gastrointestinal tract through a dynamic gastric model of digestion. MRS broth was used as delivery vehicle and survival was investigated during in vitro gastric and gastric plus duodenal digestion. Results highlighted that all tested strains showed good survival rate during both gastric and duodenal digestion. In particular, three strains exhibited a great survival showing a recovery percentage in the range between 117 and 276%. In agreement with survival data, high lactic acid production was detected for all strains, confirming their metabolic activity during digestion.     

Probiotic lactobacilli in a semi-soft cheese survive in the simulated human gastrointestinal tract

In this study, probiotics Lactobacillus acidophilus NCFM and Lactobacillus rhamnosus HN001 in cheese were studied using models simulating the human gastrointestinal tract with the aim of investigating whether the cheese matrix affected the survival and metabolic properties of these probiotic strains. Probiotics in cheese survived in the simulated upper gastrointestinal tract model, and numbers of L. acidophilus, L. rhamnosus and total lactobacilli were increased in the colonic fermentation simulations of the probiotic cheese when compared with the non-probiotic cheese used as a control. The cheese matrix also beneficially affected cyclooxygenase-gene expression of colonocytes in a cell culture model. Freeze-dried probiotics, which were also analysed in the colonic simulator, showed similar changes in Lactobacillus numbers, although gave a stronger increase and also affected other microbial groups. These results indicate that the probiotic microbes in cheese survive in the gastrointestinal tract and that the cheese matrix does not seem to affect the probiotic survival.     

Survival of probiotic lactobacilli in the upper gastrointestinal tract using an in vitro gastric model of digestion

The aim of this study was to investigate survival of three commercial probiotic strains (Lactobacillus casei subsp. shirota, L. casei subsp. immunitas, Lactobacillus acidophilus subsp. johnsonii) in the human upper gastrointestinal (GI) tract using a dynamic gastric model (DGM) of digestion followed by incubation under duodenal conditions. Water and milk were used as food matrices and survival was evaluated in both logarithmic and stationary phase. The % of recovery in logarithmic phase ranged from 1.0% to 43.8% in water for all tested strains, and from 80.5% to 197% in milk. Higher survival was observed in stationary phase for all strains. L. acidophilus subsp. johnsonii showed the highest survival rate in both water (93.9%) and milk (202.4%). Lactic acid production was higher in stationary phase, L. casei subsp. shirota producing the highest concentration (98.2 mM) after in vitro gastric plus duodenal digestion.     

Behaviour of Listeria monocytogenes isolates through gastro-intestinal tract passage simulation, before and after two sub-lethal stresses

The effects of previous exposure to sub-lethal acidic and osmotic stresses on the survival of Listeria monocytogenes during exposure to gastro-intestinal (GI) tract simulation, was investigated. Six L. monocytogenes strains isolated from cheeses were selected and exposed to high salt concentrations or acidic conditions and their viability compared in quick and slow digestions. The results demonstrated that (i) all isolates were more sensitive to the exposure to acidic than to osmotic sub-lethal conditions (ii) significant differences (p < 0.05) between the two types of digestion were observed; in slow digestion, the log reduction was higher for all the tested isolates (iii) all isolates were inhibited in the presence of bile salts for both types of digestion (iv) differences between quick and slow digestion were not observed (p > 0.05) after exposure to either osmotic or acidic stress (v) a higher cellular inactivation (p < 0.001) was observed during the passage through the GI tract simulation after exposure to osmotic than to acidic stresses and (vi) neither osmotic nor acidic sub-lethal stresses conferred resistance to simulated GI tract conditions.     

Persistence of probiotic strains in the gastrointestinal tract when administered as capsules, yoghurt, or cheese

Most clinical studies of probiotics use freeze-dried, powdered bacteria or bacteria packed in capsules. However, probiotics are commercially available in various food matrices, which may affect their persistence in the gastrointestinal tract. The objective of the study was to compare oral and faecal recovery during and after administration of a combination of Lactobacillus rhamnosus GG and LC705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium animalis subsp. lactis Bb12 as capsules, yoghurt, or cheese. This randomized, parallel-group, open-label trial (n = 36) included a 4-week run-in, 2-week intervention, and 3-week follow-up period. Participants consumed 10¹⁰ cfu/day of probiotic combination and provided saliva and faecal samples before, during, and after the intervention. Strain-specific real-time PCR was used to quantify the strains.L. rhamnosus GG was the only probiotic strain regularly recovered in saliva samples. During the intervention period it was recovered in the saliva of 88% of the volunteers at least once. No difference was found between the yoghurt and cheese groups. At the end of the intervention, L. rhamnosus GG and LC705 counts were high in faecal samples of all product groups (8.08 and 8.67 log₁₀ genome copies/g, respectively). There was no matrix effect on strain quantity in faeces or the recovery time after ceasing the intervention. For P. freudenreichii subsp. shermanii JS and B. animalis subsp. lactis Bb12, a matrix effect was found at the end of the intervention (P < 0.01 and P < 0.001, respectively) and in the recovery time during follow-up (P < 0.05 for both). Yoghurt yielded the highest faecal quantity of JS and Bb12 strains (8.01 and 9.89 log₁₀ genome copies/g, respectively). The results showed that the administration matrix did not influence the faecal quantity of lactobacilli, but affected faecal counts of propionibacteria and bifidobacteria that were lower when consumed in cheese. Thus, the consumption of probiotics in yoghurt matrix is highly suitable for studying potential health benefits and capsules provide a comparable means of administration when the viability of the strain in the capsule product is confirmed.     

Survival of potentially probiotic enterococci in dairy matrices and in the human gastrointestinal tract

The aim of this study was to investigate the stability of Enterococcus strains isolated from a traditional Portuguese cheese and previously proved to be safe, in dairy matrices, and to assess survival of the best strains in the human gastrointestinal (GI) tract. Enterococcus faecium 32 and Enterococcus durans 37 were added to yoghurt that was ingested by 4 healthy adults. Detection of the enterococcal strains was performed with RAPD-PCR. The intervention trial showed transient colonisation with both strains, via presence in faeces during the ingestion period and disappearance by 10 d post-ingestion. Viable numbers of enterococci increased during the consumption period by 1.8–4.4 log-values, and returned to baseline level during the follow-up period. Based on data of the dairy matrix stability trials and human intervention study involving yoghurt ingestion, E. faecium 32 survived well both in the food matrix and in the human GI tract, thus showing probiotic potential.     

Protective effect of whey cheese matrix on probiotic strains exposed to simulated gastrointestinal conditions

A probiotic whey cheese added with Lactobacillus casei LAFTI®L26, Lactobacillus acidophilus LAFTI®L10 or Bifidobacterium animalis Bo was subject in vitro to sequential conditions that parallel the four major steps of digestion: mouth (artificial saliva), oesophagus-stomach (artificial gastric juice), duodenum (artificial intestinal juice) and ileum; its manufacture followed the traditional cheesemaking protocol of Portuguese Requeijão. MRS broth was inoculated in parallel as reference medium, to ascertain the protective effect of the whey cheese matrix itself upon those strains in every digestion step. Mouth conditions had an almost negligible effect upon all three strains, whereas oesophagus-stomach, duodenum and ileum conditions decreased the viable numbers of L. casei and L. acidophilus; in both systems, B. animalis suffered only slight decreases in viable numbers; and L. casei and L. acidophilus behaved likewise in MRS exposed to duodenum and ileum conditions. Whey cheese matrices thus appeared to protect the aforementioned three strains during transit throughout the simulated gastrointestinal system, so they are promising carriers of those probiotic bacteria.     

Influence on cheese proteolysis and sensory characteristics of non-starter lactobacilli strains with probiotic potential

The contribution to cheese proteolysis and sensory profile of four potentially probiotic non-starter lactobacilli strains was assessed in two different models: soft and semi-hard cheeses. All the strains were able to grow in both types of cheese, where they maintained high levels during ripening. Overall, adjunct cultures of Lactobacillus rhamnosus showed the strongest influence in peptidolysis, which was verified by changes in peptide profiles and increase of free amino acids concentration. Nevertheless, some cheeses treated with L. rhamnosus also showed post-acidification during ripening and decreased sensory characteristics compared to controls. Cheeses with adjunct cultures of Lactobacillus casei I90 and Lactobacillus plantarum I91 exhibited, in general, an intermediate level of peptidolysis between control and L. rhamnosus-added cheeses; their sensory characteristics were preserved or improved, and they did not show any defects. Adjunct cultures showed similar trends in both models, confirming L. casei I90 and L. plantarum I91 as the most performing adjunct cultures for cheese-making among the strains.     

Microbiological,immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese containing Lactobacillus rhamnosus EM1107

Cheeses are able to serve as suitable matrices for supplying probiotics to consumers, enabling appropriate conditions for bacteria to survive gastric transit and reach the gut, where they are assumed to promote beneficial processes. The present study aimed to evaluate the microbiological, immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese supplemented with the probiotic strain Lactobacillus rhamnosus EM1107. Thirty male albino Wistar rats were randomly distributed into 5 experimental groups with 6 animals each: negative (NC) and positive (PtC) control groups, control goat cheese (CCh), goat cheese added with L. rhamnosus EM1107 (LrCh), and L. rhamnosus EM1107 only (EM1107). All animals, except NC group were challenged with Salmonella Enteritidis (10⁹ cfu in 1 mL of saline through oral gavage). Microbial composition was assessed with high-throughput 16S rRNA sequencing by means of Illumina MiSeq (Illumina, San Diego, CA). Nuclear factor kappa B (NF-κB) from the animal cecum tissue was determined by real-time PCR and interleukins (TNF-α, IL-1β, IL-10, and IFN-γ) by means of ELISA. Myeloperoxidase and malondialdehyde levels were determined biochemically. The administration of the L. rhamnosus EM1107 probiotic strain, either as a pure culture or added to a cheese matrix, was able to reduce Salmonella colonization in the intestinal lumen and lessen tissue damage compared with rats from PtC group. In addition, the use of cheese for the probiotic strain delivery (LrCh) was associated with a marked shift in the gut microbiota composition toward the increase of beneficial organisms such as Blautia and Lactobacillus and a reduction in NF-κB expression. These findings support our hypothesis that cheeses might be explored as functional matrices for the efficacious delivery of probiotic strains to consumers.     

Influence of whey-based fruit juice containing Lactobacillus rhamnosus on intestinal well-being and humoral immune response in healthy adults

The ability of probiotics to balance intestinal microbes and to modulate gut immune system has been reported for several probiotic strains. In the present study, Lactobacillus rhamnosus strain VTT E-97800 (E800) or L. rhamnosus Lc705 (the latter in combination with Propionibacterium freudenreichii ssp. shermanii JS) were separately administered to healthy adult volunteers (n=11 in both study groups) in a whey-based fruit juice. Survival of the potentially probiotic L. rhamnosus strains in the GI-tract and their effect on intestinal well-being and peripheral immune parameters were assessed. Both L. rhamnosus strains were recovered in high numbers in faecal samples during the consumption period and with few exceptions they were not detected after the 2 weeks follow-up period. Consumption of the juice supplemented with either of the L. rhamnosus strains did not have significant effect on faecal consistency or defecation frequency. No changes were observed in the immunological parameters (number or immunoglobulin A-, G- and M-secreting cells, expression of Fcα-receptor or complement receptors 1 and 3), except for a slight decrease in the number of IgA secreting cells during ingestion of the juice supplemented with L. rhamnosus Lc705 and P. freudenreichii ssp. shermanii JS. The results indicate that L. rhamnosus strains E800 and Lc705 had good survival ability in the GI-tract when administered in a whey-based fruit juice matrix. No adverse effects on intestinal function or on studied immunological parameters were observed during consumption of the whey based juice drink supplemented with the potential probiotic strains.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Antimicrobial properties of probiotic strains isolated from breast-fed infants

The protective and probiotic characteristics of intestinal lactobacilli, bifidobacteria and enterococci isolated from breast-fed infant faeces were investigated. Fifty-two selected isolates were identified at the species level and further characterized for their antimicrobial and safety features. Resistance to simulated gastric digestion and adherence to Caco-2 cells were also studied in the selected strains Lactobacillus paracasei INIA P272, Lactobacillus rhamnosus INIA P344, L. rhamnosus INIA P426, Lactobacillus mucosae INIA P459, Bifidobacterium pseudolongum INIA P2, Bifidobacterium breve INIA P18, Enterococcus faecium INIA P445 and Enterococcus faecalis INIA 127 due to their tolerance to GIT (gastrointestinal tract) conditions and interesting antimicrobial properties compared to the other strains investigated. A considerable diversity in the probiotic and antimicrobial properties among breast-fed infant lactic acid bacteria and bifidobacteria isolates was recorded. Some of the selected strains could be promising candidates to be exploited in the development of infant formulas or novel food products.     

Viability of Probiotics in Goat Cheese During Storage and Under Simulated Gastrointestinal Conditions

The objective of this study was to evaluate the suitability of two strains of probiotic bacteria (Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus) incorporated individually into Boursin-type goat cheese and their in vitro resistance through the passage of the gastrointestinal tract in the cheese. The viability of B. lactis and Lb. rhamnosus cultures was unaffected throughout 35 days of storage at 4 °C, with a final count of >?7 log CFU/g. No significant difference (p?>?0.05) was observed between probiotic treatments and control in relation to pH and titratable acidity. B. animalis presented greater resistance to the artificial gastric and enteric juices than Lb. rhamnosus, with mean decreases in the initial populations of 0.2 and 4.0 log CFU/g within 35 days of storage, respectively. The addition of probiotic cultures did not affect the consumer acceptance of the goat cheeses. Three segments of consumers with different liking were identified. The results demonstrated that “Boursin” goat cheese is a promising matrix for the incorporation and protection of B. animalis subsp. lactis.     

In vitro gastrointestinal digestion of liquid and semi-liquid dairy matrixes

Protein digestion in two liquid dairy matrixes with different heat treatments (pasteurized and sterilized milks) and in one semi-liquid dairy matrix (stirred-yogurt) was investigated using an in vitro gastrointestinal digestion model. After buccal digestion, significantly lower amount of soluble proteins were measured in yogurt than in both milks. This difference between dairy matrixes decreased during gastric digestion and disappeared at the end of the duodenal digestion upon the proteolytic action of pepsin and pancreatin. Electrophoresis pattern of digested mixtures showed that casein digestion began at the gastric phase and was slower for pasteurized milk than sterilized milk and yogurt. At the end of duodenal digestion, no more intact caseins were present in all the dairy matrixes while faint bands of whey proteins were still visible for pasteurized milk and yogurt. The release of free amino acids during the duodenal phase varied according to their nature (acid, basic, neutral or hydrophobic) and seems to be governed by the specificity of the enzymes. These results suggest that the severity of milk's heat treatment influences the kinetics of protein digestion, mainly during the gastric phase, and that the impact of processing has to be considered to study protein digestion in dairy products.     

A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use

Specific strains should only be regarded as probiotics if they fulfill certain safety, technological and functional criteria. The aim of this work was to study, from a comprehensive point of view (in vitro and in vivo tests), three Lactobacillus strains (Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37) isolated from feces of local newborns, determining some parameters of technological, biological and functional relevance. All strains were able to adequately grow in different economic culture media (cheese whey, buttermilk and milk), which were also suitable as cryoprotectants. As selective media, LP-MRS was more effective than B-MRS for the enumeration of all strains. The strains were resistant to different technological (frozen storage, high salt content) and biological (simulated gastrointestinal digestion after refrigerated storage in acidified milk, bile exposure) challenges. L. rhamnosus 64 and L. gasseri 37, in particular, were sensible to chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin, increased the phagocytic activity of peritoneal macrophage and induced the proliferation of IgA producing cells in small intestine when administered to mice. Even when clinical trails are still needed, both strains fulfilled the main criteria proposed by FAO/WHO to consider them as potential probiotics for the formulation of new foods.     

A survey of non-starter lactic acid bacteria in traditional cheeses: Culture dependent identification and survival to simulated gastrointestinal transit

Cultivable NSLAB in traditional Pasta filata and ewes' milk cheeses were studied by both PCR-DGGE of cells from Rogosa agar and by isolation and molecular identification after a simulated gastric juice (SGJ) treatment of the cheese. Two to six species were retrieved from each sample. The majority of isolates were identified as Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus rhamnosus, Enterococcus or Pediococcus. Bile tolerance and bile salt hydrolase (BSH) activity were tested on 88 strains: 64% were able to grow with ≥0.15% bile and 40% were BSH positive. The effect of simulated digestion was tested on 15 strains. Inactivation ranged from 0.15 to 2.93 log cycles; most of the lethality was associated with pancreatic juice treatment. Although SGJ treatment alone may not provide a correct estimate of tolerance to gastrointestinal transit, it allowed selection of strains with a high tolerance to gastric juice, which may be tested as probiotic candidates.     

Technological and probiotic role of adjunct cultures of non-starter lactobacilli in soft cheeses

The influence of two cheese-isolated Lactobacillus strains on cheese composition, acceptability and probiotic capacity was assessed. Soft cheeses with and without the addition of Lactobacillus plantarum I91 or Lactobacillus paracasei I90 were prepared. Gross composition was assessed and secondary proteolysis was described by soluble fractions and free amino acids profiles. Acceptability was determined by a panel of 98 non-trained consumers. Cheeses harboring added Lactobacillus strains were also studied in vivo to evaluate their probiotic capacity. Gross composition of the cheeses was similar for control and treated (Lactobacillus-added) cheeses. Peptidolysis increased in cheeses with added lactobacilli, which was evidenced by a higher free amino acid content. Overall, the acceptability of the cheeses was good: 65%–80% of the consumers said that they “liked very much” or “liked” the cheeses. Cheeses with L. plantarum I91 showed the highest changes in composition and proteolysis and were the most accepted ones. On the contrary, composition of cheeses with L. paracasei I90 was similar to that of the controls, but these samples were less accepted than cheeses without lactobacilli. The oral administration of cheese containing L. plantarum I91 or L. paracasei I90 proved to be safe and able to enhance the number of IgA + cells in the small intestine lamina propria of mice. The use of selected strains of NSLAB exerted a technological and probiotic role: it contributed to the standardization of cheese quality and induced benefic health effects at the gut mucosa in vivo.     

In vitro evaluation of physiological probiotic properties of different lactic acid bacteria strains of dairy and human origin

Eleven lactic acid bacteria strains of importance to the dairy industry were subjected to in vitro analyses to determine their probiotic potential. Seven strains were isolated from ewe’s and cow’s milk (Enterococcus faecalis – five –, Lactococcus lactis and Lactobacillus paracasei). Four were obtained from American Type Culture Collection (ATCC), isolated from cheese (Lactobacillus casei 393), human feces (L. paracasei 27092 and Lactobacillus rhamnosus 53103) and used in cheese making (L. lactis 54104). Although none of the strains was able to degrade mucin, all E. faecalis showed, at least, one transferable antibiotic resistance, which excluded them as candidates for addition to foods. Of the remaining six safe strains, L. lactis strains were more tolerant to low pH than Lactobacillus spp.; all were tolerant to pancreatin and bile salts and showed antibacterial activity. The highest level of adhesion to Caco-2 cells was observed with L. lactis 660, even higher than L. rhamnosus ATCC 53103 (recognized probiotic and used as control). The physiological probiotic properties of these strains, mainly isolated from dairy sources, are interesting in view of their use in cheese productions as starter and non starter cultures. The five LAB safe strains studied may have potential as novel probiotics in the dairy foods.     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.