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.     

Addition of probiotic bacteria in a semi-hard goat cheese (coalho): Survival to simulated gastrointestinal conditions and inhibitory effect against pathogenic bacteria

In this study, the survival of the probiotics Lactobacillus acidophilus (LA-5), Lactobacillus casei subsp. paracasei (L. casei 01) and Bifidobacterium lactis (BB12) incorporated in a Brazilian semi-hard goat cheese (coalho) when exposed to in vitro simulated conditions of digestion was assessed. The inhibitory effects of these probiotic bacteria were also evaluated against Listeria monocytogenes and Staphylococcus aureus in the goat coalho cheese during refrigerated storage. At the end of the in vitro digestion, all of the probiotic tested strains presented decreased (p < 0.05) viable cell counts (5.5–6.0 log cfu/g) with respect to those determined before exposure to the mouth conditions (7–8 log cfu/g). L. casei subsp. paracasei presented inhibition rate of 7.87% and 23.63% against S. aureus on the 14th and 21st day of storage at 10 °C, respectively; against L. monocytogenes these values were 12.96 and 32.99%. Positive inhibition rates of B. lactis toward S. aureus were found on the 1st, 14th and 21st days of storage (16.32%, 10.12% and 3.67%, respectively); and against L. monocytogenes only on the 1st day of storage (3.28%). From these results, goat coalho cheese could be an interesting carrier of probiotic strains of L. acidophilus, L. casei subsp. paracasei and B. lactis. Moreover, L. casei subsp. paracasei, could be used as protective culture for delaying the growth of S. aureus and L. monocytogenes in goat coalho cheese.     

Acidification rates of probiotic bacteria in Minas frescal cheese whey

The acidification rates of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bifidobacterium animalis subsp. lactis (Bl) in co-culture with Streptococcus thermophilus (St) were studied in Minas frescal cheese whey. Effects of the co-culture composition and the final pH values on the kinetic parameters of acidification, post-acidification and counts of health promoting micro-organisms were also studied. Fermentation time to reach pH 4.5 was longer when St-Lr co-culture was used, while St-Lb had the shortest fermentation time when compared with the other co-culture combinations. All products showed development of acidity during the storage period and lowest values had been observed employing St-Bl co-culture. The technological interest of using M. frescal cheese whey for the production of a probiotic lactic beverage is discussed in this article.     

Incorporation of probiotic bacteria in whey cheese: decreasing the risk of microbial contamination

For dairy products that are consumed fresh, contamination by spoilage microorganisms and pathogens from the environment is a major concern. Contamination has been associated with a number of outbreaks of foodborne illnesses; however, consistent data pertaining to the microbial safety of whey cheeses specifically have not been reported. Hence, the goals of this research effort were (i) to manufacture a probiotic whey cheese with Bifidobacterium animalis and Lactobacillus casei and (ii) to assess the antimicrobial activity of these probiotics against a set of foodborne pathogens (Listeria innocua, Salmonella Enteritidis, and Staphylococcus aureus) and food spoilage microorganisms (Pseudomonas aeruginosa and Escherichia coli). Three ranges of these microbial contaminants were used for inoculation of cheeses: 10(3) to 10(4), 10(4) to 10(6), and 10(6) to 10(8) CFU/g. Inoculation in plain culture medium served as a control. The inhibition produced by the probiotics was calculated, and the major effect was found to be bacteriostatic. In specific cases, full inhibition was observed, i.e., by B. animalis against P. aeruginosa and by L. casei against Salmonella Enteritidis and L. innocua. Conversely, the least inhibition was detected for L. casei against P. aeruginosa. Our results suggest that use of these probiotic strains can extend the shelf life of whey cheeses and make them safer by delaying or preventing growth of common contaminant bacteria.     

Survival of probiotic bacteria nanoencapsulated within biopolymers in a simulated gastrointestinal model

The objective of this work was to fabricate electrospun nanofiber mats (nano-scale in diameter) using a combination of corn starch (CS) and sodium alginate (SA) and encapsulate probiotic strains of lactobacilli (Lactobacillus acidophilus (LA5) and Lactobacillus rhamnosus 23,527 LGG) and bifidobacteria (Bifidobacterium bifidum and Bifidobacterium animalis) to improve their survival in simulated gastrointestinal fluids. The viability of the lactobacilli and bifidobacteria (determined using plate count method) after electrospinning was 94.1% and 89.4% of the initial population. Upon exposure to in vitro condition of gastric fluid (HCl and pepsin, at 37 °C), the population (starting level of 9 log CFU/mL) of nanoencapsulated lactobacilli and bifidobacteria decreased only by 1.58 and 1.03 log CFU at 120 min. Treated with in vitro prepared intestinal fluid (dipotassium hydrogen phosphate, sodium hydroxide, bovine bile salt, and trypsin) no cell was detected at 30 min and the number of coated lactobacilli and bifidobacteria decreased by 2.90 and 2.23 log CFU at 120 min in comparison to nonencapsulated control. After 180-min exposure to simulated gastrointestinal fluid, population of encapsulated lactobacilli and bifidobacteria decreased by 3.02 and 2.55 log CFU at 180 min. The viability of the probiotic bacteria in simulated gastrointestinal conditions was enhanced significantly (81–100% of the initial population) by nanoencapsulation within nanofiber mats of CS/SA.     

Evaluation of cheddar cheese as a food carrier for delivery of a probiotic strain to the gastrointestinal tract.

Cheddar cheese was evaluated as a food carrier for the delivery of viable microorganisms of Enterococcus faecium (Fargo 688; Quest Int., Naarden, The Netherlands) to the gastrointestinal tract. This strain had previously been shown to possess properties required of a probiotic microorganism including the ability to relieve irritable bowel syndrome. The strain was found to survive to high numbers in Cheddar cheese during ripening at 8 degrees C for 15 mo (4 x 10(8) cfu/g) and in yogurt during storage at 4 degrees C for 21 d (4 x 10(7) cfu/g). In an in vitro model system, Cheddar cheese was found to have a greater protective effect than yogurt upon exposure of the probiotic culture to porcine gastric juice at pH 2. Subsequently, a feeding trial involving 8 pigs per group was performed in which a rifampicin-resistant variant of the probiotic strain was fed for 21 d at a mean daily intake of 1.3 x 10(10) cfu/d from Cheddar cheese or 3.7 x 10(9) cfu/d from yogurt. During the feeding period, Cheddar cheese yielded a significantly higher mean fecal probiotic count (2 x 10(6) cfu/g of feces) than did yogurt (5.2 x 10(5) cfu/g of feces). These data indicate that mature Cheddar cheese compares very favorably with fresh yogurt as a delivery system for viable probiotic microorganisms to the gastrointestinal tract.     

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.     

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.     

Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese

Two probiotic strains, Lactobacillus acidophilus and Lactobacillus paracasei subsp. paracasei, were used as adjunct cultures in semi-hard cheesemaking experiments, in order to study their influence on proteolysis during ripening. Cheeses with and without probiotic bacteria were manufactured. The population of probiotics remained above 10⁷ cfu g⁻¹ during all ripening, and they did not influence primary proteolysis. However, L. acidophilus produced a significant increase in the level of low molecular weight nitrogen compounds and individual free amino acids; the amino acid profiles were also different. Multivariate analysis of peptide profiles showed that samples were grouped mainly by ripening time, although the impact of probiotics was also noticeable. L. acidophilus showed a clear influence on secondary proteolysis, while a minor effect of L. paracasei was evidenced at the end of the ripening. These results showed that the tested strains influenced distinctly proteolysis of cheeses, probably as a consequence of their different proteolytic systems and their activity via the alimentary matrix (cheese).     

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.     

Influence of probiotic strains added to cottage cheese on generation of potentially antioxidant peptides,anti-listerial activity,and survival of probiotic microorganisms in simulated gastrointestinal conditions

Our objective was to evaluate the viability of probiotic microorganisms added to cottage cheese under simulated gastrointestinal conditions, the release of potentially-antioxidant peptides, and their antimicrobial effect on Listeria monocytogenes. Cottage cheeses were prepared in triplicate, incorporating Lactobacillus casei, Lactobacillus rhamnosus GG, the commercial mix YO-MIX™ 205, or a control without probiotic addition. The probiotic population remained at >10⁶ cfu g⁻¹ during 28 days of storage at 8 °C. Cheeses made with added probiotics showed an increased metabolic activity with higher levels of lactic and acetic acids. Higher numbers of potentially bioactive peptides were observed in cheeses added with probiotics. L. monocytogenes population was reduced by about one log cycle after 20 days of storage, in cheeses with probiotics added. Our results indicate that cottage cheese is a good vehicle for probiotic bacteria.     

The influence of probiotic bacteria on the properties of Iranian white cheese

The viability of Bifidobacterium animalis (ATCC 25527) and Lactobacillus rhamnosus (ATCC 7469), study of interaction between probiotic and starter and their effect on the properties of Iranian white cheese were investigated during 60 days of ripening. The results indicated that probiotics did not exert any effect on the growth of commercial starters, but starters showed a synergistic effect on the growth of probiotics, and probiotics survived above the recommended level for the therapeutic minimum (10⁶–10⁷ cfu/g) after 60 days. Addition of probiotic adjunct did not alter the chemical composition except for moisture and protein. Also, the highest pH value was in probiotic cheeses without starter.     

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.     

Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt

A modified method using calcium alginate for the microencapsulation of probiotic bacteria is reported in this study. Incorporation of Hi-Maize starch (a prebiotic) improved encapsulation of viable bacteria as compared to when the bacteria were encapsulated without the starch. Inclusion of glycerol (a cryo-protectant) with alginate mix increased the survival of bacteria when frozen at -20 degrees C. The acidification kinetics of encapsulated bacteria showed that the rate of acid produced was lower than that of free cultures. The encapsulated bacteria, however, did not demonstrate a significant increase in survival when subjected to in vitro high acid and bile salt conditions. A preliminary study was carried out in order to monitor the effects of encapsulation on the survival of Lactobacillus acidophilus and Bifidobacterium spp. in yoghurt over a period of 8 weeks. This study showed that the survival of encapsulated cultures of L. acidophilus and Bifidobacterium spp. showed a decline in viable count of about 0.5 log over a period of 8 weeks while there was a decline of about 1 log in cultures which were incorporated as free cells in yoghurt. The encapsulation method used in this study did not result in uniform bead size, and hence additional experiments need to be designed using uniform bead size in order to assess the role of different encapsulation parameters, such as bead size and alginate concentration, in providing protection to the bacteria.     

Survival and metabolic activity of probiotic bacteria in green tea

The aim of this study was to determine the enzymatic activity and survival of three probiotic strains (Lactobacillus paracasei LAFTI-L26, Lactobacillus acidophilus LAFTI-L10 and Bifidobacterium animalis ssp. lactis LAFTI-B94) during incubation in six different varieties of green tea extracts. The polyphenol content, antioxidant, antimicrobial and antihypertensive properties in green tea (variety Wu Lu Mountain) and in two standards (epigallocatechin-3-gallate and rutin) were also investigated before and after incubation with B. animalis B94. The green tea extracts permitted the survival of the selected probiotic strains better than the saline solution, with B. animalis B94 maintaining the highest levels of viable cells. These selected probiotic bacteria exhibited β-glucosidase, β-galactosidase and α-rhamnosidase activity. The antioxidant and antihypertensive properties of standard solutions of epigallocatechin-3-gallate and rutin increased after incubation with B. animalis B94, caused by polyphenol content reduction and the formation of other more highly biologically active metabolites. However, during the incubation of green tea with B. animalis B94, changes in the concentration of the most abundant green tea polyphenols did not enhance the biological activity.     

Microecology of the gastrointestinal tract in relation to lactic acid bacteria

The distal small bowel and the large bowel of humans, even in health, are inhabited by a complex collection of microbes (mostly bacteria) known as the normal microflora. Gram-positive, lactic acid-producing bacteria are represented in this colection (bifidobacteria, lactobacilli, enterococci, eubacteria, peptostreptococci). The influence of the normal microflora as a whole on the host is well known from studies comparing germfree and conventional animals. More recently, using an unique experimental animal system (lactobacillus-free mice), lactobacilli have been shown to influence markedly the biochemistry of the intestinal tract (azoreductase,β-glucuronidase, bile salt hydrolase, proportions of unconjugated/conjugated bile acids). The effect of the consumption of lactic acid-producing bacteria on the normal microflora and the intestinal milieu of humans is therefore of interest. Detailed analysis of the composition, at the level of bacterial strains, of the normal microflora should be possible using DNA probes and ribotyping. These studies are necessary to provide a basis for the study of the effects of ingested lactic acid bacteria on the large bowel ecosystem, and for the derivation and use of genetically modified bacteria that will have a desirable impact on human health.