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 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.     

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.     

Probiotic strains: survival under simulated gastrointestinal conditions,in vitro adhesion to Caco-2 cells and effect on cytokine secretion

This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screening of dairy yeast strains for probiotic applications

To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.     

The survival and persistence in the human gastrointestinal tract of five potential probiotic lactobacilli consumed as freeze-dried cultures or as probiotic sausage

Among five lactobacilli (L. plantarum MF1291, MF1298, DC13, L. pentosus MF1300 and L. salivarius DC5) which were administrated as freeze-dried cultures for 17 volunteers, MF1298 and DC13 were the most frequently reisolated strains in faeces demonstrating the human gastric survival of these strains. Furthermore, MF1298 and DC13 persisted in the same volunteer after ended intake, suggesting host-specific persistence behaviour. When MF1298 was administrated as sausage fermented with this strain, the number of volunteers harbouring MF1298 increased from 4 to 10 indicating that the sausage matrix protects the survival through the gastrointestinal tract (GIT).     

Effects of encapsulation on the viability of probiotic strains exposed to lethal conditions

The effect of microencapsulation on the viability of Lactobacillus casei, L. paracasei, L. acidophilus Ki and Bifidobacterium animalis BB‐12 during exposure to lethal conditions (25% NaCl, pH 3.0 and 55–60 °C) was evaluated. Results demonstrated that survival of probiotic strains to the imposed lethal stress conditions was strain dependent. With the exception of exposure to 25% (w/v) NaCl, L. acidophilus Ki (free and encapsulated cells) demonstrated the highest survival rates through exposure to lethal conditions of temperature and pH. For this probiotic strain exposed to heat, microencapsulated cells expressed a higher heat tolerance at 55 °C than free cells. For the other tested bacteria, in general, encapsulation had no positive effect on survival through the tested lethal 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.     

Proteomic response of Saccharomyces boulardii to simulated gastrointestinal conditions and encapsulation

Food Science and Biotechnology - Probiotics are live microorganisms conferring health benefits when administered in adequate amounts. However, the passage through the gastrointestinal tract...     

Effects of bile salt deconjugation by probiotic strains on the survival of antibiotic-resistant foodborne pathogens under simulated gastric conditions

This study was designed to evaluate the effects of bile acid deconjugation by probiotic strains on the antibiotic susceptibility of antibiotic-sensitive and multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus. Eight probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus brevis KACC 10553, Lactobacillus casei KACC 12413, Lactobacillus paracasei ATCC 25598, Lactobacillus rhamnosus GG, Leuconostoc mesenteroides KACC 12312, and Pediococcus acidilactici KACC 12307, were used to examine bile acid tolerance. The ability to deconjugate bile acids was evaluated using both thin-layer chromatography and high-performance liquid chromatography. The antibiotic susceptibility testing was carried out to determine the synergistic inhibitory activity of deconjugated bile acids. L. acidophilus, L. brevis, and P. acidilactici showed the most tolerance to the conjugated bile acids. P. acidilactici deconjugated glycocholic acid and glycodeoxycholate from 3.18 and 3.09 mM to the detection limits, respectively. The antibiotic susceptibility of selected foodborne pathogens was increased by increasing the concentration of deconjugated bile acids. The study results are useful for understanding the relationship between bile acid deconjugation by probiotic strains and antibiotic susceptibility in the presence of deconjugated bile acids, and they may be useful for designing new probiotic-antibiotic combination therapy based on bile acid deconjugation.     

Potential probiotic Lactobacillus strains from fermented sausages: Further investigations on their probiotic properties

A rational selection of probiotic microorganisms is an important challenge and requires the definition of fundamental information about the physiology and genetics of candidate strains. In this study, selected Lactobacillus (Lact.) strains already characterized in a previous study for their capability to resist low pH and to grow in conditions simulating the intestinal environment, were further investigated to explore their probiotic properties, such as the adhesion capability to intestinal human Caco-2 cell lines and their growth behaviour in the presence of various prebiotic carbohydrates. At first 25 Lactobacillus strains were characterized by pulsed field gel electrophoresis using the endonuclease NotI. Among them, 13 strains belonging to the Lact. plantarum-group were identified at species level by a multiplex PCR assay. Subsequently 11 Lactobacillus strains showing different PFGE restriction pattern and the best acid- and bile-resistances, were chosen to investigate their in vitro adhesion capability to human intestinal epithelial cells and their fermentation properties of five prebiotic substances (FOS, Inulin, IMO, GOS and lactulose) at a concentration of 2%. The 11 strains analysed in this study possessed good adhesion capability to Caco-2 cell layers and, in particular, the eight strains belonging to the Lact. plantarum-group showed the higher final number of viable adhering cells. Moreover a species-related fermentative behaviour was pointed out and the strain Lact. paracasei EL7 was the only one able to grow in the presence of all prebiotics tested. In conclusion the strains of Lactobacillus studied in this research could be further investigated to assess possible in vivo human health benefits.     

Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions

The main challenge to probiotics, during their passage through the gastrointestinal tract, are the acidic gastric secretions of the stomach, and the bile salts released into the duodenum. The survival of the strains, in this phase, is strongly influenced by the food used for their delivery.     

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.     

Camel milk-derived probiotic strains encapsulated in camel casein and gelatin complex microcapsules: Stability against thermal challenge and simulated gastrointestinal digestion conditions

Probiotics have received increased attention due to their nutritional and health-promoting benefits. However, their viability is often impeded during food processing as well as during their gastrointestinal transit before reaching the colon. In this study, probiotic strains Lactobacillus rhamnosus MF00960, Pediococcus pentosaceus MF000967, and Lactobacillus paracasei DSM20258 were encapsulated within sodium alginate, camel casein (CC), camel skin gelatin (CSG) and CC:CSG (1:1 wt/wt) wall materials. All 3 strains in encapsulated form showed an enhanced survival rate upon simulated gastrointestinal digestion compared with free cells. Among the encapsulating matrices, probiotics embedded in CC showed higher viability and is attributed to less porous structure of CC that provided more protection to entrapped probiotics cells. Similarly, thermal tolerance at 50°C and 70°C of all 3 probiotic strains were significantly higher upon encapsulation in CC and CC:CSG. Scanning electron microscope micrographs showed probiotic strains embedded in the dense protein matrix of CC and CSG. Fourier-transform infrared spectroscopy showed that CC- and CSG-encapsulated probiotic strains exhibited the amide bands with varying intensity with no significant change in the structural conformation. Probiotic strains encapsulated in CC and CC:CSG showed higher retention of inhibitory properties against α-glucosidase, α-amylase, dipeptidyl peptidase-IV, pancreatic lipase, and cholesteryl esterase compared with free cells upon exposure to simulated gastrointestinal digestion conditions. Therefore, CC alone or in combination with CSG as wall materials provided effective protection to cells, retained their bioactive properties, which was comparable to sodium alginate as wall materials. Thus, CC and CC:CSG can be an efficient wall material for encapsulation of probiotics for food applications.     

Effect of different matrices on probiotic resistance to in vitro simulated gastrointestinal conditions

In this study, we evaluated the effect of different matrices (MRS, milk and milk with inulin) on the tolerance of probiotic strains (Lactobacillus acidophilus La‐5 and Bifidobacterium animalis subsp. lactis BB‐12) to simulated conditions similar to those found in the gastrointestinal tract. Both probiotic strains demonstrated a significantly lower viability after exposure to in vitro gastric and intestinal conditions, and B. animalis subsp. lactis BB‐12 showed higher survival during the test compared to L. acidophilus La‐5 in all tested matrices. Milk and inulin protected probiotics from in vitro gastrointestinal stress. These results suggest that it is critical to formulate the food matrix to be used as probiotic carrier.     

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.     

Intestinal survival and persistence of probiotic Lactobacillus and Bifidobacterium strains administered in triple-strain yoghurt

The aim of the present study was to investigate the intestinal survival and persistence of probiotic strains Lactobacillus F19, Lactobacillus acidophilus NCFB 1748, and Bifidobacterium animalis subsp. lactis Bb-12 consumed in a yoghurt (ABC product), and also their effect on the intestinal microbiota. Based on the results of culture studies and strain-level analysis by randomly amplified polymorphic DNA (RAPD) fingerprinting Lactobacillus F19 and B. animalis subsp. lactis Bb-12 survived well through the human gastrointestinal tract; they were detected in reasonable numbers in the faeces of 100% and 79% of the study subjects, respectively. Ingestion of the probiotic yoghurt increased transiently the numbers of bifidobacteria and lactobacilli. For lactobacilli the increase was due to the detection of the ingested probiotic strains in faeces, while in bifidobacteria the increase was likely caused by the increase of indigenous bifidobacteria since the ingested Bifidobacterium strain did not comprise the predominant part of bifidobacterial population during the intervention. Probiotic strains were infrequently detected in mucosal biopsy samples. The present study indicates that developing probiotic food products with multiple probiotic strains is feasible.     

芽菜酵母菌株的发酵条件优化研究

以前期筛选、驯化的宜宾芽菜酵母菌株GD9为研究对象,在单因素实验的基础上,采用Box-Behnken实验设计和响应面法对该菌株的发酵条件进行优化。结果表明,芽菜酵母菌菌株GD9的最佳发酵条件为:发酵培养时间22h、发酵温度26℃、发酵液pH3.7,在此条件下OD620可达2.05;同时,建立的响应面模型可准确预测不同响应条件下该菌株的生长表达量。