Survival In Simulated Gastrointestinal Conditions Research Articles

Layer-by-layer coated probiotics with chitosan and liposomes demonstrate improved stability and antioxidant properties in vitro

Probiotics are of increasing interest for their potential health benefits, but their survival and adhesion in the harsh gastrointestinal environment remain a concern. This study explored a single-cell encapsulation technique to enhance probiotic survival and adhesion in the gastrointestinal tract. We encapsulated probiotics in curcumin-loaded liposomes, further coated them with polymers using layer-by-layer techniques. The coated probiotics were evaluated for survival in simulated gastrointestinal conditions, adhesion to colonic mucus, and scavenging of reactive oxygen species (ROS). The results showed that multi-layer encapsulation increased probiotic size at the nanoscale, enhancing their survival in simulated gastrointestinal conditions. Upon reaching the colon, the shedding of the coating coincided with probiotic proliferation. Additionally, the coated probiotics exhibited increased adhesion to colonic mucus. Moreover, the coating acted as a protective barrier for effectively scavenging reactive oxygen radicals, ensuring probiotic survival in inflammatory environments. This study combines the synergistic effects of probiotics and curcumin, underscoring the promise of single-cell encapsulation techniques in improving the efficacy of probiotics for addressing colitis-related diseases.

Microencapsulation of Lactiplantibacillus plantarum with inulin and evaluation of survival in simulated gastrointestinal conditions and roselle juice

Abstract This study aimed to evaluate the survivability of Lactiplantibacillus plantarum 299v encapsulated in chitosan-coated calcium alginate beads with inulin as prebiotic in simulated gastrointestinal conditions and roselle juice. The concentration of calcium chloride and inulin for L. plantarum 299v microencapsulation was optimised and the survivability of free and microencapsulated L. plantarum was assessed under simulated gastrointestinal conditions. Storage stability of the optimised encapsulated L. plantarum 299v-inulin was determined throughout four (4) weeks of storage in roselle juice at 4 °C and 25 °C. The optimized formula for L. plantarum 299v was 2.0% (w/v) of calcium chloride and 3.0% (w/v) of inulin. Optimized calcium alginate-chitosan L. plantarum 299v microbeads with inulin did not affect (p > 0.05) the bead diameter, with a mean diameter of 685.27 μm, and microencapsulation efficiency of 95%. Encapsulated L. plantarum 299v with inulin showed higher survivability (>107 CFU/mL) than free cells and encapsulated L. plantarum 299v without inulin under simulated gastrointestinal conditions and after four (4) weeks of storage in roselle juice at 4 °C. The results indicate that co-extrusion encapsulation and addition of inulin had improved the viability of L. plantarum 299v in roselle juice by protecting probiotic against unfavourable gastrointestinal conditions and prolonged storage.

The effect of improved formulation of chitosan-alginate microcapsules of Bifidobacteria on serum lipid profiles in mice

BackgroundProbiotics have been associated with many beneficial effects in human digestive physiology. The aim of this study was to evaluate the effect of improved formulation of chitosan-alginate microcapsules of Bifidobacterium strains on serum triglycerides, cholesterol, HDL, and LDL in mice. MethodsFive approved probiotic strains of Bifidobacterium were tested for anti-proliferative effect and interleukin-8 induction on HT-29 cell lines. Bifidobacterium strains plus five approved Lactobacillus were encapsulated in chitosan-alginate microcapsules and tested for its survival in simulated gastrointestinal conditions. These microcapsules were administered to 4 groups of mice (including 1. Bif (Bifidobacterium strains), 2. Lac (Lactobacillus strains), 3. Bif-Lac (Bifidobacterium plus Lactobacillus strains) and 4. Control) for 8 days. At eighth day, the blood of mice were taken and serum levels of triglycerides, cholesterol, HDL, and LDL of them were determined. ResultsAll of the Bifidobacterium strains significantly (P < 0.001) reduced secretion of IL-8 in HT-29 cells as well as maximum antiproliferative effects (P < 0.001). In addition, all microcapsules showed impressive survival rate in bile (>%94.1) and gastrointestinal (>%78.28) conditions (P < 0.05). Only Bif-Lac group displayed significantly lower serum cholesterol and LDL levels than control group (P < 0.05). Besides, all groups indicate statistically significant weight loss of mice during the 8 days in comparison with the control group (P < 0.05). ConclusionThe results of this study showed that the microencapsulated probiotics with alginate and chitosan had an effective mean of delivery of viable bacterial cells and non-pharmacological interventions use to reduce serum cholesterol and LDL levels in in-vivo condition.

Fermented yellow mombin juice using Lactobacillus acidophilus NRRL B-4495: Chemical composition, bioactive properties and survival in simulated gastrointestinal conditions.

The purpose of the present study was to evaluate yellow mombin (Spondias mombin L.) juice as a vehicle for the Lactobacillus acidophilus NRRL B-4495 probiotic. The initial pH and fermentation temperature conditions were optimized by central composite rotational design. The beverage was evaluated for its chemical composition, bioactive properties, microbiological stability, survival in simulated gastrointestinal conditions and sensory analysis. The ideal conditions for probiotic juice production were an initial pH of 6.4 and 16 h of fermentation, with maximum viability of 12.9 ± 0.4 Log CFU/mL. The fermented juice showed a total phenolic concentration of 94.90 ± 7.12 GAE/mL and antioxidant activity, as measured by DPPH (0.31 ± 0.00 μmol TE/mL) and ABTS sequestration (2.59 ± 0.30 μmol TE/mL). Antibacterial activity could also be observed against S. aureus, E. coli and K. pneumoniae. The obtained formulation showed good microbiological stability when stored at 4ºC for 28 days. In addition, there was no significant change in viability after exposure to simulated gastrointestinal conditions. The sensory analysis showed that the probiotic beverage was not well accepted. However, the Just-About-Right (JAR) ideal scale test enabled identifying the specific attributes which need to be improved from the tasters’ point of view so that it is possible to improve product acceptance.

Effect of added prebiotic (Isomalto-oligosaccharide) and Coating of Beads on the Survival of Microencapsulated Lactobacillus rhamnosus GG

Abstract The study aimed to encapsulate Lactobacillus rhamnosus GG (LGG) with the selected prebiotic, using co-extrusion technology with a poly-L-lysine (PLL) coating and evaluate probiotic survival in simulated gastrointestinal conditions. Selection of ideal prebiotic was conducted using inulin, fructo-oligosaccharide (FOS) and isomalto-oligosaccharide (IMO) and its optimal concentration to be incorporated in microencapsulation was determined. Microcapsules without coating (S1: no prebiotic and S3: with prebiotic) and with coating (S2: no prebiotic and S4: with prebiotic) produced were evaluated based on its physical properties and survival in simulated gastrointestinal environment. The IMO with a concentration of 3.0% (w/v) was selected due to its best effect in promoting growth of LGG after 24 h (8.63±0.07 log CFU/mL). The morphology analysis revealed that all microcapsules produced were spherical with a diameter ranging from 491.3 to 541.7 µm and microencapsulation efficiency ranged from 84.16 ±5.30% to 90.56±3.33%. The incorporation of IMO and coating with PLL improved the survival of LGG by 3% up to 52% after 2 h of incubation in simulated gastric digestion. Among all formulations, PLL coated microcapsules added with IMO was the most effective in protecting LGG during the first hour of simulated gastric digestion (6.52 log CFU/mL) with cell viability greater than the minimum recommended level of 106 CFU/mL.

Yoghurt added with Lactobacillus casei and sweetened with natural sweeteners and/or prebiotics: Implications on quality parameters and probiotic survival

The effect of addition of natural sugar substitutes (stevia, erythritol or xylitol) and prebiotics (oligofructose or polydextrose) on physicochemical characteristics, probiotic survival (Lactobacillus casei) and sensory acceptance of yoghurts during storage (7 °C, 28 days) was evaluated. Yoghurts with sucrose or sucralose were also prepared. Xylitol and sucralose added yoghurts had physicochemical characteristics and sensory acceptance similar to those of the product with sucrose. Xylitol was more efficient in maintenance of textural characteristics and probiotic survival in simulated gastrointestinal conditions (SGIC) than sucralose. Addition of erythritol and stevia changed the textural parameters, reduced acceptance and decreased probiotic survival in SGIC. Addition of oligofructose or polydextrose improved texture and increased probiotic survival but decreased flavour acceptance. All formulations could be considered probiotic products during shelf life (7 °C, 28 days) with counts higher than 107 cfu mL−1 in the product and 104 cfu mL−1 after SGIC.

Probiotic potential of Lactobacillus fermentum G-4 originating from the meconium of newborns

The present study was dedicated to determining probiotic potential of a human isolate G-4, originated from meconium. The isolate was identified using morphological, physiological and biochemical assays and molecular method based on 16S rRNA gene sequencing. In order to evaluate its probiotic properties in vitro tests were performed: the survival in simulated gastrointestinal conditions, adhesion to hexadecane, and antimicrobial activity. Safety aspects of the isolate were examined by testing toxicity, gastrointestinal tolerance and bacterial translocation in vivo, as well as hemolytic activity in vitro. The isolate G-4, identified as Lactobacillus fermentum, showed viability in artificial gastric and intestinal juice (low degree of cell viability reduction for 0.69 and 1.30 logCFU mL-1 units, respectively), moderate adhesion to hexadecane (39?2.1 %), and antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype Abony and Clostridium sporogenes, due to production of lactic acid (9.80 g L-1). No signs of toxicity, bacterial translocation, hemolytic activity, were observed.

Encapsulation of bacteriophage T4 in mannitol-alginate dry macrospheres and survival in simulated gastrointestinal conditions

In recent years application of bacteriophages in the reduction of bacterial pathogens that may contaminate the food supply has become of growing interest. The aim of this study was to produce dried macrospheres ensuring viability of bacteriophages during exposure to adverse conditions of the digestive system after storage for an extended period of time. The viability of phages entrapped in mannitol-alginate wet and dried formulations was investigated for up to 4 weeks of storage at room temperature. Phage-loaded beads were exposed to an acidic environment (pH 2.5) of simulated gastric fluid and directly transferred to intestinal medium to dissolve. During the period from 1 to 4 weeks the survival of bacteriophage T4 in dried macrospheres under simulated gastrointestinal conditions revealed only a slight decrease and resulted in a final survival of 5.02 log10 pfu/ml. The results of this study show that the addition of 0.3 M mannitol may improve phage protection against gastrointestinal conditions and enable a large proportion of bacteriophage T4 to remain active. The potential of the developed technology can be considered in oral application of bacteriophages to reduce intestinal colonization of pathogenic bacteria via prolonged stability of phages in a dry state.

Encapsulation of probiotic living cells in alginate-PVA based electrospun nanofibers: Evaluation of viability and survival in simulated gastrointestinal conditions

Encapsulation of probiotic living cells in alginate-PVA based electrospun nanofibers: Evaluation of viability and survival in simulated gastrointestinal conditions

Encapsulation of Bifidobacterium longum in alginate-dairy matrices and survival in simulated gastrointestinal conditions, refrigeration, cow milk and goat milk

Abstract The aim of this study was to microencapsulate Bifidobacterium longum subsp. infantis CCUG 52486 using the extrusion method in a variety of matrices, namely sodium alginate (SA), sodium alginate-cow milk (SACM), sodium alginate-goat milk (SAGM) and sodium alginate-casein hydrolysate (SACH), and to evaluate the survival of free and encapsulated bacterial cells under different conditions. The encapsulation yield, size and surface morphology of the microcapsules were evaluated. The survival of microencapsulated bacterial cells and free bacterial cells were evaluated under simulated gastrointestinal conditions as well as in refrigeration, cow milk, and goat milk during storage at 4 °C for 28 days. The average size of SACM capsules and SAGM capsules was 2.8 ± 0.3 mm and 3.1 ± 0.2 mm respectively. Goat milk and cow milk-based matrices resulted in dense microcapsules which led to better performances in simulated gastrointestinal conditions than SA and SACH microcapsules. The bacterial cells encapsulated in SAGM showed the highest survival rate in cow milk (7.61 log cfu g−1) and goat milk (8.10 log cfu g−1) after the storage of 28 days. The cells encapsulated in SA and SACH and the free cells performed poorly under the simulated gastrointestinal conditions and in all different storage conditions. This study showed that SACM and SAGM are suitable to encapsulate B. longum subsp. infantis CCUG 52486 using the extrusion technique and more specifically, SAGM has a potential to be used as a new encapsulation material for encapsulating probiotic bacteria, resulting in milk and goat milk-based products with higher probiotic cell concentrations during refrigerated storage.

In Silico and Experimental Data Claiming Safety Aspects and Beneficial Attributes of the Bacteriocinogenic Strain Enterococcus faecalis B3A-B3B.

This study aimed at comparing the genome of Enterococcus faecalis B3A-B3B, a bacteriocinogenic strain recently isolated from a healthy Iraqi infant to those of Enterococci of clinical and beneficial grades. The putative genes gelE, cpd, efaAfm, ccf, agg, and cob coding for virulence factors were detected in B3A-B3B strain, which meanwhile resulted to be non-cytotoxic, non-hemolytic, devoid of inflammatory effects, and sensitive to most of the antibiotics tested except for clindamycin and trimethoprim, which resistance is usually ascribed to intrinsic nature. B3A-B3B strain was remarkable for its hydrophobicity, auto-aggregation, adhesion to human Caco-2 cells, and survival in simulated gastrointestinal conditions, and cholesterol assimilation fulfilling therefore key beneficial attributes.

Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion

BackgroundOne of the working mechanisms of probiotic bacteria is their ability to compete with pathogens. To define the probiotic properties of seven Lactic Acid Bacteria (LAB) strains, we tested them for survival in simulated gastro-intestinal conditions, antimicrobial activities, co-aggregative abilities, and interferences studies against five human intestinal pathogens (Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644, Escherichia coli O157: H7 ATCC 35150, Cronobacter sakazakii ATCC 29544 and Campylobacter jejuni ATCC 33291).ResultsThe LAB strains were able to survive the stomach simulated conditions, and varied in their abilities to survive the small intestinal-simulated conditions. The strains showed antibiotic susceptibility profiles with values equal or below the breakpoints set by the European Food and Safety Authority. The LAB cell-free cultures supernatants showed antimicrobial activities, with inhibition zones ranging from 10.0 to 17.2 mm. All the LAB strains showed moderate auto-aggregation abilities while the greatest co-aggregation abilities were observed for Bifidobacterium bifidum W23, Lactobacillus plantarum W21 and Lactobacillus rhamnosus W71. The individual LAB strains showed strain-specific abilities to reduce the invasion of intestinal pathogens in an interference model with Caco-2 cells. Increased invasion inhibition was found when different combinations of LAB strains were used in the interference tests.ConclusionThe LAB strains examined in this study may protect the intestinal epithelium through a series of barriers (antimicrobial activity, co-aggregation with pathogens, adherence) and interference mechanisms. Consequently, these LAB strains may be considered candidates for prophylactic use to prevent intestinal infections.

Probiotic Potential of Lactic Acid Bacteria Ch-2 Isolated from Chuli Characterization of Potential Probiotic Lactic Acid Bacteria- Pediococcus acidilactici Ch-2 Isolated from Chuli- A Traditional Apricot Product of Himalayan Region for the Production of Novel Bioactive Compounds with Special Therapeutic Properties

Objectives: In the present study, Pediococcus acidilactici Ch-2 isolated from Chuli has been evaluated for its probiotic potential and its ability to produce bioactive compounds. Methodology: Isolation was done on MRS agar followed by the safety assessment of isolates. Isolates were screened on the basis of their antagonistic potential against pathogenic bacteria. Probiotic attributes of screened isolate Ch-2 i.e. acid and bile tolerance, survival in simulated gastrointestinal conditions, bile salt tolerance, autoand co-aggregation, antimicrobial potential and metabolic profiling have been performed. Results: It was observed that P. acidilactici Ch-2 was resistance to low pH and bile salts (0.3%) and simulated gastric and intestinal conditions, was able to produce bacteriocin and lactic acid against a number of serious food borne and spoilage causing microorganisms. The susceptibility to selected eleven antibiotics, inability to produce gelatinase and DNase and non-hemolytic nature revealed its safe status for further use in food and nutraceutical industry. In the present study, Squalene – a rare and therapeutic anticancerous compound has been reported for the very first time from a probiotic bacteria isolated from fermented food product and recommended for its further investigation as a potential source of Squalene. Conclusion: In conclusion, most of the results in the present work revealed P. acidilactici Ch-2 as a potential probiotic candidate with many functional properties and novel compounds.

Adsorption of Lactobacillus acidophilus on attapulgite: Kinetics and thermodynamics and survival in simulated gastrointestinal conditions

In the present study, we investigated the optimum adsorption conditions, adsorption characteristics and surviva of Lactobacillus acidophilus adsorbed on attapulgite (ATT). Results of single factor experiments showed that the optimum adsorption parameters were ATT concentration of 10%, initial pH of 6.5 and temperature of 35 °C. From the static experiments of adsorption characteristics, we found that the experimental data were fitted best to the pseudo-secondorder kinetics and Langmuir isotherm models, and the adsorption was a spontaneous and endothermic process. Bacteria survival test showed that the microencapsulated L. acidophilus stored at −18 °C for 30 days achieved the highest survival rate (83.2% ± 5.8) and obtained better protection at simulated conditions of gastric pH and at high bile salt concentrations when compared with free bacteria. The study demonstrated that microencapsulation of L. acidophilus in ATT was an effective technique of protection against low-temperature injury and under simulated gastrointestinal environment.

Co-encapsulation of Lactobacillus helveticus cells and green tea extract: Influence on cell survival in simulated gastrointestinal conditions

Probiotics and green tea extracts (GTE) have numerous potential health benefits. GTE had also positive effects on the survival of some probiotic bacteria in food products or during exposure to gastrointestinal conditions. In this study, Lactobacillus helveticus cells and GTE were co-encapsulated in calcium pectinate microparticles (MP). MP's characteristics and their ability to protect cells under gastrointestinal conditions were investigated. Small spherical MP having antioxidant activities and high bacterial cells and polyphenol contents were produced. The coating of these MP with whey proteins was an efficient way to protect cells from conditions encountered in the upper part of the gastrointestinal tract. The co-encapsulation of bacteria with 1000 µg/mL GTE provided an additional protection to the cells in gastric conditions. This study indicates that whey protein-coated pectinate MP could be a new carrier for the combined delivery of viable probiotic cells and GTE to the lower part of the gastrointestinal tract.

Probiotic Potential of Lactic Acid Bacteria from Traditional Fermented Dairy and Meat Products: Assessment by In Vitro Tests and Molecular Characterization

The aim of the present study was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from Greek traditional fermented products. A series of In vitro tests that included survival in simulated gastrointestinal conditions (resistance to low pH, bile salts resistance and bile salts hydrolysis) and safety assessment (resistance to antibiotics, haemolytic and antimicrobial activity) were performed to select potential probiotic candidates, while Lactobacillus rhamnosus GG and Lactobacillus casei Shirota were used as reference strains. Initially, a total of 255 isolates of LAB have been recovered and screened for their survival in simulated gastrointestinal tract conditions and 133 isolates that exhibited moderate or good behavior in these tests were subsequently differentiated and characterized at species level with molecular tools. Pulsed Field Gel Electrophoresis was applied for strain differentiation, while species differentiation was based on restriction analysis of the amplified 16S rRNA gene. Specific multiplex PCR assay targeting the recA genes was applied to resolve the species level of the isolates, belonged to Lb. plantarum group. From the 133 isolates, 47 different strains were recovered and were assigned to Lactobacillus sakei(14), Lactobacillus curvatus (4), Leuconostoc mesenteroides (4), Lactococcus lactis (4), Lactobacillus casei group (1), Lactobacillus brevis (1), Lb. plantarum (10), Lb. pentosus (7) and Lb. paraplantarum (2). The identified strains with good behavior to the gastrointestinal tract tests were selected and further evaluated for their safety aspect. In conclusion, 19 out of the 47 identified strains were assessed as well-behaved, under simulated gastrointestinal conditions and also considered as safe, possessing thus desirable In vitro probiotic properties similar or better to that of the reference strains. These strains may be considered as good candidates for further investigation at in vivo and in situ studies to assess their potential health benefits and their performance as novel probiotic starters or adjunct cultures.

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.

Development of a synbiotic product for newborns and infants

The capability of ten commercial fibers of selectively stimulating the growth of four Bifidobacterium strains were studied with the purpose of developing a synbiotic product for infants. Two galacto-oligosaccharides (GOS), one fructo-oligosaccharide (sc-FOS), four inulins with different polymerization degree (DP), a gluco-oligosaccharide, an arabinogalactan and a hydrolyzed guar gum were used (10 gl−1). The prebiotic score was calculated comparing the capability of the fibers of stimulating the growth of bifidobacteria compared to infant pathogens. GOS, sc-FOS, low DP inulin (oligofructose) and the gluco-oligosaccharide could stimulate growth. However, the fibers showing the highest prebiotic score were oligofructose (Orafti®HIS), sc-FOS (Actilight®950P) and the GOS Vivinal®. Lyophilized strain survival in simulated gastro-intestinal conditions was also assayed to define suitable ways of administration. Survival in gastric juice at pH 2.5 was poor, whereas it was higher at pH 4, a value closer to newborn pH. Microencapsulation in a lipid matrix ensured strain survival also at pH 2.5. Survival to 1 gl−1 bile salts was acceptable. The results allowed to conclude that Bifidobacterium breve B632 strain, in a lyophilized or microencapsulated form, has the potential for use in synbiotic products targeted to infants coupled to a mixture of GOS and FOS or oligofructose.

Milk fermented with the probiotic candidate Lactobacillus paracasei FT700 induces differentiation of monocytes toward macrophages in vitro

Lactobacillus paracasei FT700 was tested for its survival in simulated gastrointestinal conditions (in vitro) and for its sensitivity to antibiotics. The strain was used to produce fermented milk and the cell-free supernatant was freeze-dried and applied in cell cultures (monocytes and macrophages) to evaluate its cytotoxicity and immunomodulatory effects. After 180 min at pH 2.0 or in simulated gastric juice, L. paracasei FT700 concentrations were reduced by 4 and 3 log cfu/mL, respectively. Moreover, the strain was susceptible to the majority of antibiotics tested. Freeze dried cell-free fermented milk supernatant (FMS) was toxic to monocytes after 72 h of exposure at 10 mg/mL inducing their apoptosis. However, at concentrations below toxic level, FMS stimulated the differentiation of monocytes into macrophages, as observed by the increased expression of CD71 marker. This immune stimulation was not inflammatory since low production of TNF-α was observed. Overall, L. paracasei FT700 has potential to be used as probiotic in functional fermented milk.

Encapsulation of probiotic Lactobacillus bulgaricus in alginate–milk microspheres and evaluation of the survival in simulated gastrointestinal conditions

In this paper, a new encapsulation carrier was applied in order to improve the survival of Lactobacillus bulgaricus (L. bulgaricus). L. bulgaricus was encapsulated in alginate–milk microspheres prepared by extrusion method. Around 100% encapsulation yield was achieved. The tolerance of encapsulated L. bulgaricus to adverse environments such as low pH (pH 2.0 and 2.5), high concentration of bile salt (1.0% and 2.0%) and long time storage (1month), was investigated. Release characteristic of encapsulated L. bulgaricus in Simulated Intestine Fluid (SIF) was also studied. The results showed that encapsulation could improve the tolerance of L. bulgaricus to adverse environments. The viability of encapsulated L. bulgaricus did not change after 120min incubation in Simulated Gastric Fluid (SGF) pH 2.5. The viability of encapsulated L. bulgaricus could be kept more than 8logCFU/g after 120min incubation in SGF pH 2.0. The viability of encapsulated L. bulgaricus in 1% bile salt solution was reduced from 9.98logCFU/g microspheres to 9.24 and 8.48logCFU/g microspheres after 1 and 2h incubation, respectively. Around 1.3 and 2.1logCFU/g microspheres were reduced after 1 and 2h exposure in 2% bile salt solution, respectively. Full viability of encapsulated L. bulgaricus could be preserved after 1month’s storage at 4°C. L. bulgaricus encapsulated in alginate–milk microspheres could be completely released in 1h. These studies demonstrated encapsulation of L. bulgaricus in alginate–milk microspheres is an effective protection technique against extreme simulated gastrointestinal environment.