Gastrointestinal Stresses Research Articles

Probiotic Insights from the Genomic Exploration of Lacticaseibacillus paracasei Strains Isolated from Fermented Palm Sap.

This study focused on L. paracasei strains isolated from fermented palm sap in southern Thailand that exhibit potential probiotic characteristics, including antibiotic susceptibility, resistance to gastrointestinal stresses, and antimicrobial activity against various pathogens. However, a thorough investigation of the whole genome sequences of L. paracasei isolates is required to ensure their safety and probiotic properties for human applications. This study aimed to sequence the genome of L. paracasei isolated from fermented palm sap, to assess its safety profile, and to conduct a comprehensive comparative genomic analysis with other Lacticaseibacillus species. The genome sizes of the seven L. paracasei strains ranged from 3,070,747 bp to 3,131,129 bp, with a GC content between 46.11% and 46.17% supporting their classification as nomadic lactobacilli. In addition, the minimal presence of cloud genes and a significant number of core genes suggest a high degree of relatedness among the strains. Meanwhile, phylogenetic analysis of core genes revealed that the strains possessed distinct genes and were grouped into two distinct clades. Genomic analysis revealed key genes associated with probiotic functions, such as those involved in gastrointestinal, oxidative stress resistance, vitamin synthesis, and biofilm disruption. This study is consistent with previous studies that used whole-genome sequencing and bioinformatics to assess the safety and potential benefits of probiotics in various food fermentation processes. Our findings provide valuable insights into the potential use of seven L. paracasei strains isolated from fermented palm sap as probiotic and postbiotic candidates in functional foods and pharmaceuticals.

Modified Prebiotic-Based "Shield" Armed Probiotics with Enhanced Resistance of Gastrointestinal Stresses and Prolonged Intestinal Retention for Synergistic Alleviation of Colitis.

Oral administration of probiotics is a promising method to alleviate inflammatory bowel diseases (IBDs). However, gastrointestinal environmental sensitivity and inferior intestinal colonization of probiotics hinder the alleviation effect. Here, we developed a simple yet effective modified prebiotic-based "shield" (Fe-TA@mGN) composed of an Fe3+-tannic acid cross-linking network and carboxymethylated β-glucan for arming Escherichia coli Nissle 1917 (EcN@Fe-TA@mGN). The Fe-TA@mGN "shield" not only acted as a dynamic barrier to enhance the gastrointestinal stress resistance ability of EcN but also aided the intestinal colonization of EcN as well as synergized with EcN for the alleviation of dextran sulfate sodium (DSS) induced colitis. More specifically, with the protection of the Fe-TA@mGN "shield", the survival rate of armed EcN could be up to ∼1720 times higher than that of bare EcN after exposure to simulated gastric fluid. Excitingly, the intestinal retention rate of EcN@Fe-TA@mGN was as high as 47.54 ± 6.06% at 16 h post-administration, while almost all bare EcNs were excreted out at 8 h post-administration. With all of the aforementioned attributes, EcN@Fe-TA@mGN efficiently alleviated colitis, verified by the repair of the intestinal barrier and the attenuation of inflammation. Moreover, for EcN@Fe-TA@mGN, mGN synergized with EcN to positively modulate gut microbiota and promote the production of short-chain fatty acids (SCFAs, especially for butyric acid, a primary source for maintaining intestinal health), both of which would further advance the alleviation of colitis. We envision that the strategy developed here will inspire the exploitation of various prebiotics to arm probiotics for the effective alleviation of IBD.

Enhancing the Antioxidant Ability of Momordica grosvenorii Saponin to Resist Gastrointestinal Stresses via Microcapsules of Sodium Alginate and Chitosan and Its Application in Beverage

Momordica grosvenorii saponin (MGS), as a promising dietary supplement with remarkable biological properties, has poor stability under acidic conditions and thus hinders its application in functional foods. In this study, capsules of chitosan and sodium alginate were successfully prepared to enhance the stability of MGS. The optimized parameters for preparing MGS capsules were established. Sodium alginate of 20.8 mg/mL and triplication of MGS powder were added to chitosan of 4 mg/mL and calcium chloride of 10 mg/mL at a volume ratio of 3:1, stirring at 1000 r/min for 30 min to form the capsules. In this case, the fresh particles averaged 1687 μm with an encapsulation efficiency (EE) of 80.25% MGS. The capsule tolerated acidic environments better, and in vitro MGS could be controlled to release in a stimulated gastrointestinal tract system. The antioxidant activity and delayed release of MGS could be achieved by microencapsulation of chitosan/sodium alginate. Moreover, one drink containing 19 mg/mL MGS was successfully developed for the fruit.

Isolation and Characterization of Commensal Bifidobacteria Strains in Gut Microbiota of Neonates Born Preterm: A Prospective Longitudinal Study.

Bifidobacterial population dynamics were investigated using a longitudinal analysis of dominant species isolated from feces of neonates born preterm (singletons (n = 10), pairs of twins (n = 11)) from birth up to 16 months of age. We performed quantification, isolation, and identification of the dominant bifidobacteria strains. The genetic relationship of the isolates was investigated via pulsed field gel electrophoresis (PFGE) genotyping, and PCR was used to screen the specific genetic marker tet genes. Additionally, all of the isolated strains were phenotypically characterized by their response to gastro-intestinal stresses and the MIC determination of tetracycline. In the same individual, our results showed a turnover of the bifidobacteria dominant population not only at species but also at strain levels. In addition, we found clonally related strains between twins. A minority of strains were tolerant to gastric (6%) and intestinal (16%) stresses. Thirteen percent of the strains were resistant to tetracycline. This work is original as it provides insights at the strain level of the early life in vivo dynamics of gut microbiota bifidobacteria in preterm neonates. It highlights the need to take into consideration the fluctuation of bifidobacteria populations that may occur for one individual.

Compositional Quality and Possible Gastrointestinal Performance of Marketed Probiotic Supplements.

The local pharmacies and shops are brimming with various probiotic products that herald a range of health benefits. The poor quality of probiotic products in both dosage and species is symptomatic of this multi-billion-dollar market making it difficult for consumers to single out reliable ones. This study aims to fill the potential gap in the labeling accuracy of probiotic products intended for human consumption. We describe a combinatorial approach using classical culture-dependent technique to quantify and molecular techniques (16s rRNA gene sequencing, multilocus sequence, and ribotyping) for strain recognition of the microbial contents. The full gamut of probiotic characteristics including acid, bile and lysozyme tolerances, adhesiveness, anti-pathogenicity, and degree of safeness were performed. Their capacity to endure gastro-intestinal (GIT) stresses and select drugs was assessed in vitro. Our results forced us to declare that the local probiotic market is essentially unregulated. Almost none of the probiotic products tested met the label claim. Some (11%) have no viable cells, and a quarter (27%) showing significant inter-batch variation. A lower microbial count was typical with undesirables constituting a quarter of the total (~ 27%). Half of the products contained antibiotic-resistant strains; the unregulated use of these probiotics carries the risk of spreading antibiotic resistance to gut pathobionts. Poor tolerance to gut conditions and mediocre functionalism make the case worse. The current regulatory systems do not take this discrepancy into account. We recommend an evidence-based regular market surveillance of marketed probiotics to ensure the authenticity of the claims and product effectiveness.

Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology.

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

Application of the Combination of Soybean Lecithin and Whey Protein Concentrate 80 to Improve the Bile Salt and Acid Tolerance of Probiotics.

To improve the bile salt and acid tolerance of probiotics against gastrointestinal stresses, we investigated the effects of soybean lecithin and whey protein concentrate (WPC) 80 on the bile salt tolerance of Lacticaseibacillus paracasei L9 using a single-factor methodology, which was optimized using response surface methodology (RSM). The survival rate of L. paracasei L9 treated with 0.3% (w/v) bile salt for 2.5 h, and combined with soybean lecithin or WPC 80, was lower than 1%. After optimization, the survival rate of L. paracasei L9 incubated in 0.3% bile salt for 2.5 h reached 52.5% at a ratio of 0.74% soybean lecithin and 2.54% WPC 80. Moreover, this optimized method improved the survival rate of L. paracasei L9 in low pH condition and can be applied to other lactic acid bacteria (LAB) strains. Conclusively, the combination of soybean lecithin and WPC 80 significantly improved the bile salt and acid tolerance of LAB. Our study provides a novel approach for enhancing the gastrointestinal tolerance of LAB by combining food-derived components that have different properties.

Probiotic Properties of Lactobacillus Plantarum LRCC5193, a Plant-Origin Lactic Acid Bacterium Isolated from Kimchi and Its Use in Chocolates.

This study involves an investigation of the probiotic properties of lactic acid bacteria isolated from Kimchi, and their potential applications in chocolate. Lactobacillus plantarum-LRCC5193 (LP-LRCC5193) demonstrated a significantly higher degree of heat, acid, and bile acid tolerance compared to other Kimchi isolates. The intestinal adhesion assay also revealed that 84.2 log percentage of LP-LRCC5193 adhered to the Caco-2 cells after 2 h of incubation. Furthermore, the lyophilized LP-LRCC5193 maintained 92.9 log percentage and 97.2 log percentage survival rate within artificial stomach juice (pH 2.5, pepsin 0.04%) and artificial intestinal juice (oxgall 0.5%, trypsin 0.04%, and pancreatin 0.04%), respectively. Meanwhile, we also found that lyophilized LP-LRCC5193 incorporated in chocolate exhibited significantly higher survivability than lyophilized LP-LRCC5193 in both artificial gastric and intestinal juice under 1 to 3 hr incubation, where the survivability was within the range of 96.3 to 98.5 log percentage, and 98.8 to 98.9 log percentage, respectively. A 6-month storage test further revealed that LP-LRCC5193 demonstrated higher stability than the lyophilized LP-LRCC5103 in 3 different temperature ranges, where the final survival rates were 97.2 log percentage (20 °C), 89.2 log percentage (33 °C), and 94.4 log percentage (15 to 30 °C/wk). Altogether, our data suggest that chocolate can be used as a tasty delivery vehicle for delivering putative probiotic strain, LP-LRCC5193 to the gastrointestinal tract. PRACTICAL APPLICATION: Lactobacillus plantarum LRCC5193 (LP-LRCC5193) isolated from Kimchi demonstrated high stability under gastrointestinal environmental stresses and good adhesion to the intestinal epithelial cells in vitro. In addition, LP-LRCC5193 containing chocolates remained highly stable after storage at room temperature for 6 months. Chocolate containing LP-LRCC5193 can thus be considered a promising probiotic delivery system.

Analysis of technological and probiotic properties of Algerian L. mesenteroides strains isolated from dairy and non-dairy products

Six Leuconostoc mesenteroides strains isolated from camel (3) and sheep (1) milk, silage (1) and honey (1) have been investigated. All showed probiotic properties due to their resistance to gastrointestinal tract stresses, antibiogram profiles, hydrophobicity levels and antimicrobial activities against Staphylococcus aureus and Escherichia coli. Six had proteolytic, lipolytic, acidifying, and coagulation activities and five produced dextran. Metabolic flux analysis during growth in a sucrose-containing medium of two representative dextran-producing strains, and a non-producing strain revealed differences in the levels of the sucrose metabolites: fructose, glucose, lactic acid and mannitol, and a correlation between sucrose consumption and dextran synthesis for the producing strains. CM9 from camel milk showed high dextran production and the best pattern of intestinal tract colonization of gnotobiotic zebrafish embryos. These facts together with CM9’s technological and probiotic properties indicate that this strain may be useful for the production of functional dairy food.

Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential.

Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization of bacterial strains is critical for the development of novel probiotics, as variability between strains can dramatically alter bacterial physiology and functionality.

Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress.

Transfer of antibiotic resistance genes from probiotic bacteria to pathogens poses a safety concern. Orally administered probiotics are exposed to stressful conditions during gastrointestinal transit. In this study, filter mating experiments were performed to investigate the potential role of exposure of Bifidobacterium isolates to acid and bile stress on the transfer of a tetracycline resistance gene, tet(W), to Enterococcus faecalis ATCC 51299. No E. faecalis transconjugants were obtained after mating with either stressed or unstressed Bifidobacterium, thereby suggesting that tet(W) could not be transferred as a result of exposure to gastrointestinal stresses.

Variation of mucin adhesion, cell surface characteristics, and molecular mechanisms among Lactobacillus plantarum isolated from different habitats.

The adhesion ability to mucin varied greatly among 18 Lactobacillus plantarum isolates depending on their isolation habitats. Such ability remained at high level even though they were sequentially exposed to the gastrointestinal (GI) stresses. The majority of L. plantarum isolated from shrimp intestine and about half of food isolates exhibited adhesion ability (51.06-55.04%) about the same as the well-known adhesive L. plantarum 299v. Interestingly, five infant isolates of CIF17A2, CIF17A4, CIF17A5, CIF17AN2, and CIF17AN8 exhibited extremely high adhesion ranging from 62.69 to 72.06%. Such highly adhesive property correlating to distinctively high cell surface hydrophobicity was significantly weaken after pretreatment with LiCl and guanidine-HCl confirming the entailment of protein moiety. Regarding the draft genome information, all molecular structures of major cell wall-anchored proteins involved in the adhesion based on L. plantarum WCSF1, including lp_0964, lp_1643, lp_3114, lp_2486, lp_3127, and lp_3059 orthologues were detected in all isolates. Exceptionally, the gene-trait matching between yeast agglutination assay and the relevant mannose-specific adhesin (lp_1229) encoding gene confirmed the Msa absence in five infant isolates expressed distinctively high adhesion. Interestingly, the predicted flagellin encoding genes (fliC) firstly revealed in lp_1643, lp_2486, and lp_3114 orthologues may potentially contribute to such highly adhesive property of these isolates.

Exploring the relationship between exposure to technological and gastrointestinal stress and probiotic functional properties of lactobacilli and bifidobacteria.

Strains of Lactobacillus and Bifidobacterium are considered probiotic because of their associated potential health benefits. Probiotics are commonly administered orally via incorporation into food products. Microorganisms for use as probiotics encounter stress conditions, which include acid, bile, osmotic, oxidative, heat and cold stresses. These can occur during processing and storage and during passage through the gastrointestinal tract, and can affect viability. Probiotic bacteria have to remain viable to confer any health benefits. Therefore, the ability to withstand technological and gastrointestinal stresses is crucial probiotic selection criteria. While the stress tolerance mechanisms of lactobacilli and bifidobacteria are largely understood, the impact of exposure to stressful conditions on the functional properties of surviving probiotic microorganisms is not clear. This review explores the potentially positive and negative relationships between exposure to stress conditions and probiotic functional properties, such as resistance to gastric acid and bile, adhesion and colonization potential, and tolerance to antibiotics. Protective strategies can be employed to combat negative effects of stress on functional properties. However, further research is needed to ascertain synergistic relationships between exposure to stress and probiotic properties.

Lipid droplet levels vary heterogeneously in response to simulated gastrointestinal stresses in different probiotic Saccharomyces cerevisiae strains

To exert their therapeutic action, probiotic Saccharomyces cerevisiae strains must survive harsh digestive environments. Lipid droplets accumulate in cells which undergo stress-inducing situations, supposedly having a protective role. We assessed lipid droplet levels, either naturally accumulated or induced in response to digestive challenges, of probiotic strains S. boulardii, S. cerevisiae A-905, S. cerevisiae Sc47 and S. cerevisiae L11, and of non-probiotic strains S. cerevisiae BY4741 and S. cerevisiae BY4743. Strains 905 and Sc47 had lower and higher lipid droplet levels, respectively, when compared to the remaining strains, showing that higher accumulationof these neutral lipids is not a feature shared by all probiotic Saccharomyces strains. When submitted to simulated gastric or bile salts environments, lipid droplet levels increase in all tested probiotic strains, at least for one to the induced stresses, suggesting that lipid droplets participate in the protective mechanisms against gastrointestinal stresses in probiotic Saccharomyces yeasts.

Characterization of clinical and food Listeria monocytogenes isolates with different antibiotic resistance patterns through simulated gastrointestinal tract conditions and environmental stresses

Thirty-three Listeria monocytogenes isolates previously collected from two sources, food (n = 18) and human patients suffering from listeriosis (n = 15), with variable antibiotic susceptibility profiles (sensitive/resistant) to erythromycin, ciprofloxacin and nitrofurantoin, were studied for their ability to survive (i) the environmental stress provided by sequential conditions that simulate the digestive tract, and (ii) extreme pH values (1.5–12). The results showed a response that was only strain dependent. There were no variability in survival results based on type of stress (low or high pH), source (food or clinical), or sensitivity/resistance to antibiotics (p > 0.01). Some strains of L. monocytogenes are able to survive extreme acid and alkaline conditions, and conditions that mimic the sequential stressors found in the gastro-intestinal tract. The resistance to the antibiotics tested in this study by some L. monocytogenes strains did not confer any cross-protection to acid or alkaline stressors.

The Isolation, Identification and Analyses of Lactobacillus Genus Bacteria with Probiotic Potential.

103 strains of lactic acid bacteria of Lactobacillus genus were isolated from natural sources and identified for genus and species level with API tests and 16S rRNA sequencing. However, only 27 strains from isolated cultures demonstrated a high stability to gastric stress and from that - only 15 strains were highly resistant to intestinal stress. Results indicated that only some isolated cultures of lactobacilli possessed potential probiotical properties and could serve as new probiotics for dairy industry with high resistance to gastro-intestinal stresses.

Lactic Acid Bacteria in Pharmaceutical Formulations: Presence and Viability of “Healthy Microorganisms”

Many probiotic formulations are available in the market and are advertised for several preventive or curative roles. The aim of this study was the identification of microorganisms composing different lactic acid bacteria based pharmaceutical formulations and the ascertainment of their ability to survive gastro-intestinal (GI) stresses, the main requisite to produce beneficial effects. For this purpose, viable bacteria were enumerated by plate counts in different media. Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction (PCR-DGGE) analysis was applied on pure isolates and on crude formulations to confirm the composition in species. Also, crude formulations were subjected to stresses characteristic of the GI tract (GIT) to assess cell survival. Results highlighted concentrations lower than those reported in the labels in almost all the formulations. Moreover, some discrepancies were observed between reported species and those ascertained through the identification, and the use of an erroneous nomenclature was highlighted. The GI stress test revealed that bacteria are strongly injured, and this fact was evidenced by a marked reduction in viable counts after the stress. In conclusion, a widespread number of lactic acid bacteria based formulations are sold as probiotics, but their probiotic requisites are not adequately observed.

Acid adaptation to improve viability and X‐prolyl dipeptidyl aminopeptidase activity of the probiotic bacterium Lactobacillus fermentum HA6 exposed to simulated gastrointestinal tract conditions

SummaryIn this work, the viability of the probiotic bacterium Lactobacillus fermentum HA6 isolated from naturally fermented vegetables in Vietnam was improved by growing the bacterium into a mild acid condition (pH 4.0). Viability and probiotic functionality [X‐prolyl dipeptidyl aminopeptidase (PepX) activity] of the acid‐adapted bacterium exposed to simulated gastrointestinal conditions were investigated. After 180 min in the simulated gastric juice (0.3 g/L pepsin, pH 2.0), the viability of acid‐adapted L. fermentum HA6 (11.5%) was higher than that of control L. fermentum HA6 (2.2%). Specific PepX activity of acid‐adapted cells (24.5 U/mg) was higher than that of control cells (17.8 U/mg). After 180‐min exposure to the simulated small intestinal medium (0.3 g/L bile salts, 0.1 g/L pancreatin, pH 8.0), the viability of acid‐adapted L. fermentum HA6 (13.5%) was twofold as high as that of control L fermentum HA6 (8.0%). Our results suggested that acid adaptation has a key role in acquiring cross‐protection mechanism, which in this study resulted in higher survival of L. fermentum HA6 after simulated gastrointestinal stresses. The strategy of acid adaptation could be valuable for the production of robust probiotics.

Functional characterization of a novel Mn2+ dependent protein serine/threonine kinase KpnK, produced by Klebsiella pneumoniae strain MGH78578

Klebsiella pneumoniae MGH78578 contains ∼500 uncharacterized signaling proteins and in this study, we characterized the biological functions of a novel eukaryotic-like serine/threonine kinase; ESTK (KpnK). Studies demonstrated that KpnK undergoes autophosphorylation within the pH range 7.0–7.5 at 37°C in a time- and concentration- dependent manner, with Mn2+ as its cofactor. The ΔkpnK mutant exhibited higher sensitivity to gastrointestinal and oxidative stresses. Deletion of kpnK resulted in a two to threefold increased susceptibility towards imipenem, cefepime, ceftriaxone and ceftazidime. Our study has provided overall evidence for the involvement of ESTK in regulating bacterial physiology, stress response and drug resistance. This report has unmasked the occurrence of Ser/Thr kinase mediated signaling for the first time in K. pneumoniae. Structured summary of protein interactionsKpnKphosphorylatesKpnK by protein kinase assay (View interaction).

Flow cytometric assessment of the protectants for enhanced in vitro survival of probiotic lactic acid bacteria through simulated human gastro-intestinal stresses

The aim of this study was to apply flow cytometric (FCM) analysis to assess the use of sucrose and lecithin vesicles for the protection of probiotic lactic acid bacteria in response to the challenge of gastric acidity and bile salts. FCM analysis in combination with fluorescent probes carboxyfluorescein (cF) and propidium iodide was used to reveal the physiological heterogeneity in the stressed bacteria population. Three subpopulations (intact, stressed, and damaged) were differentiated by FCM in all six examined strains. Significant changes were observed in the presence of the selected protectants. The addition of 20mM sucrose in the simulated gastric fluid substantially increased the number of intact cells over 20 folds and reduced the damaged subpopulation by half. The presence of 2% (w/v) lecithin vesicles was shown to protect 50% more intact cells from the challenge of bile salts. The improved survival as evaluated by FCM analysis was further assessed for the proliferation capacity by sorting a number of cells from each subpopulation on nutrient agar plate. The result confirmed conformity between the proliferation-based cultivability and the probe-indicated viability in the samples of the intact and the damaged subpopulations. However, it also revealed the complexities of the stressed (injured) subpopulation. In conclusion, FCM analysis confirmed that the selected protectants could improve the survival of the probiotic strains in the simulated GI environments. The FCM analysis also proved to be a useful analytical tool for the probiotics research.