Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus.

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that αS2-casein, ß-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.

Cysteine protected cells from H2O2-induced damage and promoted long-chain fatty acids synthesis in vivo to improve γ-aminobutyric acid production in Levilactobacillus brevis.

Levilactobacillus brevis NPS-QW-145 isolated from kimchi is deficient in glutamate dehydrogenase-encoding gene (gdhA) to form glutamate, hence it required exogenous supplementation of glutamate/monosodium glutamate (MSG) for decarboxylation reaction to produce γ-aminobutyric acid (GABA). However, GABA conversion rate from MSG was relatively low. The individual effect of 20 amino acids on regulating GABA biosynthesis was investigated. Cysteine was selected to significantly improve GABA production from MSG. It was found that Lb. brevis was capable of producing H2O2, cysteine protected Lb. brevis against H2O2-induced oxidative damage to increase cell viability for the enhancement of GABA production. Moreover, cysteine promoted glucose consumption to produce acetyl-CoA for synthesizing long-chain fatty acids to significantly up-regulate GABA biosynthesis. These findings deciphered antioxidative capability of cysteine in Lb. brevis 145 and provided a theoretical basis for fatty acids synthesis-mediated GABA synthesis in Lb. brevis 145, and possibly in other lactic acid bacteria.

Invited review: Characterization of new probiotics from dairy and nondairy products-Insights into acid tolerance, bile metabolism and tolerance, and adhesion capability.

The selection of potential probiotic strains that possess the physiological capacity of performing successfully in the gastrointestinal tract (GIT) is a critical challenge. Probiotic microorganisms must tolerate the deleterious effects of various stresses to survive passage and function in the human GIT. Adhesion to the intestinal mucosa is also an important aspect. Recently, numerous studies have been performed concerning the selection and evaluation of novel probiotic microorganisms, mainly probiotic bacteria isolated from dairy and nondairy products. Therefore, it would be crucial to critically review the assessment methods employed to select the potential probiotics. This article aims to review and discuss the recent approaches, methods used for the selection, and outcomes of the evaluation of novel probiotic strains with the main purpose of supporting future probiotic microbial assessment studies. The findings and approaches used for assessing acid tolerance, bile metabolism and tolerance, and adhesion capability are the focus of this review. In addition, probiotic bile deconjugation and bile salt hydrolysis are explored. The selection of a new probiotic strain has mainly been based on the in vitro tolerance of physiologically related stresses including low pH and bile, to ensure that the potential probiotic microorganism can survive the harsh conditions of the GIT. However, the varied experimental conditions used in these studies (different types of media, bile, pH, and incubation time) hamper the comparison of the results of these investigations. Therefore, standardization of experimental conditions for characterizing and selecting probiotics is warranted.

Comparative Peptidomic and Metatranscriptomic Analyses Reveal Improved Gamma-Amino Butyric Acid Production Machinery in Levilactobacillus brevis Strain NPS-QW 145 Cocultured with Streptococcus thermophilus Strain ASCC1275 during Milk Fermentation.

The high-gamma-amino butyric acid (GABA)-producing bacterium Levilactobacillus brevis strain NPS-QW 145, along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity), enhances GABA production in milk. However, a mechanistic understanding of how Levilactobacillus brevis cooperates with S. thermophilus to stimulate GABA production has been lacking. Comparative peptidomic and metatranscriptomic analyses were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the coculture. We found that particular peptides hydrolyzed by S. thermophilus ASCC1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was metabolized as the main carbon source by Lb. brevis 145 for ATP production. In the stationary phase, under acidic conditions due to the accumulation of lactic acid produced by S. thermophilus 1275, the expression of genes involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 was induced for GABA production.SIGNIFICANCE A huge market for GABA-rich milk as a dietary therapy for the management of hypertension is anticipated. The novelty of this work lies in applying peptide profiles supported by metatranscriptomics to elucidate (i) the pattern of casein hydrolysis by S. thermophilus 1275, (ii) the supply of peptides and glucose by S. thermophilus 1275 to Lb. brevis 145, (iii) the transportation of peptides in Lb. brevis and the degradation of peptides by this organism, which was reported to be nonproteolytic, and (iv) GABA production by Lb. brevis 145 under acidic conditions. Based on the widely reported contribution of lactic acid bacteria (LAB) and GABA to human health, the elucidation of interactions between the two groups of bacterial communities in the production of GABA-rich milk is important for promoting the development of functional dairy food and may provide new insight into the development of industrial GABA production.

Structural characterization of exopolysaccharide from Streptococcus thermophilus ASCC 1275.

In this study, we purified and characterized exopolysaccharide (EPS) produced by a high-EPS-producing dairy starter bacterium, Streptococcus thermophilus ASCC 1275. Crude EPS was extracted from S. thermophilus ASCC 1275 and partially purified using dialysis. Further purification and fractionation of exopolysaccharide was conducted using HPLC on a Superose 6 column (Cytiva/Global Life Sciences Solutions, Marlborough, MA). Glycosyl composition analysis, linkage analysis along with 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy were performed to deduce the structure of EPS. Three fractions (F) obtained from gel permeation chromatography were termed F1 (2.6%), F2 (45.8%), and F3 (51.6%) with average molecular weights of approximately 511, 40, and 5 kDa, respectively. Monosaccharide composition analysis revealed the dominance of glucose, galactose, and mannose in all 3 fractions. Major linkages observed in F3 were terminal galactopyranosyl (t-Gal), 3-linked glucopyranosyl (3-Glc), 3-linked galactofuranosyl (3-Galf), and 3,6-linked glucopyranosyl (3,6-Glc) and major linkages present in F2 were 4-Glc (48 mol%), followed by terminal mannopyranosyl (t-Man), 2- + 3-linked mannopyranosyl (2-Man+3-Man), and 2,6-linked mannopyranosyl (2,6-Man; total ∼28 mol%). The 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy revealed that F2 comprised mannans linked by (1→2) linkages and F3 consisted of linear chains of α-d-glucopyranosyl (α-d-Glcp), ß-d-glucopyranosyl (ß-d-Glcp), and ß-d-galactofuranosyl (ß-d-Galf) connected by (1→3) linkages; branching was through (1→6) linkage in F3. A possible structure of EPS in F2 and F3 was proposed.

Enterococcus hirae WEHI01 isolated from a healthy Chinese infant ameliorates the symptoms of type 2 diabetes by elevating the abundance of Lactobacillales in rats.

Enterococcus hirae WEHI01 is a potential probiotic strain isolated from a healthy Chinese infant. This strain has previously been characterized as having cholesterol-lowering potential and good dairy fermentation performance. In this study, we used rat models with obesity and type 2 diabetes mellitus (T2DM) induced by a high fat and sucrose diet and low-dose streptozotocin, respectively, and we evaluated the effect of E. hirae WEHI01 on glycolipid metabolism, glycolipid-related gene expression, organ histopathology, and intestinal flora changes in the 2 models. Our results showed that administration of 5.0 × 109 cfu of E. hirae WEHI01 for 4 wk decreased serum lipid levels and regulated glycolipid metabolism in the liver of obese rats. Following continuous administration of the same concentration of E. hirae WEHI01 to a T2DM rat model for another 5 wk, E. hirae WEHI01 improved glucose tolerance, recovered body weight loss, and led to significant decreases in tumor necrosis factor-α, IL-6, IL-10, and total bile acid in serum. We also found that E. hirae WEHI01 restored the morphology of the pancreas, kidney, and liver, and changed the composition of the gut microbiota (i.e., decreased the Shannon index, increased the Simpson index, and substantially increased the abundance of Lactobacillales). Combining the results for the obese model and the T2DM model, we speculated that beneficial effects of E. hirae WEHI01 on T2DM could be due to (1) a significant increase in PPARA expression and a tendency for increased CYP7A1 expression in the liver of obese rats, promoting the conversion of cholesterol into bile acid and reducing serum total bile acid levels in T2DM model rats; or (2) a change in gut microbial diversity, especially elevated Lactobacillales abundance, which reduced the total bile acid in T2DM model rats. These results demonstrated that E. hirae WEHI01 has the potential to ameliorate type 2 diabetes in rats and provide a promising rationale for further research into the prevention and treatment of T2DM.

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses.

Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature, low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.

Sulfonation of Lactobacillus plantarum WLPL04 exopolysaccharide amplifies its antioxidant activities in vitro and in a Caco-2 cell model.

Exopolysaccharide (EPS) of Lactobacillus plantarum WLPL04 and its sulfated EPS were systematically investigated for their antioxidant activities and effects on protecting the oxidative damage of Caco-2 cells from H2O2. Exopolysaccharide was successfully sulfonated from purified EPS as confirmed by Fourier-transform infrared spectroscopy, and the degree of sulfonation was 0.30. Both EPS and sulfated EPS showed antioxidant activities in vitro determined by 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radical scavenging tests, and those activities of sulfated EPS were significantly enhanced at 1,000 µg/mL. Cell viabilities of Caco-2 in the range of 1 to 100 µg/mL of EPS and sulfated EPS showed no significant difference. In H2O2-damaged Caco-2 cells models, EPS and sulfated EPS significantly inhibited the enhancement of reactive oxygen species and malondialdehyde levels, and sulfated EPS enhanced the effects by 40.86% and 61.11% when compared with the purified EPS at the same concentration of 100 µg/mL, respectively. For the activities of antioxidant-related enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and expression of genes (SOD2, GPX2, MT1M) on Caco-2 cells, strong protection abilities against the oxidative stress were displayed from both EPS and sulfated EPS, and sulfated EPS exhibited significant enhancement as compared with either EPS or control groups. In summary, sulfonation is an effective strategy for improving the antioxidant activities of EPS from L. plantarum WLPL04 in vitro and on Caco-2 cells.

Restoration of GABA production machinery in Lactobacillus brevis by accessible carbohydrates, anaerobiosis and early acidification.

Lactobacillus brevis is an efficient cell factory for producing bioactive γ-aminobutyric acid (GABA) by its gad operon-encoded glutamic acid decarboxylase (GAD) system. However, little mechanistic insights have been reported on the effects of carbohydrate, oxygen and early acidification on GABA production machinery in Lb. brevis. In the present study, GABA production from Lb. brevis was enhanced by accessible carbohydrates. Fast growth of this organism was stimulated by maltose and xylose. However, its GABA production was highly suppressed by oxygen exposure, but was fully restored by anaerobiosis that up-regulated the expression of gad operon in Lb. brevis cells. Although the level of cytosolic acidity was suitable for the functioning of GadA and GadB, early acidification of the medium (ipH 5 and ipH 4) restored GABA synthesis strictly in aerated cells of Lb. brevis because the expression of gad operon was not up-regulated in them. We conclude that GABA production machinery in Lb. brevis could be restored by accessible carbohydrates, anaerobiosis and early acidification. This will be of interest for controlling fermentation for synthesis of GABA and manufacturing GABA-rich fermented vegetables.

Contemporary nucleic acid-based molecular techniques for detection, identification, and characterization of Bifidobacterium.

Bifidobacteria are one of the most important bacterial groups found in the gastrointestinal tract of humans. Medical and food industry researchers have focused on bifidobacteria because of their health-promoting properties. Researchers have historically relied on classic phenotypic approaches (culture and biochemical tests) for detection and identification of bifidobacteria. Those approaches still have values for the identification and detection of some bifidobacterial species, but they are often labor-intensive and time-consuming and can be problematic in differentiating closely related species. Rapid, accurate, and reliable methods for detection, identification, and characterization of bifidobacteria in a mixed bacterial population have become a major challenge. The advent of nucleic acid-based molecular techniques has significantly advanced isolation and detection of bifidobacteria. Diverse nucleic acid-based molecular techniques have been employed, including hybridization, target amplification, and fingerprinting. Certain techniques enable the detection, characterization, and identification at genus-, species-, and strains-levels, whereas others allow typing of species or strains of bifidobacteria. In this review, an overview of methodological principle, technique complexity, and application of various nucleic acid-based molecular techniques for detection, identification, and characterization of bifidobacteria is presented. Advantages and limitations of each technique are discussed, and significant findings based on particular techniques are also highlighted.

Integrating omics to unravel the stress-response mechanisms in probiotic bacteria: Approaches, challenges, and prospects.

Identifying the stress-response mechanism of probiotic bacteria has always captivated the interest of food producers. It is crucial to identify probiotic bacteria that have increased stress tolerance to survive during production, processing, and storage of food products. However, in order to achieve high resistance to environmental factors, there is a need to better understand stress-induced responses and adaptive mechanisms. With advances in bacterial genomics, there has been an upsurge in the application of other omic platforms such as transcriptomics, proteomics, metabolomics, and some more recent ones such as interactomics, fluxomics, and phenomics. These omic technologies have revolutionized the functional genomics and their application. There have been several studies implementing various omic technologies to investigate the stress responses of probiotic bacteria. Integrated omics has the potential to provide in-depth information about the mechanisms of stress-induced responses in bacteria. However, there remain challenges in integrating information from different omic platforms. This review discusses current omic techniques and challenges faced in integrating various omic platforms with focus on their use in stress-response studies.

High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter.

γ-Aminobutyric acid (GABA) and GABA-rich foods have shown anti-hypertensive and anti-depressant activities as the major functions in humans and animals. Hence, high GABA-producing lactic acid bacteria (LAB) could be used as functional starters for manufacturing novel fermented dairy foods. Glutamic acid decarboxylases (GADs) from LAB are highly conserved at the species level based on the phylogenetic tree of GADs from LAB. Moreover, two functionally distinct GADs and one intact gad operon were observed in all the completely sequenced Lactobacillus brevis strains suggesting its common capability to synthesize GABA. Difficulties and strategies for the manufacture of GABA-rich fermented dairy foods have been discussed and proposed, respectively. In addition, a genetic survey on the sequenced LAB strains demonstrated the absence of cell envelope proteinases in the majority of LAB including Lb. brevis, which diminishes their cell viabilities in milk environments due to their non-proteolytic nature. Thus, several strategies have been proposed to overcome the non-proteolytic nature of Lb. brevis in order to produce GABA-rich dairy foods.

The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods.

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and ß-galactosidase assay suggest that PTSGal system, PTSLac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway.

Mutual growth-promoting effect between Bifidobacterium bifidum WBBI03 and Listeria monocytogenes CMCC 54001.

In this study, Bifidobacterium bifidum WBBI03 and Listeria monocytogenes CMCC 54001 were selected to detect the changes in their growth pattern after mutual interaction between them. The proteomic changes after the interaction between the 2 bacteria were detected by the isobaric tags for relative and absolute quantitation method. The proteins related to the biosynthesis and cell reproduction were selected, and their changes at the transcriptional level were monitored by fluorescent quantitative PCR. Also, 3 other types of probiotic organisms and opportunistic pathogens were used to verify the results mentioned above. The results showed that growing the 2 organisms together could promote the growth of each other, resulting in earlier entry into the logarithmic phase. The results also showed that the expression of these proteins mostly tended to be upregulated at the translational and transcriptional level. The increase in the expression of these proteins might help promote the growth and reproduction of B. bifidum WBBI03 and L. monocytogenes CMCC 54001. One aspect of the biological significance of their presence in the normal intestine may be that the opportunistic pathogens promote the growth of the probiotics.

Health-promoting benefits of low-fat akawi cheese made by exopolysaccharide-producing probiotic Lactobacillus plantarum isolated from camel milk.

Lactic acid bacteria isolated from camel milk exhibit remarkable probiotic and exopolysaccharide (EPS)-producing characteristics. The health-promoting benefits of exopolysaccharide-producing probiotic Lactobacillus plantarum isolated from camel milk used for making low-fat akawi cheese were investigated. Three low-fat akawi cheeses were made using traditional culture (non-EPS-producing, EPS-), commercial EPS-producing (MEPS+), and camel milk EPS-producing (CEPS+) cultures. α-Amylase and α-glucosidase inhibitory activities, antioxidant activities, angiotensin-converting enzyme (ACE) inhibition, and antiproliferative activity were determined. Cheese made with CEPS+ culture exhibited comparable α-amylase inhibition to that of cheeses made with MEPS+. Scavenging rates of cheese made with EPS+ cultures were higher than those of cheese made with EPS- cultures. The percentage of α-glucosidase inhibition ranged from >45% at 0 d to ∼55% at 21 d of storage. After 7 d of storage, the scavenging rate in CEPS+ cheese increased >60% by ABTS assay [2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)] and >20% by DPPH assay (1,1-diphenyl-2-picrylhydrazyl). Throughout storage, cheese made with EPS+ cultures showed higher ACE-inhibition activity compared with EPS- cultures. Cheese made with CEPS+ showed ACE inhibition >70% after 7 d of storage. Antiproliferation activity of CEPS+ cheese increased from 38 to 48% during 7 d of storage and was maintained above 45% with prolonged storage. Low-fat akawi cheese produced with these cultures exhibited similar or greater health-promoting benefits compared with cheese made using commercial starter cultures. Therefore, incorporation of these cultures in food is promising for commercial uses.

Beneficial effects of probiotic cholesterol-lowering strain of Enterococcus faecium WEFA23 from infants on diet-induced metabolic syndrome in rats.

The aim of this study was to select probiotic Enterococcus strains that have the potential to improve metabolic syndrome (MS). Ten Enterococcus strains isolated from healthy infants were evaluated for their probiotic properties in vitro, and Enterococcus faecium WEFA23 was selected due to its cholesterol removal ability (1.89 ± 0.07 mg/1010 cfu), highest glycodeoxycholic acid-hydrolase activity (1.86 ± 0.01 U/mg), and strong adhesion capacity to Caco-2 cells (17.90 ± 0.19%). The safety of E. faecium WEFA23 was verified by acute oral administration in mice, and it was found to have no adverse effects on general health status, bacterial translocation, and gut mucosal histology. Moreover, the beneficial effects of E. faecium WEFA23 on high-fat diet-induced MS in rats were investigated, and we found WEFA23 significantly decreased body weight, serum lipid levels (total cholesterol, triacylglycerols, and low-density lipoprotein cholesterol), blood glucose level, and insulin resistance in rats fed with a high-fat diet. This indicated that administration of E. faecium WEFA23 improved almost all key markers of MS, including obesity, hyperlipidemia, hyperglycemia, and insulin resistance. Our results supported E. faecium WEFA23 as a candidate for cholesterol-lowering dairy products and improvement of MS. Our research provided novel insights on Enterococcus as a strategy to combat MS.

Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review.

The use of live probiotic bacteria as food supplement has become popular. Capability of probiotic bacteria to be kept at room temperature becomes necessary for customer's convenience and manufacturer's cost reduction. Hence, production of dried form of probiotic bacteria is important. Two common drying methods commonly used for microencapsulation are freeze drying and spray drying. In spite of their benefits, both methods have adverse effects on cell membrane integrity and protein structures resulting in decrease in bacterial viability. Microencapsulation of probiotic bacteria has been a promising technology to ensure bacterial stability during the drying process and to preserve their viability during storage without significantly losing their functional properties such acid tolerance, bile tolerance, surface hydrophobicity, and enzyme activities. Storage at room temperatures instead of freezing or low temperature storage is preferable for minimizing costs of handling, transportation, and storage. Concepts of water activity and glass transition become important in terms of determination of bacterial survival during the storage. The effectiveness of microencapsulation is also affected by microcapsule materials. Carbohydrate- and protein-based microencapsulants and their combination are discussed in terms of their protecting effect on probiotic bacteria during dehydration, during exposure to harsh gastrointestinal transit and small intestine transit and during storage.

Synergistic in vitro and in vivo antimicrobial effect of a mixture of ZnO nanoparticles and Lactobacillus fermentation liquor.

In this study, we investigated the antibacterial activity of ZnO nanoparticles (NPs) and Lactobacillus-fermentation liquor (LFL) against two pathogenic bacteria in vitro and in vivo. Bactericidal tests were performed on solid agar plates and quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE) techniques were used to examine the antibacterial activity of the mixture of ZnO NPs and LFL in vivo. The results showed that the mixture exhibited higher antibacterial activity against Salmonella typhimurium in vitro in comparison with ZnO NPs alone. The results showed that ZnO NPs and LFL significantly enhanced microbial diversity in mouse intestine which suggested a synergistic antibacterial activity against the tested pathogenic bacteria that could be used for the control of the spread and persistence of bacterial infections.

Effect of salt stress on morphology and membrane composition of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum, and their adhesion to human intestinal epithelial-like Caco-2 cells.

The effects of NaCl reduction (10.0, 7.5, 5.0, 2.5, and 0% NaCl) and its substitution with KCl (50% substitution at each given concentration) on morphology of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum was investigated using transmission electron microscopy. Changes in membrane composition, including fatty acids and phospholipids, were investigated using gas chromatography and thin layer chromatography. Adhesion ability of these bacteria to human intestinal epithelial-like Caco-2 cells, as affected by NaCl and its substitution with KCl, was also evaluated. Bacteria appeared elongated and the intracellular content appeared contracted when subjected to salt stress, as observed by transmission electron microscopy. Fatty acid content was altered with an increase in the ratio of unsaturated to saturated fatty acid content on increasing the NaCl-induced stress. Among the phospholipids, phosphatidylglycerol was reduced, whereas phosphatidylinositol and cardioplipin were increased when the bacteria were subjected to salt stress. There was a significant reduction in adhesion ability of the bacteria to Caco-2 cells when cultured in media supplemented with NaCl; however, the adhesion ability was improved on substitution with KCl at a given total salt concentration. The findings provide insights into bacterial membrane damage caused by NaCl.

Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens.

Lactobacillus plantarum ZDY2013 is a potential probiotic isolated from fermented bean acid. In this study, we aimed to evaluate the in vitro antimicrobial activity of this organism against Bacillus cereus in milk fermentation, the antiadhesion ability on intestinal epithelial cells, as well as its ability to abrogate the cytotoxic effect and expression levels of genes. We found no antimicrobial activity produced by L. plantarum once the pH was adjusted to 6.0 and 7.0. The pH decreased continuously when L. plantarum and B. cereus were co-incubated during milk fermentation, which caused a decrease in the B. cereus counts. Antiadhesion assays showed that L. plantarum can significantly inhibit the adhesion of enterotoxin-producing B. cereus ATCC14579 and pathogenic B. cereus HN001 by inhibition, competition, and displacement. The supernatants of B. cereus, either alone or in conjunction with L. plantarum, caused damage to the membrane integrity of Caco-2 cells to release lactate dehydrogenase. In addition, L. plantarum tended to attenuate proinflammatory cytokine and oxidative stress gene expression on Caco-2 cells, inducing with B. cereus HN001 supernatants. This study provided systematic insights into the antagonistic effect of L. plantarum ZDY2013, and the information may be helpful to explore potential control measures for preventing food poisoning by lactic acid bacteria.