Bifidobacterium longum K5 Prevents Enterohaemorrhagic Escherichia coli O157:H7 Infection in Mice through the Modulation of the Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1ß was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.

Effects of Viable and Heat-Inactivated Bifidobacterium longum D42 on Proliferation and Apoptosis of HT-29 Human Colon Cancer Cells.

Bifidobacterium longum is a common probiotic; both viable and heat-inactivated Bifidobacterium longum have many probiotic effects, such as anticancer effects. But some mechanisms of anticancer effects are still unclear, especially for heat-inactivated probiotics. In this study, we analyzed the effects of viable and heat-inactivated Bifidobacterium longum D42 on human colon cancer cells (HT-29). Cell proliferation, membrane permeability and apoptosis were detected by using the CCK-8 method, LDH method and Annexin V-FITC/PI kits. The ROS level and mitochondrial membrane potential were examined using the fluorescent probes DCFH-DA and JC-1. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the expression of mitochondrial apoptosis pathway genes and proteins. The results showed that viable and heat-inactivated Bifidobacterium longum D42 at concentrations of 1 × 106 CFU/mL significantly inhibited the proliferation of and increased the level of LDH release of HT-29 colon cancer cells. We found that they could increase the apoptosis rate of HT-29 cells. Moreover, they could also induce apoptosis by inducing cells to produce ROS and destroying the mitochondrial membrane potential of cells. Further studies found that they could increase the mRNA transcription and protein expression levels of the Caspase-3, Caspase-9 and Bax genes in cells, and reduce the mRNA transcription and protein expression levels of the Bcl-2 gene. In summary, our findings revealed that viable and heat-inactivated Bifidobacterium longum D42 have inhibitory effects on proliferation and promote the apoptosis of human colon cancer cells, and also have certain adjuvant drug therapeutic effects and have potential application value in the adjuvant treatment of colon cancer.

Sulfated hyaluronic acid gel for the treatment of rheumatoid arthritis in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. NSAIDS, cyclophosphamide and glucocorticoid were commonly used to treat RA in clinical application, which long-term administration of these drugs caused serious adverse reactions. Therefore, sulfated hyaluronic acid (sHA) gel (SG) was prepared to firstly treat the RA and avoid the problem of toxic side effect caused by long-term application. In vitro evaluation showed that sHA inhibited the level of reactive oxygen species and TNF-α, IL-1ß, and IL-6, and decreased the ratio of macrophage M1/M2 type, which exerted better anti-inflammatory capacity. In vivo studies showed that the injection of SG into the joint cavity of collagen-induced rheumatoid arthritis (CIA) rats could effectively treat joint swelling and reduce the level of inflammatory factors in the serum. Immunofluorescence showed that SG exerted its anti-inflammatory activity by decreasing the ratio of M1/M2 type macrophages in synovial tissue. Cartilage tissue sections showed that SG reduced bone erosion and elevated chondrocyte expression. These results confirmed that sHA is expected to be developed as a drug to treat or relieve RA, which could effectively regulate the level of macrophages in rat RA, alleviate the physiological state of inflammatory over-excitation, and improve its anti-inflammatory and antioxidant capacity.

Genome Analysis of Bifidobacterium Bifidum E3, Structural Characteristics, and Antioxidant Properties of Exopolysaccharides.

In this study, the antioxidant properties of intact cells (IC), cell-free supernatant (CFS), and cell-free extracts (CFE) and whole genome sequencing of Bifidobacterium bifidum E3 (B. bifidum E3), as well as the structural characteristics and antioxidant properties of EPS-1, EPS-2, and EPS-3, were evaluated. The results revealed that intact cells (IC), cell-free supernatant (CFS), and cell-free extracts (CFE) had potent DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), hydroxyl, and superoxide anion radical scavenging capacities, among which CFS was the best. At the genetic level, we identified a strong carbohydrate metabolism capacity, an EPS synthesis gene cluster, and five sugar nucleotides in B. bifidum E3. Therefore, we extracted cEPS from B. bifidum E3 and purified it to obtain EPS-1, EPS-2, and EPS-3. EPS-1, EPS-2, and EPS-3 were heteropolysaccharides with an average molecular weight of 4.15 × 104 Da, 3.67 × 104 Da, and 5.89 × 104 Da, respectively. The EPS-1 and EPS-2 are mainly comprised of mannose and glucose, and the EPS-3 is mainly comprised of rhamnose, mannose, and glucose. The typical characteristic absorption peaks of polysaccharides were shown in Fourier transform infrared spectroscopy (FT-IR spectroscopy). The microstructural study showed a rough surface structure for EPS-1, EPS-2, and EPS-3. Furthermore, EPS-1, EPS-2, and EPS-3 exhibited potent DPPH, hydroxyl, and superoxide anion radical scavenging capacities. Correlation analysis identified that antioxidant capacities may be influenced by various factors, especially molecular weight, chemical compositions, and monosaccharide compositions. In summary, the EPS that was produced by B. bifidum E3 may provide insights into health-promoting benefits in humans.

Bifidobacterium bifidum E3 Combined with Bifidobacterium longum subsp. infantis E4 Improves LPS-Induced Intestinal Injury by Inhibiting the TLR4/NF-κB and MAPK Signaling Pathways In Vivo.

Changes in the functions of the intestinal barrier occur in parallel with the development of sepsis. The protection by Bifidobacterium strains (BB, BL, BB12, and BLBB) was evaluated in mice injected with lipopolysaccharide (LPS). The results revealed an increase in the ratio of ileal villus length to crypt depth in the BLBB group compared with that in the LPS group, as were the number of IgA+ plasma, CD4+/CD8+ T, and dendritic cells. The levels of diamine oxidase (DAO) and d-lactic acid in the serum were lessened in the BLBB group after LPS injection compared with that in the LPS group. In addition, the BLBB group exhibited an increased expression level of tight junction proteins (zonula occludens-1, occludin, and claudin-1), mucin (MUC2) mRNA, reduced NFκB/MAPK signaling pathways, and decreased expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α). The BLBB group enriched the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, Clostridia_Ucg-014, and Alistipes, resulting in an increase in strains producing short-chain fatty acids. Furthermore, the BLBB group leads to higher levels of deoxycholic acid and biosynthesized linoleate. This study suggested that the BLBB group could enhance the capacity of the intestinal barrier and intestinal mucosal immunity, reduce intestinal inflammation, and improve the composition of gut microbiota. Bifidobacterium bifidum E3 combined with Bifidobacterium longum subsp. infantis E4 may thus serve as a probiotic against the intestinal injury caused by LPS.

Sodium hyaluronates applied in the face affects the diversity of skin microbiota in healthy people.

OBJECTIVE: A healthy and stable microbiome has many beneficial effects on the host, while an unbalanced or disordered microbiome can lead to various skin diseases. Hyaluronic acid is widely used in the cosmetics and pharmaceutical industries; however, specific reports on its effect on the skin microflora of healthy people have not been published. This study aimed to determine the effect of sodium hyaluronate on the facial microflora of healthy individuals. METHODS: Face of 20 healthy female volunteers between 18 and 24 years was smeared with sodium hyaluronate solution once per day. Cotton swabs were used to retrieve samples on days 0, 14, and 28, and high-throughput sequencing of 16 S rRNA was used to determine the changes in bacterial community composition. RESULTS: Facial application of HA can reduce the abundance of pathogenic bacteria, such as Cutibacterium and S. aureus, and increase the colonization of beneficial bacteria. CONCLUSION: This is the first intuitive report to demonstrate the effect of hyaluronic acid on facial microflora in healthy people. Accordingly, sodium hyaluronate was found to have a positive effect on facial skin health.

Analysis of the complete genome sequence of Lactobacillus delbrueckii ssp. bulgaricus with post-acidification capacity and its influence on yogurt in storage.

In recent years, yogurt has been one of the most popular fermented dairy products and is sold worldwide. In this study, pH and titrated acid changes of 4 strains of Lactobacillus delbrueckii ssp. bulgaricus fermented milk during storage were detected. The difference between L. bulgaricus KLDS1.1011 and KLDS1.0207 was significant, with the latter exhibiting reduced acidity levels. Therefore, we determined the complete genome sequence of the 2 strains. Then the expression of specific genes and common genes related to glucose metabolism and proteolysis of L. bulgaricus KLDS1.1011 and KLDS1.0207 were detected by quantitative real-time reverse-transcription PCR. Analysis indicated that the key enzymes in glycometabolism and proteolysis of L. bulgaricus KLDS1.1011 were significantly different than those of L. bulgaricus KLDS1.0207. The contents of lactose and glucose decreased during storage of L. bulgaricus fermented milk, as determined by HPLC, and the contents of lactic acid and galactose increased, with L. bulgaricus KLDS1.1011 increasing less. With skim milk as a raw material, L. bulgaricus KLDS1.1011, KLDS1.0207, and Streptococcus thermophilus S1 were used as fermentation strains to yield yogurt at 42°C, and sensory evaluation was compared with yogurt fermented by commercial starter cultures. Yogurt from L. bulgaricus KLDS1.1011 was the highest-rated. Therefore, the study may provide guidelines for the development of yogurt starters.

Gut microbiota modulation and anti-inflammatory properties of mixed lactobacilli in dextran sodium sulfate-induced colitis in mice.

Correlations between gut microbiota activities and inflammatory bowel disease (IBD) treatment are gaining research interest. In our previous study, Lactobacillus acidophilus KLDS 1.0901, Lactobacillus helveticus KLDS 1.8701, and Lactobacillus plantarum KLDS 1.0318 showed antibacterial, antioxidant, and immunomodulatory activities. In the current study, we evaluated the effects of three tested strains and their mixture on dextran sulfate sodium (DSS)-induced colitis in C57BL/6J mice. The three tested strains and their mixture significantly decreased the disease activity index (DAI), colon shortening, and myeloperoxidase (MPO) activity. Additionally, the three tested strains and their mixture improved the histological damage, increased the colonic mucous layer integrity, and exhibited lower levels of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), while up-regulating colonic anti-inflammatory cytokine IL-10 levels, tight junction proteins (E-cadherin, zonulae occludens (ZO)-1, occludin and claudin-1) and mucin (MUC1 and MUC2) mRNA expressions to some extent. In addition, mixed lactobacilli showed better anti-inflammatory effects than single-strain treatment. Our study further revealed that mixed lactobacilli increased bacterial diversity and improved gut microbiota composition, increasing short-chain fatty acid (SCFA) production. These results indicated that mixed lactobacilli supplementation could attenuate DSS-induced colitis by modulating the gut microbiota and repairing the intestinal barrier, which provided a scientific basis for its clinical application in the future.

Effects of Lactobacillus acidophilus KLDS1.0901 on Proliferation and Apoptosis of Colon Cancer Cells.

Colon cancer is the most common type of malignant tumor. The cytotoxicity effect of lactic acid bacteria may be active by inhibiting cancer cell proliferation, producing anticancer compounds, and inducing apoptosis in cancer cells, but the mechanism is unclear. Our previous study revealed that Lactobacillus acidophilus KLDS1.0901 has good probiotic properties. In this study, We screened out the highest inhibition rate of L. acidophilus KLDS1.0901 and assessed the effects on the proliferation of HT-29, Caco-2, and IEC-6 cells. Then, the apoptosis mechanism of HT-29 cells was studied when treated with L. acidophilus KLDS1.0901. Results showed that L. acidophilus KLDS1.0901 inhibited the proliferation of HT-29 and Caco-2 cells in a dose-dependent manner and reached the maximum under the condition of multiplicity of infection (MOI) = 100 (rate of Lactobacillus to cells) at 48 h. With the increase in time and MOI, reactive oxygen species in HT-29 cells, the apoptosis rates of HT-29 cells were increased, and the amount of blue fluorescence of the cells was also increased after Hoechst 33258 staining. Furthermore, L. acidophilus KLDS1.0901 reduced the mitochondrial membrane potential of HT-29 cells. Notably, 1,133 differentially expressed genes were screened by transcriptomics research, including 531 up-regulated genes and 602 down-regulated genes. These genes were involved in the nuclear factor κB and PI3K-AKT signaling pathways related to the apoptosis of HT-29 cells. These findings suggested that L. acidophilus KLDS1.0901 has the potential to be used in the development of a new type of functional foods for adjuvant treatment of colon cancer.

Distinct Gut Microbiota and Metabolite Profiles Induced by Different Feeding Methods in Healthy Chinese Infants.

Human milk is closely correlated with infant gut microbiota and is important for infant development. However, most infants receive exclusively insufficient breast milk, and the discordance between effects of commercial formula and human milk exists. To elucidate the differences induced by various feeding methods, we determined microbiota and metabolites composition in fecal samples from 77 healthy infants in Northeast China and identified the differences in various feeding methods. Bacterial 16S rRNA gene sequence analysis demonstrated that the fecal samples of exclusively breastfed (BF) infants were abundant in Bifidobacterium and Lactobacillus; the mixed-fed (MF) infants had the highest abundance of Veillonella and Klebsiella; the exclusively formula-fed (FF) infants were enriched in Bacteroides and Blautia; and the complementary food-fed (CF) infants were associated with higher relative abundance of Lachnoclostridium and Akkermansia. Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics data revealed that the fecal samples of BF infants had the highest abundance of dl-citrulline, threonine, l-proline, l-glutamine, guanine, and l-arginine; the MF infants were abundant in d-maltose, stearidonic acid, capric acid, and myristic acid; the FF infants were enriched in itaconic acid, 4-pyridoxic acid, prostaglandin B2, thymine, dl-α-hydroxybutyric acid, and orotic acid; and the CF infants were associated with higher relative abundance of taurine, l-tyrosine, adenine, and uric acid. Furthermore, compared with the BF infants, the MF and FF infants were more abundant in fatty acid biosynthesis. Collectively, these findings will provide probable explanations for some of the risks and benefits related to infant feeding methods and will support a theoretical basis for the development of infant formula.

Lactobacillus acidophilus alleviates type 2 diabetes by regulating hepatic glucose, lipid metabolism and gut microbiota in mice.

Diabetes, an endocrine and metabolic disorder, has become the third most non-infectious chronic disease that threatens human health. Type 2 diabetes (T2D) accounts for more than 90% of diabetic patients, mainly caused by environmental factors. Lactic acid bacteria (LAB) exhibit several health benefits to the host including regulating glucose and lipid metabolism and improving oxidative stress and inflammatory response. However, the anti-diabetic mechanism of probiotics has not been elucidated clearly. In this study, the anti-diabetic effects of Lactobacillus acidophilus KLDS1.1003 and KLDS1.0901 on T2D mice were assessed. Oral administration of L. acidophilus KLDS1.1003 and KLDS1.0901 for 6 weeks significantly improved the epithelial barrier function, which in turn lowered inflammation cytokines, including IL-8, TNF-α and IL-1ß in liver and colon tissue, and prevented liver and colon tissue injuries to some extent. Additionally, L. acidophilus treatment regulated the expression genes that are related to glucose and lipid metabolism. The two tested strains down-regulated the expression of glycogen synthase kinase 3ß (GSK-3ß), fatty acid synthase (FAS) and sterol regulatory element-binding transcription factor 1c (SREBP-1c), and up-regulated the expression of protein kinase B (Akt). However, L. acidophilus KLDS1.0901 is better for improving T2D than L. acidophilus KLDS1.1003. Further research showed that L. acidophilus KLDS1.0901 supplementation could reshape gut microbiota, increasing short chain fatty acid-producing bacteria (Blautia, Roseburia and Anaerotruncus) and the level of SCFAs and decreasing the relative abundance of Gram-negative bacteria such as Desulfovibrio, Alistipes and Bacteroides. Notably, L. acidophilus KLDS1.0901 treatment restored the structure of gut microbiota similar to the control group. These findings suggested that L. acidophilus KLDS1.0901 might be used as a new type of antidiabetic drug candidate.

Screening for Potential Novel Probiotics With Dipeptidyl Peptidase IV-Inhibiting Activity for Type 2 Diabetes Attenuation in vitro and in vivo.

Diabetes has become the second most severe disease to human health. Probiotics are important for maintaining gastrointestinal homeostasis and energy balance and have been demonstrated to play a positive role in the prevention and treatment of metabolic syndromes, such as obesity, inflammation, dyslipidemia, and hyperglycemia. The objective of this study was to screen potential antidiabetic strains in vitro and evaluate its effects in vivo. For the in vitro section, dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant activities of 14 candidate Lactobacillus spp. strains were tested. Then hydrophobicity and acid and bile salt tolerance assays were determined. The most promising in vitro strain was further evaluated for its antidiabetic properties in vivo using type 2 diabetes mice induced by high-fat diet and intraperitoneal injection of streptozotocin (STZ). The reference strain for this study was Lactobacillus rhamnosus GG. Results showed that cell-free excretory supernatants and cell-free extracts of Lactobacillus acidophilus KLDS1.0901 had better DPP-IV inhibitory activity, antioxidative activities, and biological characteristics than other strains. At the end of the treatment, we found that L. acidophilus KLDS1.0901 administration decreased the levels of fasting blood glucose (FBG), glycosylated hemoglobin, insulin in serum and AUCglucose, and increased the level of glucagon-like peptide 1 in serum compared with diabetic mice (p < 0.05). Moreover, L. acidophilus KLDS1.0901 supplementation increased the activities of superoxide dismutase, glutathione peroxidase, the level of glutathione, and reduced the level of malondialdehyde in serum. These results indicated that L. acidophilus KLDS1.0901 could be used as a potential antidiabetic strain; its application as food supplement and drug ingredient is thus recommended.