Lactobacillus paracasei TK1501 fermented soybeans alleviate dextran sulfate sodium-induced colitis by regulating intestinal cell function.

BACKGROUND: Probiotic food provide health benefits by regulating intestinal floras via live bacteria, but the shelf life is short and the preservation condition is demanding due to the bacteria being fragile. Owing to these problems, we have tried to find a fermented food that is helpful for inflammatory bowel disease treatment but independent of live bacteria. In addition, the mechanisms of fermented food were investigated. Dextran sulfate sodium was used to model inflammatory bowel disease in mice, and Lactobacillus paracasei TK1501 fermented soybeans and their metabolites were used to treat inflammatory bowel disease. RESULTS: In this study, TK1501 fermented soybean alleviated colitis. However, the efficacy was associated with bacterial metabolites but not live or dead bacteria. Compositional analysis of soybean before and after fermentation shows that soy carbohydrates were used for bacteria growth and produced functional substances. Further, we display the main active ingredient was lipoteichoic acid and peptidoglycan, because lipoteichoic acid reduced the colonic macrophage and peptidoglycan may increase the mucin-2 expression. A cell experiment displayed that lipoteichoic acid could enhance the phagocytosis of macrophages. CONCLUSION: In general, TK1501 fermented soybean alleviating colitis is dependent on metabolites of TK1501, particularly lipoteichoic acid and peptidoglycan. The fermented food may have a long shelf life and lax storage condition. © 2023 Society of Chemical Industry.

Complete Genome Sequence of a Novel Lactobacillus paracasei TK1501 and Its Application in the Biosynthesis of Isoflavone Aglycones.

Lactobacillus strains are considered safe and healthy probiotics for manufacturing "natural food" products; this is due to their ability to produce bioactive compounds that reduce the incidence of various human diseases. Lactobacillus paracasei TK1501 is a novel probiotic strain isolated from naturally fermented congee; and can produce a high yield of genistein, one of the most widely studied isoflavone aglycones with plenty of physiological functions. To better understand the molecular basis of isoflavone aglycones biosynthesis, the complete 2,942,538 bp genome of L. paracasei TK1501 was sequenced and assembled; a group of genes that are involved in isoflavone aglycones production were identified. Of note, a ß-glucosidase was analyzed in the L. paracasei TK1501. Moreover, we also found that L. paracasei TK1501 could be used in soymilk fermentation; which would remarkably increase the contents of genistein, daidzein, and glycitein. This work was meaningful to the application of L. paracasei TK1501 and the molecular mechanism analysis of isoflavone aglycones biosynthesis in Lactobacillus strains.

Oligosaccharide and short-chain fatty acid: A double-edged sword in obese mice by regulating food intake and fat synthesis.

Dietary fiber has been used to prevent obesity by regulating the intestinal flora and promoting the production of short-chain fatty acids (SCFAs). However, it is insufficient to conclude the decisive role of microbiota and SCFAs by adding oligosaccharides to foods without caloric balance. In this study, the effects of oligosaccharides and their regulated microflora on the development of obesity in mice were studied by designing a high-fat diet with equal calories but different contents of oligosaccharides. Isocaloric diets demonstrated that appropriate rather than excess oligosaccharides prevent obesity by regulating appetite. Such an appetite was inhibited by oligosaccharides but promoted by SCFAs. Furthermore, promoted appetite was tightly related to decreased insulin and increased acyl-CoA binding protein, which was correlated with SCFA-induced fat degradation. Interestingly, drinking butyrate alleviated obesity even with higher calorie intake. Molecular docking demonstrated that conversion of butyrate to butyryl-CoA converted from butyrate, as a structural analog of acetyl-CoA, inhibits the activity of acetyl-CoA carboxylase. Together, these findings demonstrate that fermentable fiber supplements may have limits in obesity treatment, and we provide possible obesity therapeutic targets that inhibit bacterial fermentation or increase the ratio of butyrate/acetate.

Dysregulation of intestinal flora: excess prepackaged soluble fibers damage the mucus layer and induce intestinal inflammation.

Soluble fiber is commonly used as a dietary supplement to improve intestinal flora, and many prepackaged products are sold in the market. However, whether these prepared soluble fibers are harmless for intestinal flora has not been systematically evaluated. Here, we assessed the dose-effect of fructooligosaccharides (FOSs) on obesity and intestinal flora using a mouse model. Gavage of low- and medium-dose FOS improved the microbiota in high-fat diet fed mice, but high-dose FOS leads to intestinal flatulence, diarrhea and flora disorders, including an increase in Akkermansia muciniphila and Clostridium difficile, which disrupt the mucus barrier and cause intestinal inflammation. Besides, a high dose of xylooligosaccharide by gavage induces symptoms similar to those of FOS in mice. These adverse effects can be alleviated by regulating intestinal flora. In addition, we experimentally proved that supplementary probiotics protect against the negative effects of FOS in obese mice. Therefore, prepackaged soluble fiber supplements need to be taken with caution, and excessive consumption of soluble fibers results in intestinal dysfunction and even induces intestinal inflammation. Combining probiotics and soluble fiber can be considered if necessary.

Dietary soybeans worsen dextran sodium sulfate-induced colitis by disrupting intestinal ecology.

Food mediates susceptibility to inflammatory bowel diseases (IBDs) associated with the microbiome. Existing studies suggest that a high-sugar and high-fat diet promotes IBDs, but whether a plant-based diet is fully harmless to IBD improvement remains unknown. In this study, for the first time, we assessed the effect of soybean and its carbohydrates on dextran sodium sulfate (DSS)-induced colitis. In a DSS-induced colitis mouse model (BALB/C WT), the oral administration of soybeans worsened colitis, which was associated with higher disease activity index, histology score and expression of pro-inflammatory cytokines, and lower expression of anti-inflammatory cytokines. Here, 16S rRNA sequencing and elimination of gut bacteria by antibiotics showed that the exacerbating colitis caused by soybeans depends on the changes in the intestinal flora. Furthermore, the gavage of soybean carbohydrates such as sucrose and raffinose-family oligosaccharides altered the intestinal microbiota and worsened inflammation. When co-cultured with macrophages (RAW 264.7), the metabolites of the disordered intestinal flora, isolated Escherichia coli and purified LPS showed high macrophage toxicity to inhibit pathogen clearance. These results indicate that the intake of soybeans and soybean carbohydrates is not conducive to recovery from IBDs based on changes in gut microbiota and metabolites affecting the activities of macrophages.

Development of a Novel Nomogram for Predicting Premature Rupture of Membrane in Pregnant Women With Vulvovaginal Candidiasis.

Objective: The aim of this study was to develop a nomogram to predict the risk of premature rupture of membrane (PROM) in pregnant women with vulvovaginal candidiasis (VVC). Patients and methods: We developed a prediction model based on a training dataset of 417 gravidas with VVC, the data were collected from January 2013 to December 2020. The least absolute shrinkage and selection operator regression model was used to optimize feature selection for the model. Multivariable logistic regression analysis was applied to build a prediction model incorporating the feature selected in the least absolute shrinkage and selection operator regression model. Discrimination, calibration, and clinical usefulness of the prediction model were assessed using the C-index, calibration plot, and decision curve analysis. Internal validation was assessed using bootstrapping validation. Results: Predictors contained in the prediction nomogram included age, regular perinatal visits, history of VVC before pregnancy, symptoms with VVC, cured of VVC during pregnancy, and bacterial vaginitis. The model displayed discrimination with a C-index of 0.684 (95% confidence interval: 0.631-0.737). Decision curve analysis showed that the PROM nomogram was clinically useful when intervention was decided at a PROM possibility threshold of 13%. Conclusion: This novel PROM nomogram incorporating age, regular perinatal visits, history of VVC before pregnancy, symptoms with VVC, cured of VVC during pregnancy, and bacterial vaginitis could be conveniently used to facilitate PROM risk prediction in gravidas.

Co-fermentation of lentils using lactic acid bacteria and Bacillus subtilis natto increases functional and antioxidant components.

We identified lentil products with both nutritional value and antioxidant capacity by studying the changes of probiotics and functional substances during single fermentation with lactic acid bacteria (LAB) or co-fermentation using LAB and Bacillus subtilis natto. After fermentation, the best growth of LAB was observed in anaerobic solid-state co-fermentation, whereby the viable counts of Lactobacillus plantarum TK9 and Lactobacillus paracasei TK1501 reached 2.77 × 109 and 2.78 × 109 CFU/g, respectively. Furthermore, the total phenol and genistin content produced by the two mixed groups, respectively, increased by 0.52- and 0.66-fold, as well as 0.63- and 0.64-fold, compared with unfermented samples. Similarly, the free amino acid content increased by 0.53- and 0.49-fold, respectively. The 50% inhibitory concentrations for the radical-scavenging against 1,1-diphenyl-2-picrylhydrazyl and α-glucosidase inhibitory activity were lower following anaerobic co-fermentation. Consistently, products of anaerobic mixed solid-state fermentation had higher oxygen radical absorbance capacity. Therefore, anaerobic solid-state co-fermentation of lentils using B. subtilis natto may promote the multiplication of LAB and enhance the antioxidant activity of fermented lentil products. PRACTICAL APPLICATION: Simple and efficient food handling is more suitable for industrial production. Co-fermentation is a good method to optimize the fermentation process. Co-culture technology has high potential in terms of functionality and antioxidant capacity.

Rational design of a Yarrowia lipolytica derived lipase for improved thermostability.

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 °C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 °C, which was about 5.0 °C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5 U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.

Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy.

To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type.

Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing.

A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp-deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amyL and chiA (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis, and offers promise for applications in genetic modification of other industrially relevant Bacillus species.

A Novel Antidiabetic Food Produced via Solid-State Fermentation of Tartary Buckwheat by L. plantarum TK9 and L. paracasei TK1501.

Diabetes is a chronic metabolic disease characterized by hyperglycaemia and a number of potential complications that significantly reduce the patient's quality of life. In this study, we produced an antidiabetic functional food from Tartary buckwheat fermented by Lactobacillus plantarum TK9 and L. paracasei TK1501. The results of an orthogonal experimental design indicated that the three factors with the largest effects on the growth of L. plantarum TK9 and L. paracasei TK1501 in solid-state fermentation (SSF) were in the order: water ratio>inoculum size>time of fermentation. Under the optimal fermentation conditions comprising a 1:1.5 water ratio, 24 h of SSF and a 107 CFU/g inoculum, the Tartary buckwheat fermented by L. plantarum TK9 and L. paracasei TK1501 yielded viable probiotic counts of (2.3±0.7)·109 and (3.3±0.4)·109 CFU/g, respectively. The nutritional potential, as well as antioxidant and antidiabetic properties of ethanolic extracts from fermented Tartary buckwheat were investigated. The highest α-glucosidase inhibitory activity, with an IC50 of 0.51 mg/mL, was present in Tartary buckwheat fermented by L. plantarum TK9. However, Tartary buckwheat fermented by L. paracasei TK1501 had the highest dipeptidyl peptidase IV (DPP-IV) inhibition, with an IC50 of 2.47 mg/mL. Therefore, fermentation by both L. plantarum TK9 and L. paracasei TK1501 has the potential to yield a product that can help regulate the levels of blood glucose as part of a diabetic diet.

Manipulating the Bacterial Cell Cycle and Cell Size by Titrating the Expression of Ribonucleotide Reductase.

Understanding how bacteria coordinate growth with cell cycle events to maintain cell size homeostasis remains a grand challenge in biology. The period of chromosome replication (C period) is a key stage in the bacterial cell cycle. However, the mechanism of in vivo regulation of the C period remains unclear. In this study, we found that titration of the expression of ribonucleotide reductase (RNR), which changes the intracellular deoxynucleoside triphosphate (dNTP) pools, enables significant perturbations of the C period, leading to a substantial change in cell size and DNA content. Our work demonstrates that the intracellular dNTP pool is indeed an important parameter that controls the progression of chromosome replication. Specially, RNR overexpression leads to a shortened C period compared with that of a wild-type strain growing under different nutrient conditions, indicating that the dNTP substrate levels are subsaturated under physiological conditions. In addition, perturbing the C period does not significantly change the D period, indicating that these two processes are largely independent from each other. Overall, titration of ribonucleotide reductase expression can serve as a standard model system for studying the coordination between chromosome replication, cell division, and cell size.IMPORTANCE Bacteria must coordinate growth with cell cycle progression to maintain cell size hemostasis. Cell cycle and cell size regulation is a fundamental concern in biology. The period required for chromosome replication (the C period) is a key stage in the bacterial cell cycle. However, how the C period is controlled in vivo remains largely an open question in this field of bacterial cell cycle regulation. Through introducing a genetic circuit into Escherichia coli for titrating the expression of ribonucleotide reductase, we achieve substantial perturbation of the C period and cell size. Our work demonstrates that the intracellular dNTP pool is an important parameter that controls the progression of chromosome replication. Moreover, our work indicates that bacterial cells manage to maintain subsaturated dNTP levels under different nutrient conditions, leading to a submaximal speed of DNA replication fork movement.

Identification and characterization of the ficellomycin biosynthesis gene cluster from Streptomyces ficellus.

Ficellomycin is a peptide-like antibiotic which exhibits potent in vitro activity against Penicillium oxalicum and Staphylococcus aureus, even against strains resistant to most clinically used antibiotics. The gene cluster responsible for ficellomycin biosynthesis was cloned from Streptomyces ficellus and sequenced. It was found to contain 26 ORFs and is located within 30 kb of contiguous DNA. Targeted disruption of the encoding genes revealed that most were involved in the functional section of ficellomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of ficellomycin 2-[4-guanidyl-1-azabicyclo[3.1.0]hexan-2-yl] glycine (2-GAHG). Within the 2-GAHG synthesis pathway, a sulfate adenylyltransferase appears to be involved in the synthesis of the pharmaceutically important 1-azabicyclo[3.1.0]hexane ring moiety, which has been reported to cause DNA cross-linking or impairment of semiconservative DNA replication.

Solid-state fermentation of whole oats to yield a synbiotic food rich in lactic acid bacteria and prebiotics.

This study developed a synbiotic food through the fermentation of whole oat flour with Lactobacillus plantarum TK9 and Bifidobacterium animalis subsp. lactis V9. The physicochemical properties, changes in ingredients and peptide molecular weight distributions were determined during the whole oat fermentation. The highest viable counts of the fermented oats were 2.85 × 10(9) CFU g(-1) (L. plantarum TK9) and 3.17 × 10(8) CFU g(-1) (Bif. animalis subsp. lactis V9), with the titratable acidity increased to 10.01 and 8.40 mL at the end of the fermentation. By comparing the nutrition compositions between the fermented and non-fermented oat flour, we found that there was almost no change in the soluble dietary fiber and ß-glucan content. However, the amounts of free amino nitrogen increased from 110.84 to 154.62 mg per 100 g (L. plantarum TK9) and 82.16 to 104.83 mg per 100 g (Bif. animalis subsp. lactis V9). The levels of oat peptides with molecular weights less than 6000 Da increased by 4.4 and 5.96%, respectively. The results suggest that the fermented whole oat flour has good potential for application in the production of a novel synbiotic food rich in lactic acid bacteria and ß-glucan prebiotics.

Comparison of two nutrition assessment tools in surgical elderly inpatients in Northern China.

BACKGROUND & OBJECTIVE: Nutrition assessment enables early identification of malnourished patients and those at risk of malnutrition. To determine the prevalence of malnutrition, to analyze the correlation between short-form Mini Nutritional Assessment (MNA-SF) and Nutritional Risk Screening 2002 (NRS2002) with classical nutritional markers among elderly hospitalized patients in surgery departments, with a view to improving nutrition advice for these patients. METHODS: A total of 142 elderly patients admitted for surgery were enrolled in the study. Within 48 hours of admission, MNA-SF and NRS2002 scale, anthropometric measures and biochemical tests were carried out to assess the nutritional status of each patient. RESULTS: The prevalence of malnutrition classified by MNA-SF, NRS2002, BMI, serum albumin, hemoglobin, total lymphocyte count, handgrip strength, calf circumference and mid-arm circumference were 45%, 38%, 17%, 22%, 24%, 71%, 36%, 12 % and 15 %, respectively. As the nutritional status classified by both MNA-SF and NRS2002 deteriorated, BMI, serum albumin, hemoglobin, handgrip strength, mid-arm circumference and calf circumference of patients with malnutrition were lower (P < 0.05). MNA-SF and NRS2002 had a unanimous correlation with classical nutritional markers (P < 0.05) except total lymphocyte count (P > 0.05). MNA-SF results showed a moderate agreement (P < 0.001) with NRS2002. Malnourished patients were older than well-nourished patients with NRS2002 (P < 0.05). Digestive disease patients tend to suffer from malnutrition, evaluated by MNA-SF (P < 0.05). CONCLUSIONS: The results show a relatively high prevalence of malnutrition among elderly patients in our general surgery department, especially in patients with digestive disease. NRS2002 and MNA-SF on elderly patients showed great consistency but significant difference in elderly patients with digestive disease. Both MNA-SF and NRS2002 correlated with each other and with BMI, serum albumin, hemoglobin, handgrip strength, calf circumference and mid-arm circumference. MNA-SF may be a more suitable tool for the nutrition assessment of surgical elderly inpatients.

Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus microsporus var. tuberosus.

Extracellular fibrinolytic enzyme from Rhizopus microsporus var. tuberosus was purified and characterised. The microorganism was isolated in a distillery from daqu, a fermentative agent used in the production of Chinese liquor and vinegar at different temperatures. The fibrinolytic enzyme was partially purified by ammonium sulphate precipitation, dialysis, DEAE Sepharose® Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of the fibrinolytic enzyme was estimated to be 24.5 kDa by SDS-PAGE. The purified enzyme showed optimal activity at pH=7.0 and 37 °C by fibrin plate method. It showed stronger resistance to the inhibition by trypsin and was stable at 37 °C retaining 96.1% residual activity after 4 h of incubation. The fibrinolytic activity of the enzyme was enhanced by Na+, Ca2+, Mg2+ and Mn2+. Conversely, Zn2+ and Cu2+ partly inhibited enzymatic activity. Using fibrin plate method, we found that the enzyme not only degrades fibrin directly, but also activates plasminogen into plasmin to degrade fibrin. The results indicate that the pure enzyme has a potential in dissolving blood clot, and the possibility for application in the treatment of thrombosis.

A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation.

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.