Metagenomics Reveals the Diversity and Taxonomy of Carbohydrate-Active Enzymes and Antibiotic Resistance Genes in Suancai Bacterial Communities.

Suancai, as a traditional fermented food in China with reputed health benefits, has piqued global attention for many years. In some circumstances, the microbial-driven fermentation may confer health (e.g., probiotics) or harm (e.g., antibiotic resistance genes) to the consumers. To better utilize beneficial traits, a deeper comprehension of the composition and functionality of the bacterial species harboring enzymes of catalytically active is required. On the other hand, ingestion of fermented food increases the likelihood of microbial antibiotic resistance genes (ARGs) spreading in the human gastrointestinal tract. Besides, the diversity and taxonomic origin of ARGs in suancai are little known. In our study, a metagenomic approach was employed to investigate distribution structures of CAZymes and ARGs in main bacterial species in suancai. Functional annotation using the CAZy database identified a total of 8796 CAZymes in metagenomic data. A total of 83 ARGs were detected against the CARD database. The most predominant ARG category is multidrug-resistant genes. The ARGs of antibiotic efflux mechanism are mostly in Proteobacteria. The resistance mechanism of ARGs in Firmicutes is primarily antibiotic inactivation, followed by antibiotic efflux. Due to the abundance of species with different ARGs, strict quality control including microbial species, particularly those with lots of ARGs, is vital for decreasing the risk of ARG absorption via consumption. Ultimately, we significantly widen the understanding of suancai microbiomes by using metagenomic sequencing to offer comprehensive information on the microbial functional potential (including CAZymes and ARGs content) of household suancai.

Metagenomic insights into Chinese northeast suancai: Predominance and diversity of genes associated with nitrogen metabolism in traditional household suancai fermentation.

Chinese northeast suancai represents a typical and valuable food product that has been handed down by traditional household procedures over centuries. Nitrite is formed and accumulated during the suancai fermentation process and commonly causes food safety problems. The biogeochemical cycle of nitrite may provide a reference and guidance for the enzymatic degradation of nitrite in fermented food. The potential nitrogen metabolic pathways in the microbially driven suancai fermentation were reasonably inferred through monitoring nitrogen conversions and detecting the genes of different functional enzymes. Complex microbial metabolism is responsible for the unique nitrogen conversions during suancai fermentation. The metagenomic results showed that Pseudomonas with nitrate reductase genes (narG, narH, narI) and nitrite reductase genes (nirB, nirD) contributed the most to both nitrite reduction and nitrate reduction. The majority of the sequences of nitrate reductase and nitrite reductase were derived from the families of Pseudomonadaceae, Erwiniaceae and Yersiniaceae. According to the physicochemical analysis, the nitrite concentration of the fermentation broth reached the peak value (0.48 mM) and gradually decreased to the minimum (0.02 mM). The downward trend of the pH and nitrite concentration were closely associated with the nitrite enzymatic degradation period before the acid degradation period. Our results indicated that nitrite removal in suancai fermentation involved the reduction of nitrite to ammonia and denitrification, which were mainly contributed by the reduction of nitrite to ammonia mediated by the nirB/nirD enzyme (Indentified ECs: 1.7.1.15). This research offers new insights into the metagenome-based bioinformatic roles of the previously unstudied microorganisms in spontaneous suancai fermentation for the enzymatic degradation of nitrite. It provides helpful basis for the detection and even elimination of nitrite in suancai and for improving the safety level of suancai.

Laminar Inflammation Responses in the Oligofructose Overload Induced Model of Bovine Laminitis.

Bovine laminitis causes substantial economic losses and animal welfare problems in dairy farms worldwide. Previously published studies have reported that the inflammatory response plays a central role in the pathogenesis of the disease. To our knowledge, inflammation associated with bovine laminitis induced by high levels of exposure to oligofructose (OF) has not been reported and characterized. In fact, the disease manifestations in this model closely approximate those of clinical laminitis. The objective of this study was to characterize the inflammatory response in OF-induced bovine laminitis. A total of 12 Chinese Holstein dairy heifers were utilized in this study. The heifers were randomly divided into two groups, treatment (n = 6) and control (n = 6). The treatment group heifers were administered OF solutions via a stomach tube (dose: 17 g/kg of body weight). Upon development of a lameness score of 2 with consecutive positive reactions in the same claw, they would be humanely euthanized. Control heifers were administered deionized water (dose: 2 L/100 kg of body weight) and humanely euthanized at 72 h. Real-time quantitative PCR (qPCR) assays were performed to determine the messenger RNA (mRNA) concentrations of inflammatory mediators in the lamellae. Concentrations of interleukin (IL)-1ß, IL-6, IL-8, C-X-C motif chemokine ligand-1 (CXCL-1), macrophage cationic peptide-2 (MCP-2), E-selectin, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase-1 (iNOS-1), and plasminogen activator inhibitor-1 (PAI-1) were significantly increased (P < 0.05) in the treatment group. No significant difference was found for tumor necrosis factor alpha (TNF-α), IL-10, CXCL-6, and MCP-1. These results demonstrated and characterized the laminar inflammatory response leading to the pathogenesis of bovine laminitis at the early stages.

Dynamic ROS Production and Gene Expression of Heifers Blood Neutrophil in a Oligofructose Overload Model.

Alimentary oligofructose (OF) overload can induce several diseases in cattle, such as ruminal acidosis, laminitis, and synovitis. The role of blood polymorphonuclear neutrophil (PMN) remains unclear during OF overload. The aim of this study was to investigate the dynamic changes in reactive oxygen species (ROS) production and the expression profile of genes in blood PMN in a model of OF overload. Twelve clinically healthy and non-pregnant Chinese Holstein heifers, aged between 18 and 26 mo, weighing 335-403 kg, BCS (5-point scale) ranges 2.7-3.3 were used for the experiments. OF heifers (n = 6) received 17 g/kg of BW oligofructose dissolved in 2 L/100 kg of BW tap water and the CON heifers (n = 6) received 2 L/100 kg of BW tap water. Blood PMN was isolated for each heifer 0, 6, 12, 18, 24, 36, 48, 60, and 72 h after administration. PMN was analyzed either by endogenous and phorbol myristate acetate (PMA)-induced ROS production or by quantitative real-time PCR. After 12 h, PMA-induced ROS production decreased, which was sustained until 48 h. The expressions of inflammation markers (IL1α, IL1ß, IL6, IL10, TNFα, STAT3, TLR4, MMP9, and HP) and eicosanoids (ALOX5, ALOX5AP, and PLA2G4A) were upregulated. The expression of adhesion and migration (CXCR2, CXCL8, CD62L, ITGA4, ITGAM, and ITGB2) in OF heifers was increased compared with CON heifers. The expression of oxidative stress (SOD2 and S100A8) was upregulated, while SOD1 and MPO were downregulated. In metabolism and receptor genes, the expressions of GRα and INSR decreased after 12 h, while Fas increased until 6 h and then decreased at 18 h. The expression of LDHA and PANX1 did not show any differences after OF overload. These findings indicate that OF overload induced systemic activation of PMN, which provides a step toward a better understanding of the role of innate immune responses in response to oral OF administration.

The changes of inflammatory mediators and vasoactive substances in dairy cows' plasma with pasture-associated laminitis.

BACKGROUND: Hoof disease is one of the three major diseases that often occur in dairy cows. The impact of this disease on dairy farming is second only to mastitis. Laminitis is a diffuse, aseptic, serous, non-purulent inflammation of the dermal papillae and vascular layers of the cow's hoof wall. In the pasture, laminitis occurs mostly in the laminae, that is, inside the hoof shell. No lesions can be seen on the surface. Therefore, laminitis cannot attract the attention of veterinarians. However, laminitis has become a major factor that seriously affects the health and welfare of dairy cows, making it an important cause of hindering the performance of dairy cows. METHODS: The study was conducted at a dairy farm in Harbin, Heilongjiang province, China. We selected a sample of the laminitis cows based on the veterinary diagnosis, took blood from the jugular vein and then separated the plasma, and measured the index with the Elisa kit. In this study, the markers of inflammatory and vasoactive substances status in dairy cows consisted of subclinical laminitis (SCL, n = 20), chronic laminitis (CL, n = 20) and healthy dairy cows (CON, n = 20) under the local management conditions were investigated. RESULTS: Compared with healthy cattle, HIS, IL-6, LPS, and TNF-α in subclinical laminitis group significantly increased (P < 0.05), especially HIS, LPS, TNF-α (P < 0.01); in chronic laminitis cows, COX-2, HIS, IL-6, LPS, and TNF-α increased significantly (P < 0.05), especially COX-2, HIS, TNF-α (P < 0.01). iNOS (P < 0.05), TXB2 (P < 0.01) in chronic laminitis cows had significantly increased. CONCLUSION: This study reported for the first time that pasture laminitis was divided into subclinical laminitis and clinical chronic laminitis. Through research on the inflammatory factors and vasoactive substances of dairy cows, it is found that there is a close relationship between them, which affects the metabolic cycle of dairy cows. These indicators are abnormally expressed and cause hoof microcirculation disorders.

Purification and immobilization of α-amylase in one step by gram-positive enhancer matrix (GEM) particles from the soluble protein and the inclusion body.

Immobilization of the enzyme benefits the catalytic industry a lot. The gram-positive enhancer matrix (GEM) particles could purify and immobilize the recombinant α-amylase in one step without changing the enzymatic character. The enzyme immobilized by GEM particles exhibited good reusability and storage stability. The denaturants dissolved some of the GEM particles and a part of the GEM particles could bear the denaturants. The GEM particles had strong binding ability to the recombination protein with the AcmA tag even when the denaturants existed. The inclusion body was dissolved by urea and then bound by the GEM particles. The GEM particles binding the recombination protein were separated by centrifugation and resuspended in the renaturation solution. GEM particles were recycled by repeating the boiling procedure used in preparing them. The recombination α-amylase without any tag was obtained by digestion and separated via centrifugation. Altogether, our findings suggest that GEM particles have the potential to function as both immobilization and purification materials to bind the soluble recombinant protein with the AcmA tag and the inclusion body dissolved in the denaturants.

Purification and immobilization of the soluble and insoluble portions of recombinant lipase by gram-positive enhancer matrix (GEM) particles.

Lipases are important enzyme for industries. In this work, the recombinant lipase with an AcmA tag working as purification and immobilization tag was expressed in Escherichia coli. Gram-positive enhancer matrix (GEM) particles work to purify and immobilize the recombinant lipase. GEM particles are produced by boiling the cells of Lactococcus lactis NZ9000 to remove the DNA and most proteins. GEM particles specifically bind protein with the AcmA tag in the C-terminal. The recombinant lipase was in two forms, the soluble part and the inclusion body. GEM particles could purify and immobilize the lipase from the soluble part in one step. After the inclusion body being dissolved by 8 M urea, the enzyme activity was recycled by the GEM particles. The GEM particles could immobilize over 75% of the enzyme activity. The lipase immobilized was a basophilla enzyme with the optimal temperature was 30 °C. The activity of the lipase immobilized was 47.1U/OD600 GEM particles at optimal conditions. The enzyme catalysis did not need the ions added to improve the activity. The GEM particles had excellent enzyme activity reusability.

Production optimization, partial characterization and properties of an exopolysaccharide from Lactobacillus sakei L3.

The production optimization, partial characterization and properties of an exopolysaccharide (EPS) from Lactobacillus sakei L3 were performed. Sucrose concentration, initial pH and inoculation volume were the most significant variables that improved the L3 EPS production in a three-level Plackett-Burman design. The maximum EPS yield of 69.65 g/L (2.16-fold greater than the original yield) was obtained under optimal conditions of 127.80 g/L sucrose, initial pH 6.87 and 3.15% inoculation volume. Chemical analyses revealed that the observed EPS bioactivity was mainly due to the sugar moieties. The chain conformation was characterized by the Congo red test, a ß-elimination reaction and circular dichroism, which indicated that the L3 EPS exhibited a random coil structure and O-linkages in aqueous solution, and the EPS concentration did not alter the EPS chain conformation but did modify the hydrogen-bond interactions and chirality of the polysaccharide. The purified L3 EPS exhibited high water solubility, probiotic effects and emulsification activity. The L3 EPS at 2.0 mg/mL formed a highly active and stable emulsion with sunflower oil, with EA, ES24, ES48 and ES72 indices of 62.30 ±â€¯0.06%, 57.95 ±â€¯0.18%, 42.73 ±â€¯0.26% and 43.05 ±â€¯0.08%, respectively. These results collectively describe a high-yield EPS with unique characteristics for exploitation in large-scale industrial food applications.

Isolation and characterization of dextran produced by Lactobacillus sakei L3 from Hubei sausage.

An exopolysaccharide (EPS)-producing bacterial strain L3 was isolated from Hubei sausage and identified as Lactobacillus sakei via morphological, physiological, biochemical and 16S rDNA analysis. FT-IR spectroscopy and NMR revealed that L3 EPS was a dextran containing d-glucose residues with α-1,6 glycosidic linkage. Rheological studies showed that it had high viscosity at high concentration, low temperature, and acidic pH (pH 3.0). Scanning electron microscopy of the L3 dextran demonstrated a porous and branched morphology, and atomic force microscopy showed lumps of varying height on the rough surface of the L3 EPS polymer. The EPS was thermally stable up to 272°C and could coagulate sucrose-supplemented milk. Together, these results suggested that L3 EPS might have potential applications in food processing and other areas.

Secretion of the recombination α-amylase in Escherichia coli and purification by the gram-positive enhancer matrix (GEM) particles.

α-Amylases are important enzymes in industry. A recombinant α-amylase with a secretion signal peptide and an AcmA tag was expressed in Escherichia coli to improve the yield. The induction concentrations were optimized, and the temperature had a significant influence on soluble expression and secretion. A visible band could be obtained when the induction was conducted at 16 °C. The gram-positive enhancer matrix (GEM) particles could separate and purify the recombinant α-amylase with the AcmA tag, and no visible band could be seen in the culture even after the culture was concentrated ten times. The solution and concentration of the recombinant α-amylase could be adjusted by GEM particles. The recombinant untagged α-amylase was obtained after digestion. The α-amylase was characterized. The recombinant α-amylase was a thermophilic enzyme with a broad pH tolerance. In addition, the enzyme activity of the recombinant α-amylase was independent of Ca2+. The recombinant α-amylase contained the OmpA signal peptide and the AcmA tag and was expressed and purified quickly and easily.

Optimization and partial characterization of ca-independent α-amylase from Bacillus amyloliquefaciens BH1.

Strain Bacillus amyloliquefaciens BH1 was evaluated for the generation of α-amylase. Culture conditions and medium components were optimized by a statistical approach for the optimal generation of α-amylase with response surface methodology (RSM) method. The Plackett-Burman (PB) design was executed to select the fermentation variables and Central composite design (CCD) for optimizing significant factors influencing production. The optimum levels for highest generation of α-amylase activity (198.26 ± 3.54 U/mL) were measured. A 1.69-fold improve generation was acquired in comparison with the non-optimized. Partial characterization of the α-amylase indicated optimal pH and temperature at 7.0 and 40 °C, respectively. Crude α-amylase maintained a constant pH range 5.0-8.0 and 30-70 °C. The α-amylase was independent of Ca2+, and the activity was inhibited by Fe3+, Co2+, Cu2+, and Hg2+. The thermo and pH stability of the α-amylase indicate its extensive application in the food and pharmaceutical industries.

Purification, characterization and antioxidant activity of dextran produced by Leuconostoc pseudomesenteroides from homemade wine.

An exopolysaccharide (EPS) was produced by Leuconostoc pseudomesenteroides DRP-5 isolated from homemade wine. The EPS was obtained with ethanol extraction, which was further purified by chromatography of Sephadex G-100 to get a purified fraction. The monosaccharide composition of the EPS was glucose, and its molecular weight (Mw) was 6.23 × 106 Da, as determined by gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance spectra (NMR) showed that the EPS was a linear glucan with α-(1→6)-linked glucosidic bonds. The water holding capacity (WHC), water solubility index (WSI) and emulsifying activity (EA) of DRP-5 EPS were 296.76 ±â€¯18.93%, 98.62 ±â€¯3.57% and 87.22 ±â€¯2.18%, respectively. DRP-5 EPS have a higher degradation temperature of 278.36 °C, suggesting high thermal stability of the EPS. Also, DRP-5 EPS was found to have moderate 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide anion radical, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical, Fe2+ scavenging activities and reducing power. All these characteristics suggest that DRP-5 EPS might have potential applications in the pharmaceutical, cosmetic and food industries.

Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH072.

In the present study, a novel α-amylase produced by Bacillus amyloliquefaciens BH072 was purified and characterized. The molecular weight of purified α-amylase was approximately 68 kDa, determined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and ten amino acid of N-terminal was NSGLNGYLTH. The kinetic parameters Km and Vmax were 4.27 ±â€¯0.21 mg/mL and 987.34 ±â€¯23.34 U/mg, respectively. Purified α-amylase showed maximal activity at pH 7 and 60 °C. Enzyme remained stable in pH range 6.0-7.0 and 50-80 °C. The activity of the α-amylase was Ca2+ independent and stability in the presence of surfactant, oxidizing and bleaching agents. The ß-mercaptoethanol and EDTA greatly enhanced and reduced α-amylase activity, respectively. This enzyme has high hydrolysis rate toward corn, wheat and potato starch and hydrolyzes soluble starch to glucose, maltose, maltotriose and maltotetraose, indicating that the α-amylase represents a promising candidate for applications in the food industry.