Fermentation of millet bran with Bacillus natto: enhancement of bioactivity levels and the bioactivity of bran extract.

BACKGROUND: Millet bran (MB), a byproduct of millet production, is rich in functional components but it is underutilized. In recent years, researchers have shown that fermentation can improve the biological activity of cereals and their byproducts. This study used Bacillus natto to ferment millet bran to improve its added value and broaden the application of MB. The bioactive component content, physicochemical properties, and functional activity of millet bran extract (MBE) from fermented millet bran were determined. RESULTS: After fermentation, the soluble dietary fiber (SDF) content increased by 92.0%, the ß-glucan content by 164.4%, the polypeptide content by 111.4%, the polyphenol content by 32.5%, the flavone content by 16.4%, and the total amino acid content by 95.4%. Scanning electron microscopy revealed that the microscopic morphology of MBE changed from complete and dense blocks to loosely porous shapes after fermentation. After fermentation, the solubility, water-holding capacity, and viscosity significantly increased and the particle size decreased. Moreover, the glucose adsorption capacity (2.1 mmol g-1), glucose dialysis retardation index (75.3%), and α-glucosidase inhibitory (71.4%, mixed reversible inhibition) activity of the fermented MBE (FMBE) were greater than those of the unfermented MBE (0.99 mmol g-1, 32.1%, and 35.1%, respectively). The FMBE presented better cholesterol and sodium cholate (SC) adsorption properties and the adsorption was considered inhomogeneous surface adsorption. CONCLUSION: Fermentation increased the bioactive component content and improved the physicochemical properties of MBE, thereby improving its hypoglycemic and hypolipidemic properties. This study not only resolves the problem of millet bran waste but also encourages the development of higher value-added application methods for millet bran. © 2024 Society of Chemical Industry.

Prevention of high-fat-diet-induced obesity in mice by soluble dietary fiber from fermented and unfermented millet bran.

Obesity-related diabetes, cardiovascular disease, and hypertension pose many risks to human health. Thus, mice on a high-fat diet were gavaged with millet bran (unfermented/fermented) soluble dietary fiber (RSDF/FSDF, 500 mg·kg-1) for 10 weeks in current research, and then evaluated the various biological indicators. These findings revealed that RSDF and FSDF supplements could prevent fat synthesis by inhibiting sterol regulatory element-binding protein-1c gene expression. The RSDF supplements can also accelerate fat catabolism through enhanced the mRNA expression levels of adipose triglyceride lipase and peroxisome proliferator-activated receptor α. FSDF supplements can prevent obesity by decreasing 3-hydroxy-3-methyl-glutaryl-CoA reductase expression and increasing cholesterol 7α-hydroxylase expression. Moreover, FSDF also controls obesity development by lowering total cholesterol and low-density lipoprotein cholesterol levels in the blood, triglyceride, total cholesterol, and bile acid levels in the liver. Notably, FSDF supplements can promote Bacteroides and Prevotella propagation; excretive propionic acid binds to free fatty acid receptor 2/3 and then stimulates intestinal epithelial cells to generate glucagon-like-peptide-1 and peptide YY, which can reduce food and energy intake and ultimately prevent obesity. All evidence suggests that FSDF supplements play a crucial role in preventing obesity.

Effects of monolauroyl-galactosylglycerol on membrane fatty acids and properties of Bacillus cereus.

The purpose of this study was to provide new ideas for the antibacterial mechanism of monolauroyl-galactosylglycerol (MLGG) from the perspective of cell membranes. The changes in cell membrane properties of Bacillus cereus (B. cereus) CMCC 66,301 exposed to different concentrations (1 × MIC (minimum inhibitory concentration), 2 × MIC, 1 × MBC (minimum bacterial concentration)) of MLGG were evaluated. It was found that the lag phase of B. cereus cells was prolonged at low concentration MLGG (1 × MIC and 2 × MIC), while about 2 log CFU/mL reduction in B. cereus populations were observed when exposed to high concentration MLGG (1 × MBC). MLGG treated B. cereus displayed obvious membrane depolarization, while membrane permeability had no change using PI (propidium iodide) staining. Significant increase in the membrane fluidity in response to MLGG exposure occurred, which was consistent with the modification of membrane fatty acids compositions, where the relative content of straight-chain fatty acids (SCFAs) and unsaturated fatty acids (UFAs) increased, while branched-chain fatty acids (BCFAs) decreased significantly. The decreased transition Tm value and cell surface hydrophobicity was also observed. Additionally, effect of MLGG on bacterial membrane compositions were explored at the submolecular level by infrared spectroscopy. Resistance tests of B. cereus to MLGG had demonstrated the advantages of MLGG as a bacteriostatic agent. Collectively, these studies indicate that modifying the fatty acid composition and properties of cellular membranes through MLGG exposure is crucial for inhibiting bacteria growth, providing new insights into the antimicrobial mechanisms of MLGG. KEY POINTS: • Monolauroyl-galactosylglycerol inserted into B. cereus lipid bilayer membrane • Monolauroyl-galactosylglycerol treatment caused B. cereus membrane depolarization • Monolauroyl-galactosylglycerol resulted in B. cereus membrane fatty acids alteration.

Construction and optimization of a multiplex PMAxx-qPCR assay for viable Bacillus cereus and development of a detection kit.

In this study, a PMAxx-qPCR method for the detection and quantification of viable Bacillus cereus (B. cereus) was established based on the cesA gene that is involved in cereulide synthesis, enterotoxin gene bceT and hemolytic enterotoxin gene hblD combined with modified propidium monoazide (PMAxx). The sensitivity detection limit of the method was as follows: the DNA extracted by the kit reached 140 fg/µL, and the bacterial suspension without enrichment reached 2.24 × 101 CFU/mL; 14 nonB. cereus strains of the 17 tested strains all tested as negative, whereas the 2 strains of B. cereus carrying the target virulence gene(s) could be accurately detected. In terms of application, we assembled the constructed PMAxx-qPCR reaction into a detection kit and evaluated its application performance. The results showed that the detection kit has high sensitivity, strong anti-interference capability, and has good application potential. The purpose of this study is to provide a reliable detection method for the prevention and traceability of B. cereus infections.

Maltose-Enhanced Exopolysaccharide Synthesis of Lactiplantibacillus plantarum through CRP-like Protein.

Carbon sources alter the synthesis of exopolysaccharides (EPS) in Lactiplantibacillus plantarum. Maltose increased the EPS production of L. plantarum 163 6.5-fold. Subsequently, EPS production, transcriptome, and proteome were analyzed using glucose or maltose to further clarify the regulatory mechanism. A cAMP receptor protein (UniProtKB: F9UNI5) has been identified to control EPS synthesis in the presence of cAMP by binding to the EPS synthesis promoter Pcps4A-J. Overexpression of the cAMP synthesis gene cyaA increased cAMP content and EPS production 4.5- and 2.2-fold, respectively. Furthermore, yogurt produced with L. plantarum 163-cyaA had a similar viscosity to that of commercial Greek yogurt; it had 20 and 83.7% greater viscosity than that produced with L. plantarum 163 with maltose and glucose, respectively. These findings indicated that L. plantarum 163-cyaA has potential applications in the production of functional fermented dairy products.

Separation, characterization and anti-inflammatory activities of galactoglycerolipids from Perilla frutescens (L.) Britton.

The study was to optimize the separation procedures, characterize the galactoglycerolipids and explore their anti-inflammatory activities. Two monogalactosyldiacylglycerols (MGDGs) and three digalactosyldiacylglycerols (DGDGs) from Perilla frutescens (L.) Britton were obtained through one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The presence of additional MGDG (1-O-9Z,12Z,15Z-octadecatrienoyl-2-O-7Z,10Z,13Z-hexadecatrienoyl-3-O-(ß-D-galactopyranosyl)-sn-glycerol) and DGDG (1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Z-octadecatrienoyl-3-O-(ß-D-galactopyranosyl-(1'→6'')-α-D-galactopyranosyl)-sn-glycerol) was concluded for the first time in perilla, by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In lipopolysaccharide (LPS)-induced RAW264.7 cells, five galactoglycerolipids exhibited good inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner, suggesting that fatty acid chain length and unsaturation degree affected their anti-inflammatory activities.

Shared signaling pathways and targeted therapy by natural bioactive compounds for obesity and type 2 diabetes.

Epidemiological evidence showed that patients suffering from obesity and T2DM are significantly at higher risk for chronic low-grade inflammation, oxidative stress, nonalcoholic fatty liver (NAFLD) and intestinal flora imbalance. Increasing evidence of pathological characteristics illustrates that some common signaling pathways participate in the occurrence, progression, treatment, and prevention of obesity and T2DM. These signaling pathways contain the pivotal players in glucose and lipid metabolism, e.g., AMPK, PI3K/AKT, FGF21, Hedgehog, Notch, and WNT; the inflammation response, for instance, Nrf2, MAPK, NF- kB, and JAK/STAT. Bioactive compounds from plants have emerged as key food components related to healthy status and disease prevention. They can act as signaling molecules to initiate or mediate signaling transduction that regulates cell function and homeostasis to repair and re-functionalize the damaged tissues and organs. Therefore, it is crucial to continuously investigate bioactive compounds as sources of new pharmaceuticals for obesity and T2DM. This review provides comprehensive information of the commonly shared signaling pathways between obesity and T2DM, and we also summarize the therapeutic bioactive compounds that may serve as anti-obesity and/or anti-diabetes therapeutics by regulating these associated pathways, which contribute to improving glucose and lipid metabolism, attenuating inflammation.

Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (Tm) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

An endolysin Salmcide-p1 from bacteriophage fmb-p1 against gram-negative bacteria.

AIMS: A novel endolysin Salmcide-p1 was developed as a promising candidate of new preservative and a supplement to effective enzyme preparations against gram-negative bacterial contaminations. METHODS AND RESULTS: Salmcide-p1 was identified by complementing the genomic sequence of a virulent Salmonella phage fmb-p1. Salmcide-p1 of 112 µg ml-1 could quickly kill Salmonella incubated with 100 mmol l-1 EDTA, with no haemolytic activity. Meanwhile, Salmcide-p1 had a high activity of lysing Salmonella cell wall peptidoglycan. At different temperatures (4-75°C), pH (4-11) and NaCl concentration (10-200 mmol l-1 ), the relative activity of Salmcide-p1 was above 60%. At 4°C, the combination of Salmcide-p1 and EDTA-2Na could inhibit the number of Salmonella Typhimurium CMCC 50115 in skim milk to less than 4 log CFU ml-1 by 3 days, and the number of Shigella flexneri CMCC 51571 was lower than 4 log CFU ml-1 by 9 days. CONCLUSIONS: Salmcide-p1 had a wide bactericidal activity against gram-negative bacteria and showed a broader anti-Salmonella spectrum than the phage fmb-p1. The combination strategy of Salmcide-p1 and EDTA-2Na could significantly inhibit the growth of gram-negative bacteria inoculated in skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage endolysin as an antibacterial agent is considered to be a new strategy against bacterial contamination.

Structure-Function Analysis of a Quinone-Dependent Dehydrogenase Capable of Deoxynivalenol Detoxification.

The pyrroloquinoline quinone (PQQ)-dependent dehydrogenase DepA detoxifies deoxynivalenol (DON) by converting the C3-OH into a keto group. Herein, two crystal structures of DepA and its complex with PQQ were determined, together with biochemical evidence confirming the interactions of DepA with PQQ and DON and revealing a unique tyrosine residue important for substrate selection. Furthermore, four loops over the active site essential for DepA activity were identified, of which three loops were stabilized by PQQ, and the fourth loop invisible in both structures was considered important for binding DON, together constituting a lid for the active site. Preliminary engineering of the loop showed its potential for enzyme improvement. This study provides structural insights into how a PQQ-dependent dehydrogenase is equipped with the function of DON conversion and for the first time shows the necessity of a lid structure for PQQ-dependent dehydrogenase activity, laying foundation for structure-based design to enhance catalysis efficiency.

The antibacterial activity of plantaricin GZ1-27 against MRSA and its bio-preservative effect on chilled pork in combination with chitosan.

Methicillin-resistant Staphylococcus aureus (MRSA) poses great threats to human health. In this research, we found that the newly discovered bacteriocin plantaricin GZ1-27 could efficiently inhibit the MRSA, with a MIC of 32 µg/mL. Comprehensive investigations were carried out by analysis of K+ leakage, propidium iodide assay, and cell ultra-structure analysis with scanning electron microscopy and transmission electron microscopy. The results consistently showed that plantaricin GZ1-27 could increase the permeability of the membrane and impair its integrity, which induced the collapse of the cell structure and thus led to cell death. Furthermore, by sensory evaluation and biochemical analysis, it was found that plantaricin GZ1-27 combined with chitosan could significantly improve the preservation of pork when applied to the surface of meat slices. Overall, our research clearly showed the anti-MRSA bactericidal mechanism of plantaricin GZ1-27, which, together with its preliminary application trials in pork, suggested plantaricin GZ1-27 could be a potential anti-MRSA agent and is promising to be applied in pork preservation to extend the shelf life.

A Novel Class IIb Bacteriocin-Plantaricin EmF Effectively Inhibits Listeria monocytogenes and Extends the Shelf Life of Beef in Combination with Chitosan.

Plantaricin EmF separated and identified from L. plantarum 163 was a novel class IIb bacteriocin. The molecular masses of plantaricin Em and F were 1638 and 3702 Da, respectively, with amino acid sequences FNRGGYNFGKSVRH and VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG, respectively. Plantaricin EmF not only exhibited broad-pH adaptability and thermostability but also showed high efficiency and broad-spectrum antibacterial activity. Its mode of action on L. monocytogenes damaged cell membrane integrity, resulting in the leakage of cytoplasm, changes in cell structure and morphology, and ultimately cell death. Additionally, plantaricin EmF inactivated L. monocytogenes in beef, effectively improving the quality indices of beef, thereby extending its shelf life, especially in combination with chitosan. Plantaricin EmF + 1.0% chitosan extended the shelf life of beef to 15 d, demonstrating its potential application value to replace chemical preservatives to control food-borne pathogenic microorganisms and extend the shelf life of meat and meat products in agriculture and the food industry.

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential.

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg-1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0-11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min-1 and 1969.25 mM-1min-1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

Acetate Activates Lactobacillus Bacteriocin Synthesis by Controlling Quorum Sensing.

Bacteriocins are useful for controlling the composition of microorganisms in fermented food. Bacteriocin synthesis is regulated by quorum sensing mediated by autoinducing peptides. In addition, short-chain fatty acids, especially acetic acid, reportedly regulate bacteriocin synthesis. Five histidine kinases that regulated the synthesis of bacteriocins were selected to verify their interactions with acetate. Acetate activated the kinase activity of PlnB, SppK, and HpK3 in vitro and increased the yield of their cognate bacteriocins plantaricin EF, sakacin A, and rhamnosin B in vivo. The antimicrobial activity against Staphylococcus aureus of the fermentation supernatants of Lactobacillus plantarum, Lactobacillus sakei, and Lactobacillus rhamnosus with addition of acetate increased to 298%, 198%, and 289%, respectively, compared with that in the absence of acetate. Our study elucidated the activation activity of acetate in bacteriocin synthesis, and it might provide a potential strategy to increase the production of bacteriocin produced by Lactobacillus. IMPORTANCE Bacteriocins produced by lactic acid bacteria (LAB) are particularly useful in food preservation and food safety. Bacteriocins might increase bacterial competitive advantage against the indigenous microbiota of the intestines; at the same time, bacteriocins could limit the growth of undesired microorganisms in yogurt and other dairy products. This study confirmed that three kinds of histidine kinases were activated by acetate and upregulated bacteriocin synthesis both in vitro and in vivo. The increasing yield of bacteriocins reduced the number of pathogens and increased the number of probiotics in milk. Bacteriocin synthesis activation by acetate may have a broad application in the preservation of dairy products and forage silage.

L. johnsonii, L. plantarum, and L. rhamnosus alleviated Enterohaemorrhagic Escherichia coli-induced diarrhoea in mice by regulating gut microbiota.

Enterohaemorrhagic Escherichia coli (EHEC) is a prominent foodborne pathogen that causes infectious intestinal diarrhoea. Lactobacillus is a recognized probiotic that inhibits intestinal pathogens and maintains the balance of the intestinal flora. The purpose of this study was to investigate the regulatory effects of three Lactobacillus strains, L. johnsonii, L. plantarum, and L. rhamnosus, on the intestinal flora of EHEC-infected mice. The initial weight and diarrhoea index of the mice were recorded. After 21 days, the faeces of the mice were subjected to 16S rDNA high-throughput sequencing. The diarrhoea index of mice treated with Lactobacillus improved, their body weight continued to rise, and their liver index gradually decreased. The α diversity analysis showed that the intestinal flora diversity and abundance were lower in mice infected with EHEC than in healthy mice. L. plantarum, L. johnsonii, and L. rhamnosus significantly improved the diversity of the flora species. In terms of flora composition, the three main phyla present were Bacteroidetes, Firmicutes, and Proteobacteria. The abundance of these three phyla was reduced to 93.81% after infection and restored to over 96.30% after treatment. At the genus level, Lactobacillus reduced the abundance of Bacteroides, Helicobacter pylori, and Shigella, while increasing the abundance of butyric acid-producing bacteria and Lactobacillus. Finally, a heat map and non-metric multidimensional scaling analysis showed that the intestinal flora structures in the L. johnsonii, L. plantarum, and L. rhamnosus treatment groups were closest to those of healthy mice. In conclusion, L. johnsonii, L. plantarum, and L. rhamnosus regulated and improved the structure of intestinal flora and relieved diarrhoea caused by EHEC infection.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Novel Bacillus Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese.

Although rennet is one of the best choices for cheese manufacturing, its production cannot meet the growing demands of the cheese industry. Thus, new milk-clotting enzymes (MCEs) with similar or better properties as/than those of calf chymosin are needed urgently. Here, three MCEs, BY-2, BY-3, and BY-4, were mined by bioinformatic analysis and then expressed in and isolated from Escherichia coli. BY-4 had the highest milk-clotting activity/proteolytic activity (238.76) with enzyme properties similar to those of calf chymosin. BY-4 cheese had a composition, appearance, consistency/texture, and overall acceptability proximate to calf chymosin cheese. The EC50 values of peptides extracted from BY-4 cheese for 2,2-diphenyl-1-picrylhydrazyl inhibition (antioxidant property), angiotensin-converting enzyme inhibition (antihypertensivity), and growth inhibition of liver cancer cells (antitumor property) were found to be 81, 49, and 238 µg/mL, respectively, which were 2.35, 2.59, and 2.12 folds higher than those of calf chymosin cheese. These results indicated the potential of BY-4 as a supplement to calf chymosin in cheese manufacturing, especially for functional and health care purposes.

Glycoglycerolipids from the leaves of Perilla frutescens (L.) Britton (Labiatae) and their anti-inflammatory activities in lipopolysaccharide-stimulated RAW264.7 cells.

A described monogalactosyldiacylglycerol (MGDG) and two undescribed digalactosyldiacylglycerols (DGDGs) were isolated from the leaves of Perilla frutescens (L.) Britton (Labiatae) by using silica gel column chromatography and semi-preparative high performance liquid chromatography. The elucidation of complete structure of these compounds were conducted by using MS and NMR techniques. The MGDG (7.5% of total lipids) was identified as 1,2-2-O-(9Z,12Z,15E-octadecatrienoyl)-3-O-(ß-D-galactopyranosyl)-sn-glycerol. The two DGDGs (2.8% and 1.0% of total lipids, respectively) were identified as 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-(6Z,9Z,12Z-octadecatrienoyl)-3-O-[ß-D-galactopyranosyl-(1″→6')-α-D-galactopyranosyl]-sn-glycerol and 1-O- hexadecanoyl -2-O-(9Z,12Z,15Z-octadecatrienoy -l)-3-O-[ß-D-galactopyranosyl-(1″→6')-α-D-galactopyranosyl]-sn-glycerol, respectively. All the isolated MGDG and DGDGs were evaluated for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated murine macrophages RAW264.7 cells. All of them showed good inhibitory activities and significantly blocked the production of LPS-induced TNF-α, (IL)-1ß and IL-6. The above results shed some light on a better understanding of the traditional anti-inflammatory effect of Perilla frutescens and reveal the potential anti-inflammatory constituents.

Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

Influence of different factors on biofilm formation of Listeria monocytogenes and the regulation of cheY gene.

In a food-processing environment, bacterial cells often adhere to surfaces and form biofilms to protect themselves from external adverse influences. Our study aimed to identify the influence of environmental factors and cell properties on Listeria monocytogenes biofilm formation. Biofilm formation was quantified through measuring the optical density at 590 nm (OD590 nm) after crystal violet staining. Neutral pH and 37oC were beneficial for biofilm formation whereas the influence of glucose (0.0-1.0%) and sodium chloride (0.0-1.0%) were strain-dependent. In general, the addition of sodium chloride and glucose increased biofilm formation in most strains compared to that in controls with no sodium chloride or glucose added. Bacteria with strong biofilm-forming capacity always produced large amounts of biofilm in most instances. Biofilm formation positively correlated with the cell surface hydrophobicity and motility but was independent from planktonic cell growth. The expression of flagella-related flaA, motB, and the two-component chemotactic system cheA/Y genes in biofilm cells increased compared to that in planktonic cells. Meanwhile, a cheY knockout mutant was constructed, and decreased biofilm-formation ability along with reduced cell-surface hydrophobicity were found in the non-motile mutant. Furthermore, the cheY knockout mutant showed no change in growth, and pH susceptibility compared to that in the wild-type strain.