Preparation and characterization of metal-polyphenol networks encapsulated in sodium alginate microbead hydrogels for catechin and vitamin C delivery.

A novel encapsulation system was designed, utilizing sodium alginate (SA) polysaccharide as the matrix and easily absorbed Fe2+ as the metal-organic framework, to construct microbead scaffolds with both high catechins (CA) and vitamin C (Vc) loading and antioxidant properties. The structure of microbead hydrocolloids was investigated using SEM, XPS, FTIR, XRD and thermogravimetry, and the antioxidant activity, in vitro digestion and the release of CA and Vc were evaluated. These results revealed that the microbead hydrocolloids SA-CA-Fe and SA-CA-Vc-Fe exhibited denser and stronger cross-linking structures, and the formation of inter- and intramolecular hydrogen and coordination bonds improved thermal stability. Moreover, SA-CA-Fe (44.9 % DPPH and 47.8 % ABTS) and SA-CA-Vc-Fe (89.9 % DPPH and 89.3 % ABTS) displayed strong antioxidant activity. Importantly, they were non-toxic in Caco2 cells. The SA-CA-Fe and SA-CA-Vc-Fe achieved significantly higher CA (56.9 and 62.7 %, respectively) and Vc (42.2 %) encapsulation efficiency while maintaining higher CA and Vc release in small intestinal environment. These results suggested that SA polysaccharide-based encapsulation system using Fe2+ framework as scaffold had greater potential for delivery and controlled release of CA and Vc than conventional hydrocolloids, which could provide new insights into the construction of high loading, safe, targeted polyphenol delivery system.

Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Hepatoprotective Effect of Annulohypoxylon stygium Melanin on Acute Alcoholic Liver Injury in Mice.

Annulohypoxylon stygium melanin (AsM) has various functional properties such as antioxidant and anti-radiation, but its biological activity in vivo has not been fully investigated. In this study, we researched the effects of AsM on the protection against acute liver injury in mice and its mechanism. The results showed that AsM had no significant effect on body weight in mice but reduced the liver index. It was able to significantly decrease the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the contents of triglyceride (TG) and total cholesterol (TC) in mice. Simultaneously, it raised the levels of superoxide dismutase (SOD), peroxidase (CAT), and glutathione peroxidase (GSH-Px), which obviously exceeded those of the EtOH group. AsM could significantly lower the levels of inflammatory factors, with inhibition rates of 68.30%, 29.0%, and 19.50% for IL-1ß, IL-6, and TNF-α, respectively. H&E and Oil red O staining also showed that AsM ameliorated liver damage and lipid accumulation in mice. The protective mechanism of AsM may be associated to the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway, which could activate the downstream antioxidant enzymes heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC). These findings confirmed that AsM had an alleviating effect on alcoholic liver injury and provided new thoughts for the development of natural product.

Phellinus linteus polysaccharides: A review on their preparation, structure-activity relationships, and drug delivery systems.

Phellinus linteus polysaccharides exhibit antitumor, immunomodulatory, anti-inflammatory, and antioxidant properties, mitigate insulin resistance, and enhance the diversity and abundance of gut microbiota. However, the bioactivities of P. linteus polysaccharides vary owing to the complex structure, thereby, limiting their application. Various processing strategies have been employed to modify them for improving the functional properties and yield. Herein, we compare the primary modes of extraction and purification employed to improve the yield and purity, review the structure-activity relationships, and discuss the application of P. linteus polysaccharides using nano-carriers for the encapsulation and delivery of various drugs to improve bioactivity. The limitations and future perspectives are also discussed. Exploring the bioactivity, structure-activity relationship, processing methods, and delivery routes of P. linteus polysaccharides will facilitate the development of functional foods and dietary supplements rich in P. linteus polysaccharides.

Melanin of fungi: from classification to application.

Melanin is a secondary metabolite composed of complex heterogeneous polymers. Fungal melanin is considered to be a sustainable and biodegradable natural pigment and has a variety of functional properties and biological activities. On one hand, due to its own specific properties it can play the role of antioxidant, anti-radiation, adsorption, and photoprotection. On the other hand, it has good biological activities such as hepatoprotective effect, hypolipidemic effect and anti-cancer. Therefore, it is widely used in various fields of daily life, including dyeing, food, biomedical and commercial industry. It is conducive to environmental protection and human health. However, the insolubility of fungal melanin in water, acids and organic solvents has been an obstacle to its commercial applications. Thus, the chemical modification methods of fungal melanin are summarized to increase its solubility and expand the application fields. Although fungal melanin has been used in many industries, as the structure and function of fungal melanin and modified melanin are further studied, more functional properties and bioactivities are expected to be discovered for a wide range of applications in the future.

A comprehensive review of spermidine: Safety, health effects, absorption and metabolism, food materials evaluation, physical and chemical processing, and bioprocessing.

Spermidine, a natural autophagy inducer, has a variety of health effects, such as antitumor, antiaging, anti-inflammation, cardiovascular protection, and neuromodulation. It has been a hot topic in the field of food processing, and current research findings suggest that spermidine-rich foods may be used in intervention and prevention of age-related diseases. In this article, recent findings on the safety, health effects, absorption and metabolism of spermidine were reviewed, and advances in food processing, including the raw materials evaluation, physical and chemical processing, and biological processing of spermidine, were highlighted. In particular, the core metabolic pathways, key gene targets, and efficient metabolic engineering strategies involved in the biosynthesis of spermidine and its precursors were discussed. Moreover, limitations and future perspectives of spermidine research were proposed. The purpose of this review is to provide new insights on spermidine from its safety to its food processing, which will advance the commercial production and applications of spermidine-rich foods and nutraceuticals.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression.

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

Prebiotic, Probiotic, Antimicrobial, and Functional Food Applications of Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens belongs to the genus Bacillus and family Baciliaceae. It is ubiquitously found in food, plants, animals, soil, and in different environments. In this review, the application of B. amyloliquefaciens in probiotic and prebiotic microbes in fermentation, synthesis, and hydrolysis of food compounds is discussed as well as further insights into its potential application and gaps. B. amyloliquefaciens is also a potential microbe in the synthesis of bioactive compounds including peptides and exopolysaccharides. In addition, it can synthesize antimicrobial compounds (e.g., Fengycin, and Bacillomycin Lb), which makes its novelty in the food sector greater. Moreover, it imparts and improves the functional, sensory, and shelf life of the end products. The hydrolysis of complex compounds including insoluble proteins, carbohydrates, fibers, hemicellulose, and lignans also shows that B. amyloliquefaciens is a multifunctional and potential microbe which can be applied in the food industry and in functional food processing.

Enhancement of S-adenosylmethionine production by deleting thrB gene and overexpressing SAM2 gene in Bacillus amyloliquefaciens.

OBJECTIVES: To improve the S-adenosylmethionine (SAM) production in methionine-free medium, effects of deleting genes of SAM decarboxylase (speD) and homoserine kinase (thrB) on SAM titers were investigated, and the SAM synthetase gene (SAM2) was also overexpressed. RESULTS: In B. amyloliquefaciens HSAM2, deleting speD to block the SAM utilization pathway significantly reduced the SAM titer. After knockout of thrB to block the branched pathway, the resulted mutant HSAM4 produced 143.93 mg/L SAM, increasing by 42% than HSAM2. Further plasmid-based expression of SAM2 improved the SAM titer to 226.92 mg/L, and final optimization of key fermentation parameters resulted in the maximum SAM titer of 412.01 mg/L in flasks batch fermentation. CONCLUSIONS: Deleting thrB and overexpressing SAM2 gene were efficient for enhanced SAM production in B. amyloliquefaciens. The maximum SAM titer in flasks batch fermentation was much higher than that of previous reports.

Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle.

BACKGROUND: S-Adenosylmethionine (SAM) is a critical cofactor involved in many biochemical reactions. However, the low fermentation titer of SAM in methionine-free medium hampers commercial-scale production. The SAM synthesis pathway is specially related to the tricarboxylic acid (TCA) cycle in Bacillus amyloliquefaciens. Therefore, the SAM synthesis pathway was engineered and coupled with the TCA cycle in B. amyloliquefaciens to improve SAM production in methionine-free medium. RESULTS: Four genes were found to significantly affect SAM production, including SAM2 from Saccharomyces cerevisiae, metA and metB from Escherichia coli, and native mccA. These four genes were combined to engineer the SAM pathway, resulting in a 1.42-fold increase in SAM titer using recombinant strain HSAM1. The engineered SAM pathway was subsequently coupled with the TCA cycle through deletion of succinyl-CoA synthetase gene sucC, and the resulted HSAM2 mutant produced a maximum SAM titer of 107.47 mg/L, representing a 0.59-fold increase over HSAM1. Expression of SAM2 in this strain via a recombinant plasmid resulted in strain HSAM3 that produced 648.99 mg/L SAM following semi-continuous flask batch fermentation, a much higher yield than previously reported for methionine-free medium. CONCLUSIONS: This study reports an efficient strategy for improving SAM production that can also be applied for generation of SAM cofactors supporting group transfer reactions, which could benefit metabolic engineering, chemical biology and synthetic biology.

Decreased tobacco-specific nitrosamines by microbial treatment with Bacillus amyloliquefaciens DA9 during the air-curing process of burley tobacco.

Tobacco specific nitrosamines (TSNA) mainly consisting of N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are a group of toxic components threatening human health. To inhibit TSNA formation in tobacco leaves, a high nitrite reductive strain with low nitrate reduction ability was isolated and applied to tobacco leaves in an attempt to lower the nitrite precursor of TSNA. By morphology, physiology, biochemistry, and 16S rDNA sequence analysis, the strain DA9 was identified as Bacillus amyloliquefaciens. Under the optimized fermentation parameters (glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L, initial pH 7.0, inoculum age 6 h, inoculum size 3%, temperature 37 °C), the maximum cell dentisity of 1.2 × 10(9) CFU/mL was obtained at 36 h. The DA9 cell suspensions were applied in the air-curing process of the Burley tobacco (Eyan 6) leaves. The treatment by DA9 cells lowered 32% of the nitrite content and 47% of total TSNA content in the tobacco leaves, and the concentrations of the NNN, NNK, and NAT were decreased by 48%, 12%, and 35%, respectively. Collectively, this study provides a promising strain and a novel strategy for decreasing TSNA during the air-curing process.

Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas.

To develop safe and cheap thrombolytic agents, a fibrinolytic enzyme productive strain of LSSE-62 was isolated from Chinese soybean paste. This strain was identified as Bacillus amyloliquefaciens by 16S rDNA sequence analysis. Nucleotide and amino acid sequence analysis showed that this fibrinolytic enzyme was identical to subtilisin DJ-4. Chickpeas were used as the substrate for fibrinolytic enzyme production from B. amyloliquefaciens in solid-state fermentation. Under the optimized conditions (34 °C and 50% initial moisture content), the fibrinolytic activity of fermented chickpeas reached 39.28 fibrin degradation units (FU)/g. Additionally, the fermented chickpeas showed anticoagulant activity, and the purified anticoagulant component showed higher anticoagulant activity than heparin sodium. After fermentation, the total phenolic and total flavonoid contents increased by 222 and 71%, respectively, and then the antioxidant activities were improved significantly. This study provided a novel method for the preparation of multifunctional food of chickpeas or raw materials for the preparation of functional food additives and potential drugs.