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Although the microbial diversity and structure in bean-based fermented foods have been widely studied, systematic studies on functional microbiota and mechanism of community forms in multi-microbial fermentation systems were still lacking. In this work, the metabolic pathway and functional potential of microbial community in broad bean paste (BBP) were investigated by metagenomics approach, and Staphylococcus, Bacillus, Weissella, Aspergillus and Zygosaccharomyces were found to be the potential predominant populations responsible for substrate alteration and flavor biosynthesis. Among them, Staphylococcus was the most abundant and widespread functional microbe, and closely related Staphylococcus species were diverse and ubiquitously distributed, with the opportunistic pathogen S. gallinarum being the most abundant Staphylococcus specie isolated from BBP. To explain the dominance status of S. gallinarum and species distributions of Staphylococcus genus, we tested the effects of abiotic and biotic factors on three Staphylococcus species using a tractable BBP model, demonstrating that adaptation to environmental conditions (environmental parameters and other functional microbes) led to the dominant position and species coexistence of Staphylococcus, and congeneric competition among Staphylococcus species further shaped ecological distributions of closely related Staphylococcus species. In general, this work revealed the metabolic potential of microbial community and distribution mechanism of Staphylococcus species during BBP fermentation, which could help traditional factories to more precisely control the safety and quality of bean-based fermented foods.

The nitrilase-mediated green synthesis of nicotinic acid, an important reaction in synthetic organic chemistry, has attracted considerable attention in recent years. However, the application potential of nitrilase is hindered by several limitations, such as low catalytic efficiency and byproduct formation. In this study, the site saturation mutagenesis of asparagine 40, phenylalanine 50, and glutamine 207 in recombinant nitrilase from Pseudomonas putida CGMCC3830 was conducted to improve the specificity of nitrilase for nicotinic acid. The resulting mutants, which contain the mutations N40G (asparagine → glycine), F50W (phenylalanine → tryptophan), and Q207E (glutamine → glutamic acid) produced higher nicotinic acid yields than the wild type. Furthermore, double and triple mutations were introduced, and four mutants containing N40G/F50W, N40G/Q207E, F50W/Q207E, and N40G/F50W/Q207E were obtained and evaluated for their capacity to produce nicotinic acid. The double mutant F50W/Q207E and triple mutant N40G/F50W/Q207E displayed the highest activity, which was nearly two-fold higher than that of the wild type. The kinetics analysis of nicotinic acid synthesis with the mutant nitrilase revealed the higher catalytic capability of all the mutants compared with the wild type. These results provide new insights into the catalytic performance of P. putida nitrilase toward 3-cyanopyridine to promote nicotinic acid production.

Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Background Armillaria mellea (A. mellea) is a traditional Chinese medicinal and edible mushroom, which is proved to possess a lot of biological activities, including anti-oxidation, immunopotentiation, anti-vertigo and anti-aging activities. However, little information is available in regard to its neuroprotection activity in inflammation-mediated neurodegenerative diseases. Purpose We have found that A. mellea has an anti-inflammatory activity in LPS-induced RAW264.7 cells in our previous study. The objective of this study is to investigate the anti-neuroinflammatory mechanism of a bioassay-guided fractionation (Fr.2) and its active components/compounds. Methods Compounds were isolated by preparative high performance liquid chromatography (pre-HPLC) and their structures were established by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analyses. The anti-neuroinflammatory effect of Fr.2 and each compounds were investigated in lipopolysaccharide (LPS)-stimulated murine microglia cell line BV-2. Results We demonstrated that Fr.2 significantly decreased the production of inflammation mediator nitric oxide (NO) and inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) in a dose-dependent manner (10, 30, 100 µg/ml). In addition, Fr.2 markedly down-regulated the phosphorylation levels of nuclear factor kappa B p65 (NF-κB p65), inhibitory κB-α (IκB-α) and c-Jun N-terminal kinases (JNKs) pathways. Sevens compounds were isolated from Fr.2, among them, three compounds, 5-hydroxymethylfurfural (CP1), vanillic acid (CP4) and syringate (CP5) were reported for the first time in A. mellea. NO and inflammatory cytokines (TNF-α, IL-6, IL-1β) secretion indicated that daidzein (CP6) and genistein (CP7) showed a more outstanding anti-inflammation potential at non-toxic concentrations (10, 30, 100 µM) than the other five compounds. Conclusions In conclusion, Fr.2 may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and suppress inflammation pathway in activated microglia. Daidzein and genistein may serve as the effective anti-inflammation compounds of Fr.2.

A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26 kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55 °C, respectively. It could remain stable at pH 5.0–12.0 and 40–60 °C. Metal ions such as Ca2+, Mn2+, Ag+, Na+, Mg2+, Li+, Sn2+ (1 mM) displayed positive influence on keratinase, and particularly, Ca2+ exhibited remarkable improvement effect by 2.6 folds. It was strongly inhibited by PMSF as a protease inhibitor. On the contrary, it could be obviously activated by various surfactants, such as Tween 40 and Triton X-114. The recombinant keratinase showed high specificity towards casein, soluble keratin, BSA, and wool. The keratinase could efficiently degrade the feathers, which demonstrated its applicable potential for biodegradation of keratin wastes and regeneration of soluble protein.

Cicada flowers, which are edible and medicinal mushrooms, are the fruiting bodies of

A novel Gram-stain-positive, rod-shaped, obligately anaerobic, non-motile, spore-forming and binary fission encapsulated bacterium, designated strain JN500901T, was isolated from a mud cellar which has been continuously used for the fermentation of Chinese strong-flavour baijiu for over 100 years. Growth of JN500901Toccurred at pH 4.5–8.0 (optimum, pH 5.0), 20–40 °C (37 °C), 0–2 % (w/v) NaCl and 0–10 % (v/v) ethanol. The Biolog assay revealed that strain JN500901T metabolized d-fructose, l-fucose, isomaltulose and l-rhamnose among the 95 studied carbon sources. p-Cresol was the predominant volatile metabolite in the fermentation broth of strain JN500901T incubated in liquid reinforced clostridial medium under anaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JN500901T belongs to Clostridium sensu stricto, and shared the highest sequence similarity to Clostridium carboxidivorans DSM 15243T (94.2 %), followed by Clostridium scatologenes DSM 757T (94.1 %). The dominant cellular fatty acids (>10 %) were C16 : 0 FAME (36.6 %), C19 : 0 cyc 9,10 DMA (19.8 %) and C16 : 1 cis 9 DMA (11.8 %). The complete genome of strain JN500901T contained a circular chromosome of 2.812 Mb with 2611 genes and 31.0 mol% G+C content. Comparative genome analysis of the strain JN500901T, Clostridium carboxidivorans DSM 15243T and Clostridium scatologenes DSM 757T revealed 74.5 and 74.8 % average nucleotide identity, respectively. Based on the phenotypic, biochemical and phylogenetic analyses presented here, strain JN500901T is considered to be a novel species of the genus Clostridium sensustricto, for which the name Clostridium fermenticellae sp. nov. is proposed. The type strain is JN500901T (=CICC 24501T=JCM 32827T).

Baijiu is a highly-valued traditional Chinese distilled alcoholic liquor and maturation markedly improves its sensory qualities and market value. Identification of age-markers is challenging because of the complexity of the endogenous chemical profile and the absence of exogenous components added during maturation. Here, freshly-distilled raw Lu-flavor Baijiu samples were stored for two years and non-targeted screening for age-markers based on the volatile profile characterized by solid-phase micro-extraction assisted GC-MS. The resulting volatile datasets were analyzed by partial least-squares discriminant analysis, Spearman's correlation and Random-forest decision-tree, to identify potential age-markers. The contents of long chain fatty acid ethyl esters varied the most during maturation, mostly being absent in freshly-distilled Baijiu, but appearing at different times. Similar behavior was observed in bottled samples matured up to four years. In addition, a slight increase in short/medium chain fatty acid ester content was observed. Unsupervised PCA analysis, applied only to the fatty acid ethyl ester dataset, was able to distinguish between bottled Baijiu samples matured for one, two, three, or four years. These age-markers may help establish a reliable model for verifying the maturation time of Lu-flavor Baijiu. This screening approach should be adaptable to other alcoholic liquors, for quality assessment and authenticity testing.

As a widely used Asian starter culture, the quality of daqu can significantly affect the organoleptic characteristics of the final products, yet the microbial metabolic network involved in flavor development remains unclear. This study aims to investigate that network based on the dynamics of physicochemical properties, microbial community, and volatile compounds in medium-temperature daqu (MT-daqu) during spontaneous fermentation. Analyses using the metagenomic data set facilitated the gene repertoire overview of this ecosystem, indicating that Lactobacillales (mainly Weissella, Lactobacillus, and Pediococcus), Mucorales (mainly Lichtheimia), and Eurotiales (mainly Aspergillus, Rasamsonia and Byssochlamys) were the potential predominant populations successively responsible for the production of lytic enzymes and flavor precursors/compounds in MT-daqu. Flavor-relevant pathways were found to exist in multiple species, but only bacteria showed the potential to participate in butane-2,3-diol (e.g. Weissella, Lactobacillus, and Staphylococcus) and butanoate (Thermoactinomyces) metabolism, and only fungi were potentially involved in biosynthesis of guaiacol (Byssochlamys) and 4-vinylguaiacol (Aspergillus). Furthermore, a combined analysis revealed that the acidic thermal environment present in early phases was mainly due to the catabolic activities of Lactobacillales and Lichtheimia, which could contribute to the effective self-domestication of microbiota. The study helps elucidate the different metabolic roles of microorganisms and disclose the formation mechanism of daqu's partial functions, namely providing various aromatic substances/precursors and enzymes.

An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45 °C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K+ , Na+ , and Mg2+ ions can enhance the enzyme activities, while Ba2+ , Ni+ , Cu2+ , Mn2+ , Zn2+ , Ag+ , and ethylenediaminetetraacetic acid decreased the activities. It displayed high salt-tolerant ability; 0.8 M NaCl or 1.5 M KCl significantly enhanced the enzyme activity. Furthermore, Aly-W02 mainly released disaccharide, trisaccharide, and tetrasaccharid from alginate. It showed potential in producing low molecular weight alginate oligosaccharides.

Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. However, the specific microorganisms responsible for acetoin formation in this centuries-long repeated batch fermentation have not yet been clearly identified. Here, the microbial distribution discrepancy in the diacetyl/acetoin metabolic pathway of vinegar microbiota was revealed at the species level by a combination of metagenomic sequencing and clone library analysis. The results showed that Acetobacter pasteurianus and 4 Lactobacillus species ( Lactobacillus buchneri , Lactobacillus reuteri , Lactobacillus fermentum , and Lactobacillus brevis ) might be functional producers of acetoin from 2-acetolactate in vinegar microbiota. Furthermore, A. pasteurianus G3-2, L. brevis 4-22, L. fermentum M10-3, and L. buchneri F2-5 were isolated from vinegar microbiota by a culture-dependent method. The acetoin concentrations in two cocultures ( L. brevis 4-22 plus A. pasteurianus G3-2 and L. fermentum M10-3 plus A. pasteurianus G3-2) were obviously higher than those in monocultures of lactic acid bacteria (LAB), while L. buchneri F2-5 did not produce more acetoin when coinoculated with A. pasteurianus G3-2. Last, the acetoin-producing function of vinegar microbiota was regulated in situ via augmentation with functional species in vinegar Pei . After 72 h of fermentation, augmentation with A. pasteurianus G3-2 plus L. brevis 4-22, L. fermentum M10-3, or L. buchneri F2-5 significantly increased the acetoin content in vinegar Pei compared with the control group. This study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota. IMPORTANCE Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. Thus, it is of interest to understand which microbes are driving the formation of acetoin to elucidate the microbial distribution discrepancy in the acetoin metabolic pathway and to regulate the metabolic function of functional microbial groups in vinegar microbiota. Our study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota.

Liver fibrosis is a pathological process with intrahepatic diffused deposition of the excess extracellular matrix, which leads to various chronic liver diseases. Drugs with high efficacy and low toxicity for liver fibrosis are still unavailable. Antrodia camphorata has antioxidant, antivirus, antitumor and anti-inflammation roles, and has been used to treat liver diseases in the population. However, the hepatoprotective effects of A. camphorata spores and the mechanisms behind it have not been investigated. In this study, we evaluate the hepatoprotective effect of spore powder of A. camphorata (SP, 100 mg/kg/day or 200 mg/kg/day) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. SP groups reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities compared with the CCl4 group. SP also showed a decrease in hydroxyproline (Hyp) content in liver tissues. SP improved cell damage and reduced collagen deposition by H&amp, E, Sirius red and Masson staining. Furthermore, SP down-regulated the mRNA levels of &alpha, SMA and Col 1, and the protein expression of &alpha, smooth muscle actin (&alpha, SMA), collagen I (Col 1), tumor necrosis factor alpha (TNF-&alpha, ), toll like receptor 4 (TLR4) and nuclear factor-&Kappa, b (NF-&kappa, B) p65. In summary, SP has an ameliorative effect on hepatic fibrosis, probably by inhibiting the activation of hepatic stellate cells, reducing the synthesis of extracellular matrix.

A novel Gram-stain-positive, non-motile, non-spore-forming, rod-shaped, facultatively anaerobic, designated strain HSLZ-75T, was isolated from the solid-state vinegar culture of Zhenjiang aromatic vinegar. Strain HSLZ-75T grew at 20–40 °C (optimum 35 °C), pH 3.0–5.0 (optimum pH 4.0) and 0–5% (w/v) NaCl (optimum 0%). Heterolactic fermentation characterised the metabolism of strain HSLZ-75T. d- and l-lactic acid were produced from glucose in a ratio of 91:9. The major cellular fatty acids ( > 10%) consisted of C16:0, C18:1ω9c, summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid and six unknown lipids. The cell wall was found to contain meso-diaminopimelic acid-type peptidoglycan. The 16S rRNA gene sequence of strain HSLZ-75T showed the highest similarity of 88.0% with Lactobacillus fructivorans DSM 20203T. Phylogenetic analysis indicated that strain HSLZ-75T belonged to family Lactobacillaceae and formed a distinct lineage with the type strain of Lactobacillus caviae. The complete genome of strain HSLZ-75T contained a circular chromosome of 1,616,430 bp with 1570 genes and 39.7 mol% G + C content. The average nucleotide identity values between strain HSLZ-75T and the reference type strains Lactobacillus fructivorans DSM 20203T and Lactobacillus rossiae DSM 15814T were 66.4% and 65.7%, respectively. On the basis of phenotypic, chemotaxonomic, phylogenetic and genotypic characteristics, strain HSLZ-75T should be classified as a novel species of the genus Lactobacillus in the family Lactobacillaceae of the order Lactobacillales, for which the name Lactobacillus jinshani sp. nov. is proposed. The type strain is HSLZ-75T ( = CICC 6269T = JCM 33270T).

Butyrate and its derivates pertain to the key aroma contributors of strong-flavour baijiu, a kind of Chinese liquors, that is produced from grains by solid-state multispecies anaerobic fermentation in a mud cellar. Microbes inhabiting in the fermentation pit mud largely determines baijiu's flavour and quality. In order to shed light on the microbial functional groups driving butyrate production in pit mud, clone library analysis was firstly performed and the results demonstrated that Clostridia (relative abundance: 50%) and Bacilli (37%) were major groups possessing butyrate kinase (buk) pathway and Clostridia (98%) dominated butyryl-CoA:acetate CoA-transferase (but) pathway. According to Clostridial specific-16S rRNA gene sequencing analysis, we found the resilience character of Clostridial community in pit mud. Amongst Clostridial groups, 32.0% of the sequences were grouped into Clostridiales incertae sedis, followed by Heliobacteriaceae (18.3%) and Clostridiaceae 1 (8.4%). Moreover, Hydrogenispora, Sedimentibacter and Clostridium were the top three abundant genera. Relative abundance of Hydrogenispora was higher in the late days of fermentation, while Sedimentibacter exhibited higher proportion in the early days. Different from the previous studies using universal bacterial primer sets, Hydrogenispora was first reported as one dominant genus in pit mud. As for the reported potential butyrate producer Clostridium, nineteen species were obtained and ten of them were first isolated from the pit mud. Amongst them, buk was identified in eleven species by PCR analysis, while but was identified in the other seven, indicating the species-specific butyrate synthesis pathways of Clostridium. This study provides a perspective on targeting and isolating specific functional microbes in baijiu microbiota with the gene sequence-based medium prediction method.

Polysaccharopeptide (PSP) from the medicinal mushroom Coriolus versicolor has been widely used in Asia as an adjunctive immunotherapy for treating cancers and liver diseases. However, the composition and structure of bioactive components in PSP remain elusive. Herein, we purified a hepatoprotective polysaccharide (PSP-1b1) with a molecular weight of 21.7 kDa from C. versicolor mycelia in submerged culture. PSP-1b1 consists of fucose, galactose, xylose, mannose, glucuronic acid and glucose at a relative molar ratio of 0.16:0.60:0.02:0.55:0.04:1.00. Structural features were investigated by methylation and gas chromatography–mass spectrometry, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The PSP-1b1 backbone consists of →4)-α-Galp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)→, with branches of α-1,6-Manp, β-1,6-Glcp, β-1,3,6-Glcp, α-1,3-Manp, α-1,6-Galp, α-1,3-Fucp, T-α-Glcp and T-α-Galp on the O-6 position of α-Manp of the main chain, and secondary branches linked to the O-6 position of β-Glcp of the major branch. Treatment with PSK-1b1 (80 and 160 mg/kg/day) resulted in hepatoprotective effects against alcohol-induced liver injury in mice by reducing oxidative stress and modulating immunity.

The gut microbiota is considered to be associated with high-fat diet (HFD)-induced obesity and metabolic syndrome (MS). Mannan-oligosaccharide (MOS) is widely used as a natural additive, and its effect on promoting fat metabolism has been reported. Here, we performed a 11-week study on C57BL/6J mice fed a high-fat diet (HFD) with/without MOS supplementation, and the results showed that MOS could attenuate high-fat diet induced metabolic syndrome, including slower body weight gain, lowered serum lipids and reduced insulin resistance. Next generation sequencing (NGS) of the gut microbiota indicated that MOS modulated the overall structure of the gut microbiome, which was highly correlated with MS parameters. Specifically, the intake of MOS decreased the Firmicutes/Bacteroidetes ratio and could reverse the changes in the relative abundance of several species caused by HFD, including Akkermansia muciniphila, Bacteroides acidifaciens, Lactobacillus gasseri and Bifidobacterium pseudolongum. Thus, MOS has the potential to be used as a new prebiotic for regulating the gut microbiota and helping in attenuating metabolic disorders.

The constitutively expression system for P. putida nitrilase was firstly constructed to improve the nicotinic acid production and reduce the production costs. High density culture strategy was employed to enhance the biomass and nitrilase production of recombinant strain. The total nitrilase activity reached up to 654 U·mL-1 without the induction. 541 g·L-1 nicotinic acid was accumulated via fed batch mode of substrate feeding through 290 min of conversion.

Most available methods for modifying the catalytic properties of enzymes are costly and time-consuming, as they rely on the information of enzyme crystal structure or require handling large amounts of mutants. This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

This study investigated the anti-inflammatory activity of ethanol extracts of Hericium erinaceus in the inflammatory bowel disease (IBD) model. Twenty C57BL/6 mice were exposed to 2% (w/v) dextran sulfate sodium (DSS) in their drinking water for 7 d to induce acute intestinal inflammation. Orally administrated ethanol extract of H. erinaceus (HEEE) (250 mg/kg/d and 500 mg/kg/d body weight) could significantly (P < 0.05) improve body weight and colon length and decreased the intestinal bleeding of DSS-treated mice compared with DSS-treated mice not given HEEE. HEEE markedly reduced DSS-induced myeloperoxidase accumulation in colon tissues, attenuated histological change in the neutrophils and lymphocyte infiltration, and protected the mucosal epithelium. Mechanistically, HEEE ameliorated colitis not only by suppressing the production of inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in colon tissues but also by adjusting the production of nitric oxide, malondialdehyde, and superoxide dismutase in serum to suppress the oxidative stress. These results suggest that HEEE can be applied as a protective agent in the treatment of IBDs.

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Large-scale submerged fermentation (SmF) of Antrodia camphorata (A. camphorata) usually encounters challenges including tedious preparation of mycelial inoculum, long fermentation period (10-14 d), and poor repeatability. Here we developed an asexual reproduction-based repeated batch fermentation (RBF) process for bioactive metabolites production by A. camphorata ATCC 200183. Compared with traditional batch fermentation, production time was shortened to 58 d from 80 d (overall time for eight cycles) using the RBF process established in this study, and accordingly, the productivities of bioactive metabolites (including antrodins) were improved by 40-60%. Kinetic parameters (α is 2.1-18.7 times as β) indicated that the cell growth was the major contribution for bioactive metabolites production. The RBF shows excellent batch-repeatability (Pearson correlation coefficient of 0.998±0.001), together with advantages of energy-efficient, low cost, and labor-saving, RBF process can be implemented to SmF by other filamentous fungi.

Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective.

β-Mannanases can efficiently hydrolyze both the polysaccharide mannan and manno-oligosaccharides. In order to characterize a novel mannanase for potential industrial application, an endo-1,4-β-mannanase from Bacillus subtilis YH12 was purified to homogeneity and its biochemical characterization was performed. The optimal conditions for the purified enzyme were pH 6.5 and 55 °C, and it remained stable at temperature up to 60 °C and pH of 4.5–7.5. The enzyme had specific activity on complex structure polysaccharide. And it showed high activity on locust bean gum, konjac powder, guar and fenugreek gums. Furthermore, it efficiently hydrolyzed xanthan, and carrageenan gums, and it exhibited no activity on starch, and xylan. The mannanase exhibited higher activity on galactomannan branched with (1→6)-linked α- d -galactose than glucomannan. The predominant products resulting from mannanase hydrolysis were 1–7 units of manno-oligosaccharides from locust bean gum, 2–7 units of manno-oligosaccharides from konjac powder, and 1–2 units of manno-oligosaccharides from xanthan gum. The B. subtilis YH12 mannanase encoding gene was successfully cloned. This study provided a novel mannanase that demonstrates potential application in food and animal feeds technology.

Aim Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) resulting from chronic liver diseases. Efficient and well-tolerated drugs for its treatment are urgently needed. This study aims to identify the active ingredients of Antrodia camphorata by a bioassay-guided fractionation approach and explore the acting mechanism by using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growth factor-β1 (TGF-β1). Methods The accumulation of collagens was evaluated using chromogenic precipitation reaction with picro-sirius red (PSR) dye solution and quantified by spectrophotometric analysis of the dissolved stain. MTT assay, cell migration assay, quantitative polymerase chain reaction and western blotting analysis were used to determine the cell viability, cell migration and gene expression. Results We established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents. Stimulation with 10 ng/mL TGF-β1 for 48 h and 200 μL PSR dye solution were optimal for the colorimetric assay in CFSC-8B cells. We used SB431542, silybin and another 11 antifibrotic reagents to verify the cellular model. Within the safe doses, they attenuated ECM production induced by TGF-β1. Bioactivity-guided fractionation led to the identification of antrodin B from A. camphorata. Antrodin B significantly ameliorated cell proliferation, cell migration, suppressed HSC activation marker α-smooth muscle actin expression and ECM components Col1, Col3 and Fn expression, and blocked the phosphorylation of Smad2/3 induced by TGF-β1 in CFSC-8B cells in a dose-dependent manner. Conclusion We developed a simple assay based on TGF-β1-induced total collagen accumulation in CFSC-8B cells and identified antrodin B which may serve as a potential candidate for treatment of liver fibrosis.

An α-keratinase producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Bacillus pumilus K9. The major amino acids liberated from the keratin degradation of wool by B. pumilus K9 were glutamic acid and leucine. The α-keratinase was purified to electrophoretic homogeneity with a molecular weight of 32000. The purified enzyme exhibits an optimum activity at 60 °C and pH=9.0. It was stable at pH values between 8 and 11. Bacillas pumilus keratinase displays a high activity towards casein, keratin, wool and feather, which indicates its wide application range. The keratinase was completely inhibited by phenylmethyl-sulfonyl fluoride(PMSF) and β-mercaptoethanol, and moderately inhibited by ethylemediamine-tetraacetic acid(EDTA), suggesting it is a metallo-cysteine keratinase. This enzyme could remain stable that could even be promoted in the presence of surfactants, including sodium dodecyl sulfate(SDS), Tween and Triton. And Tween 40 and Triton X-100 could substantially enhance the activity of the enzyme by 54% and 35%, respectively. It may indicate the prominent feature of the keratinase to tolerate surfactants. The enzymatic properties distinguish this keratinase from others in the literature. Furthermore, this enzyme is extremely stable in the presence of a commercially available detergent with 1% concentration. Detergents ARIEL, Bluemoon and WhiteCat can enhance the activity of the keratinase by 43.56%, 15.22%, and 22.48%, respectively.

Inflammatory bowel disease (IBD) is a disease caused by a dysregulated immune with unknown etiology. Hericium erinaceus (H. erinaceus) is a Chinese medicinal fungus, with the effect of prevention and treatment of gastrointestinal disorders. In this study, we have tested the anti-inflammatory effect of polysaccharide of H. erinaceus (HECP, Mw: 86.67 kDa) in the model of dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. Our data indicated that HECP could improve clinical symptoms and down-regulate key markers of oxidative stresses, including nitric oxide (NO), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and myeloperoxidase (MPO). HECP also suppressed the secretion of interleukin (IL)-6, interleukin (IL)-1β, tumor necrosis factor (TNF)-α and the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and decreased the expression of related mRNA. Meanwhile, HECP blocked phosphorylation of nuclear factor-κB (NF-κB) p65, NF-κB inhibitor alpha (IκB-α), mitogen-activated protein kinases (MAPK) and Protein kinase B (Akt) in DSS-treated mice. Moreover, HECP reversed DSS-induced gut dysbiosis and maintained intestinal barrier integrity. In conclusion, HECP ameliorates DSS-induced intestinal injury in mice, which suggests that HECP can serve as a protective dietary nutrient against IBD.

Medicinal mushroom Antrodia camphorata sporulate large numbers of arthroconidia in submerged fermentation, which is rarely reported in basidiomycetous fungi. Nevertheless, the molecular mechanisms underlying this asexual sporulation (conidiation) remain unclear. Here, we used comparative transcriptomic and proteomic approaches to elucidate possible signaling pathway relating to the asexual sporulation of A. camphorata. First, 104 differentially expressed proteins and 2586 differential cDNA sequences during the culture process of A. camphorata were identified by 2DE and RNA-seq, respectively. By applying bioinformatics analysis, a total of 67 genes which might play roles in the sporulation were obtained, and 18 of these genes, including fluG, sfgA, SfaD, flbA, flbB, flbC, flbD, nsdD, brlA, abaA, wetA, ganB, fadA, PkaA, veA, velB, vosA, and stuA might be involved in a potential FluG-mediated signaling pathway. Furthermore, the mRNA expression levels of the 18 genes in the proposed FluG-mediated signaling pathway were analyzed by quantitative real-time PCR. In summary, our study helps elucidate the molecular mechanisms underlying the asexual sporulation of A. camphorata, and provides also useful transcripts and proteome for further bioinformatics study of this valuable medicinal mushroom.

The objective of the study was to enhance the substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma (ARTP) and cell immobilization. The mutant library was constructed by ARTP and rapidly screened by an OPA-TCA microscale reaction. A mutant strain of mut-D3 was obtained and its optimum substrate concentration was improved to 150mM from 100mM. It could accumulate 189g/L nicotinic acid (NA) from 3-cyanopyridine (3-CP), which was increased by 42% compared with that of wild type (WT). Additionally, composite immobilization of mut-D3 was performed and SA-PVA immobilized cells could catalyze 250mM 3-CP each batch with finally accumulating 346g/L NA, while free cells accumulated 175g/L NA. These results indicated that the free or immobilized catalysts of mut-D3 could serve as a good choice for NA production. This is the first report on mutation breeding of nitrilase-producing microorganisms by ARTP.

Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

The I-V and C-V characteristics of Au/n-GaN Schottky diodes irradiated with 290-MeV U-238(32+) ions are presented. The U ions can penetrate the n-type GaN epi-layer with a thickness about 3 mu m grown on the c-plane of a sapphire substrate using the MOCVD technique, leaving a purely electronic energy deposition. The Au/n-GaN Schottky diodes were irradiated to successively increasing fluences from 1 x 10(9) to 5 x 10(11) ions cm(-2). The measured I-V curves show that the height of the Schottky barrier decreases after irradiation and that the Schottky barrier almost disappears when the ion fluence reaches 5 x 10(9) ions cm(-2). Meanwhile, the irradiation increases the series resistance. The C-V curves show that the capacitance drops sharply when the ion fluence reaches 5 x 10(10) ions cm(-2). The dielectric constant also decreases following the irradiation. The changes of the electrical properties are ascribed to the neutralization of the donor-like surface state and the acceptor-like surface state due to the migration of Au atoms at the interface of Au/n-GaN under energetic U ions irradiations. (C) 2014 Elsevier B.V. All rights reserved.

Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved.

Large-scale submerged fermentation (SmF) of Antrodia camphorata usually encounters engineering problems, such as long period and complex fermentation. In this study, a modified inoculation method using arthroconidia sporulated in liquid medium was developed for an efficient large-scale fermentation of A. camphorata ATCC 200183. The optimum environmental and nutritional conditions for arthroconidial germination in the dark control were estimated as follows: temperature, 26 °C; glucose, 20 g/L; soybean peptone, 10 g/L; Mg2+ concentration, 2 mmol/L; and initial pH, 3.5. All the arthroconidia rapidly germinated within 32 h after inoculation under optimal conditions. In the shaking flask, prepared according to the arthroconidial inoculation method, the fermentation period, maximal cell growth rate, and triterpenoid productivity of SmF were 0.6-, 1.7-, and 1.6-fold of those obtained from the culture prepared according to the conventional mycelial inoculation method. Furthermore, efficient fermentation was obtained in a 4000 L fermentor, with a biomass of 7.84 ± 0.20 g/L and a triterpenoid production of 61.21 ± 2.12 mg/L. Therefore, the modified inoculation method can be used in engineering applications with several advantages, including simplicity, good controllability, and high efficiency (shorten the operation time of mycelium-based inoculation from ≥240 h to 168 h).

Liver fibrosis is a wound-healing response to chronic liver injury that could lead to liver failure, but treatment remains ineffective. In this study, we investigated anti-hepatic fibrosis activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia from six commercially available medicinal mushrooms in submerged culture, namely Antrodia camphorata, Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. Their anti-fibrotic activities were evaluated via inhibition against accumulation of TGF-β1-induced collagen deposition in CFSC-8B cells. Hex, Chl, and MeOH extracts of A. camphorata and Hex extract of A. mellea significantly decreased collagen production. Bioactivity-guided fractionation led to the identification of seven compounds using UPLC-Q-TOF-MS from the Hex Fr.2 of A. camphorata. At the molecular level, Hex Fr.2 of A. camphorata suppressed α-SMA, Collagen I, Collagen III, and Fibronectin expression induced by TGF-β1 in CFSC-8B cells as indicated by qRT-PCR analysis. They also inhibited α-SMA and Collagen I protein expression according to western blot analyses. Mechanistically, Hex Fr.2 of A. camphorata negatively regulates TGF-β1/Smad2/3 signaling. Our studies demonstrate that A. camphorata has in vitro anti-hepatofibrotic activity and that there is great potential for the discovery of new drugs for the treatment of liver fibrosis by screening more medicinal mushrooms.

Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters Km and Vmax for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only

Inonotus obliquus is a medicinal mushroom used in Russian and Eastern European folk medicine for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. Previous studies in our laboratory have demonstrated that the mycelium powders of I. obliquus possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan. However, the active ingredients of mycelium powders responsible for the diabetes activity have not been identified. This study aims to identify the active ingredients of I. obliquus mycelium powders by a bioassay-guided fractionation approach and explore the mechanism of action of these active ingredients by using a well-established DPP-4 (an important enzyme as a new therapeutic target for diabetes) inhibitory assay model. The results showed the chloroform extract of mycelium was potential inhibitory against DPP-4. Bioactivity guided fractionation led to the identification of 19 compounds using UPLC-Q-TOF-MS. Molecular docking between the compounds and DPP-4 revealed that compounds 5, 8, 9, 14, 15 may be the active components responsible for the DPP-4 inhibitory activity.

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag+, Pb2+ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

Antrodia camphorata is a well-known Chinese medicinal mushroom that protects against diverse health-related conditions. Submerged fermentation of A. camphorata is an alternative choice for the effective production of bioactive metabolites, but the effects of nutrition and environment on mycelial morphology are largely unknown. In this study, we show that A. camphorata American Type Culture Collection 200183 can form arthrospores in the end of liquid fermentation. Different morphologies of A. camphorata in submerged culture were analyzed using scanning electron microscopy. The optimal carbon and nitrogen sources for sporulation were soluble starch and yeast extract. We found that a carbon-to-nitrogen ratio (C/N) of 40:1, MgSO4 (0.5 g/l), KH2PO4 (3.0 g/l), an initial pH 5.0, and an inoculum size of 1.5×10(5) spores/ml led to maximum production of arthroconidia. Our results will be useful in the regulation and optimization of A. camphorata cultures for efficient production of arthroconidia in submerged culture, which can be used as inocula in subsequent fermentation processes.

The main purpose of this study was to determine the volatile composition of Zhenjiang aromatic vinegar, one of the four famous China-style cereal vinegars, by using headspace solid-phase microextraction (HS-SPME)/gas chromatography-mass spectrum (GC-MS) and chemometrics. For this purpose, the HS-SPME sampling method for the volatile compounds of Zhenjiang aromatic vinegar was optimized by a second-order rotatable central composite experimental design (CCD). A HS extraction of the volatile compounds by incubation on a 65 µm thickness polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre during 44.2 min at 69.5°C with 1.9 g NaCl add gave the most effective and accurate extraction. By the optimized method, a total of 58 volatile compounds, including 9 alcohols, 13 acids, 16 esters, 5 aldehydes, 4 ketones and 8 heterocycle compounds, were identified from 13 aromatic vinegar samples manufactured in Zhenjiang region. By principal components analysis (PCA), the thirteen vinegar samples were classified into 3 groups, and 10 volatile compounds were chosen as characteristic compounds of Zhenjiang aromatic vinegars. Copyright © 2012 The Institute of Brewing & Distilling

Zhenjiang aromatic vinegar is one of the most famous Chinese traditional vinegars. In this study, change of the microbial community during its fermentation process was investigated. DGGE results showed that microbial community was comparatively stable, and the diversity has a disciplinary series of changes during the fermentation process. It was suggested that domestication of microbes and unique cycle-inoculation style used in the fermentation of Zhenjiang aromatic vinegar were responsible for comparatively stable of the microbial community. Furthermore, two clone libraries were constructed. The results showed that bacteria presented in the fermentation belonged to genus Lactobacillus, Acetobacter, Gluconacetobacter, Staphylococcus, Enterobacter, Pseudomonas, Flavobacterium and Sinorhizobium, while the fungi were genus Saccharomyces. DGGE combined with clone library analysis was an effective and credible technique for analyzing the microbial community during the fermentation process of Zhenjiang aromatic vinegar. Real-time PCR results suggested that the biomass showed a "system microbes self-domestication" process in the first 5 days, then reached a higher level at the 7th day before gradually decreasing until the fermentation ended at the 20th day. This is the first report to study the changes of microbial community during fermentation process of Chinese traditional solid-state fermentation of vinegar.

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu(2+), Mn(2+), and Fe(2+), respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus.

In this work a headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) and GC-olfactometry (GC-O) was developed to evaluate the profile of the volatile compounds that contribute to the aroma of Antrodia camphorata in submerged culture. For this purpose, the HS-SPME sampling method for the volatile compounds of A. camphorata in submerged culture was optimised by a D-optimal design. A HS extraction of the culture broth of A. camphorata followed by incubation on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre during 31.8min at 54.6°C gave the most effective and accurate extraction of the volatile compounds. By the optimised method, a total of 49 volatile compounds were identified in culture broth of A. camphorata, while a total of 55 volatile compounds were identified in the mycelia. A series of C(8) aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and l-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata.

Summary Zhenjiang aromatic vinegar, which is produced from sticky rice through solid-state fermentation, is highly prized as one of the four famous China-style vinegars, owing to its unique flavour. In this study, a method of supercritical fluid extraction (SFE) was used for the first time to recover aroma compounds from Zhenjiang aromatic vinegar. For this purpose, the optimal conditions for the extraction of aroma compounds by SFE were determined as follows: CO2 flow rate, 25 L h−1; extraction time, 2 h; extraction pressure, 35 MPa; and extraction temperature, 323 K. Using headspace solid-phase microextraction-gas chromatography–mass spectrometry (HS-SPME-GC-MS) analysis, a total of 49 and 44 aroma compounds were identified in Zhenjiang aromatic vinegar and its SFE extract, respectively. Acetic acid, ethyl acetate, furfural, phenethyl alcohol, tetramethyl-pyrazine, 3-hydroxybutanone and benzaldehyde were the main aroma compounds in the vinegar and its SFE extract. SFE is a fast and sensitive method to recovery aroma compounds from the vinegar.

BACKGROUND: Zhenjiang aromatic vinegar is one of the most well-known traditional fermented vinegars in China. Previously, a alkylpyrazine named ligustrazine was found as a bioactive compound in Zhenjiang aromatic vinegar. However, the generating mechanism of ligustrazine during the fermentation and storing process of vinegar was unknown. In this study, the changes in ligustrazine and its synthesis precursors (diacetyl and acetoin) in the industrial process were investigated by gas chromatography–mass spectrometry. RESULTS: During the fermentation process, ligustrazine was first detected at the fifth day, and content reached 75.90 µg g−1 when fermentation was complete. During the storage process, the content of ligustrazine increased with storage time, and the content in 6-year-old vinegar reached 696.63 mg L−1, which was about 20 times higher than raw vinegar (34.7 mg L−1). This may be attributed to the conversion of precursors to ligustrazine through the Maillard reaction as precursor contents decreased during the storage process. CONCLUSION: Results of this study showed that ligustrazine in vinegar was mainly formed during the storage process, and the fermentation process was regarded as the accumulation stage of precursors for ligustrazine synthesis. Copyright © 2011 Society of Chemical Industry

The antihyperglycaemic and antilipidperoxidative effects of the ethanol extract from the dry matter of a culture broth (DMCB) of Inonotus obliquus were investigated in alloxan-induced diabetic mice and the possible mechanism of action was also discussed. In alloxan-induced diabetic mice, treatment with the ethanol extract from DMCB of I. obliquus (30 and 60 mg kg(-1) body weight (b.w.) for 21 days) showed a significant decrease in blood glucose level: the percentage reductions on the 7th day were 11.54 and 11.15%, respectively. However, feeding of this drug for three weeks produced reduction of 22.51 and 24.32%. Furthermore, the ethanol extract from the DMCB of I. obliquus treatment significantly decreased serum contents of free fatty acids, total cholesterol, triglycerides and low-density lipoprotein-cholesterol, whereas it effectively increased high-density lipoprotein-cholesterol, insulin levels and hepatic glycogen contents in livers of diabetic mice. Besides this, the ethanol extracts from the DMCB treatment significantly increased catalase, superoxide dismutase and glutathione peroxidase activities, except for decreasing the maleic dialdehyde level in diabetic mice. Histological morphology examination showed that the ethanol extract from the DMCB of I. obliquus restored the damage of pancreatic tissues in mice with diabetes mellitus. The results showed that the ethanol extract from the DMCB of I. obliquus possesses significant antihyperglycaemic, antilipidperoxidative and antioxidant effects in alloxan-induced diabetic mice.

Niuchangchih (Antrodia camphorata (M. Zang & C.H. Su) Sheng H. Wu, Ryvarden & T.T. Chang) is a basidiomycete endemic to Taiwan. It is well known as a Traditional Chinese Medicine (TCM), and Taiwanese aborigines used this species to treat liver diseases and food and drug intoxication. The compounds identified in Niuchangchih are predominantly polysaccharides, triterpenoids, steroids, benzenoids and maleic/succinic acid derivatives. Recent research has revealed that Niuchangchih possesses extensive biological activity, such as hepatoprotective, antihypertensive, anti-hyperlipidemic, immuno-modulatory, anticancer, anti-inflammatory and antioxidant activities. The fruiting bodies and fermented products of Niuchangchih have been reported to exhibit activity when treating liver diseases, such as preventing ethanol-, CCl(4)- and cytokine-induced liver injury, inhibiting the hepatitis B virus, ameliorating fatty liver and liver fibrosis, and inhibiting liver cancer cells. This review will address the protective effects of Niuchangchih on the pathological development of liver diseases, and the underlying mechanisms of action are also discussed.