Search

Polysaccharides can be used as a potential hepatoprotective agent in the treatment of acute liver injury. However, the underlying mechanism governing how polysaccharides protect against acute liver injury induced by lipopolysaccharide/d-galactosamine (LPS/d-GalN) remains unclear. To investigate the mechanism, the anti-oxidative and anti-inflammatory action and pathways were determined. In this study, we investigated the hepatoprotective effects of Grifola frondosa polysaccharides (GFP), which are obtained from the fruiting body of Grifola frondosa, on (LPS/d-GalN)-induced liver injury in mice. Histopathological analyses showed that GFP protected against LPS/d-GalN-induced lung inflammation. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of the inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin (IL)-2, IL-6, and monocyte chemoattractant protein-1 (MCP-1) were inhibited by GFP. The LPS/d-GalN-induced myeloperoxidase (MPO) activity and malondialdehyde (MDA) content were inhibited by GFP. The levels of superoxide dismutase (SOD) and glutathione (GSH) were upregulated by GFP. The GFP-treated group showed reduced expression levels of miR-122 in liver tissue. Nrf2 has been identified as a potential target of miR-122. The western blotting results showed that GFP attenuates LPS/d-GalN-induced acute liver injury via upregulating transcription factors Nrf2, Nqo-1, and HO-1 and downregulating transcription factor Keap-1 in the Nrf2/ARE signaling pathway. In conclusion, these results indicated that GFP was highly effective in inhibiting liver injury and may be a promising potential therapeutic reagent for liver injury treatment. GFP exerts protective effects against LPS/d-GalN-induced liver injury in mice, which may be related to the regulation of the miR-122-Nrf2/ARE pathways.

The aim of this study was to investigate the immunomodulatory effect and mechanism of the glycopeptides from Paecilomyces sinensis (CPS-II) on ethanol induced ulcers in mice. In this study, histopathological evaluation (H&E staining) and the gastric ulcer score, ulcer index, total acid secretion and gastric pH value were used to determine the anti-ulcer activity. The expression levels of interleukin (IL)-6, interleukin (IL)-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA. The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and epidermal growth factor (PEG2) in serum were measured according to the instructions for the reagents. Western blotting was used to detect the effect of CPS-II on the MEK/ERK pathway. The results showed that CPS-II could inhibit the ulcer score and ulcer index compared with the disease control group. CPS-II could significantly increase gastric pH and decrease gastric acid secretion in mice. The ELISA analysis showed that the expression levels of IL-6 and TNF-α in the CPS-II treatment group were significantly decreased, while the expression levels of IL-10 were significantly increased in the CPS-II treatment group. In the resveratrol treatment group, the content of MDA in serum was decreased, and the level of PEG2 and the activity of SOD in serum were significantly increased, which indicated that CPS-II has immunoregulation and anti-ulcer properties. The CPS-II treatment group could reduce the expression level of miR-9-5p in gastric tissue. pEGFR had been identified as a potential target of miR-9-5p. Western blot analysis showed that CPS-II could up-regulate the relative protein expression of pEGFR/EGFR, pRaf/Raf, pMEK/MEK, pERK/ERK, and ZO-1. The results showed that CPS-II could reduce oxidative stress and inflammatory response by regulating the miR-9-5p-MEK/ERK signaling pathway, thus protecting the gastric mucosa and improving stress gastric ulcers.

On account of high oxidation ability of sulfate radical-based advanced oxidation processes (AOPs), the eco-friendly catalysts for peroxymonosulfate (PMS) activation have received considerable attentions. Previous studies mainly focused on Cobalt-based catalyst due to its high activation efficiency, such as Co3O4/MnO2 and FeCo-layered double hydroxide (LDH), whereas Cobalt-based catalyst usually has serious risk to environment. To avoid this risk, MnFe-LDH was primarily synthesized in this research by simple co-precipitation and subsequently utilized as an effective catalyst for peroxymonosulfate (PMS) activation to degrade organic pollutants. The experimental results demonstrated that MnFe-LDH with a lower dosage (0.20 g/L) could efficiently activate PMS to achieve 97.56% removal of target organic pollutants Acid Orange 7 (AO7). The AO7 degradation process followed the pseudo-first-order kinetic well with an activation energy of 21.32 kJ/mol. The intrinsic influencing mechanism was also investigated. The quenching experiment and electron spin resonance (ESR) indicated that sulfate and hydroxyl radicals were produced by the effective activation of PMS by MnFe-LDH, resulting in a high rate of decolorization. The possible AO7 removal pathway in the constructed MnFe-LDH/PMS system was presented on the basis of UV–vis spectrum analysis and GC–MS, which suggested that the AO7 degradation was firstly initiated by breaking azo linkages, then generated phenyl and naphthalene intermediates and finally presented as ring-opening products. This effective MnFe-LDH/PMS system showed great application potential in the purification of wastewater contaminated by refractory organic pollutants.

Polysaccharides are closely associated with immune regulation. In this study, the aim was to investigate the effect of polysaccharides from Cordyceps gunnii mycelia (PPS) in cyclophosphamide (CTX)-induced immunodeficient mice. Compared with the CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice with orally administered PPS were significantly increased, body weight loss was alleviated, and the natural killer (NK) cytotoxicity and proliferative activities of the lymphocytes were elevated. The recovery of peripheral white blood cells, red blood cells, hemoglobins and platelets was accelerated. Furthermore, the results from ELISA showed that PPS could up-regulate the serum levels of IL-2, IL-12, IFN-γ and IgG, and reduce the level of TGF-β. Histopathological analysis of the spleen revealed the protective effect of PPS against CTX-induced immunosuppression. Western blotting results showed that PPS possessed immunomodulatory activity via TLR4/TRAF6/NF-κB signalling pathways. Finally, the intestinal absorption of PPS was poor, as detected in the Caco-2 transwell system. Taken together, these findings suggest that PPS plays a crucial role in protection against immunosuppression in cyclophosphamide-treated mice and could be a potential candidate for use in immune therapy regimens.

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

The effectiveness of deacidification technologies for high-acid rice bran oil (HRBO) are still one of the main challenges in the industrialization process of rice bran oil (RBO). A novel function of glycerol-based deep eutectic solvents (DESs) as an acyl acceptor in combination with lipases for enzymatic esterification of deacidification of HRBO was described in this study. The maximum deacidification efficiency of 92.38% was obtained under the optimized reaction conditions. Molecular docking simulation indicated that the minimum binding energy of Novozym 435 was lower than those of Lipozyme TL IM and Lipozyme RM IM, which might be one of the underlying mechanisms for its better performance. Additionally, γ-oryzanols, tocopherols and tocotrienols were retained in deacidified RBO. We are convinced that the reusability of biocatalyst and DESs systems provide an alternative strategy to design a facile, environmentally-friendliness DESs towards economic feasibility of applications and processes for oil-refined industries.

// Lihua Hou 1 , Meng Meng 1 , Yuanyuan Chen 1 and Chunling Wang 1 1 Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China Correspondence to: Chunling Wang, email: wangchunling@tust.edu.cn Keywords: water-soluble polysaccharide, immunomodulatory, RAW264.7 macrophages, TLR4, NF-κB Received: May 27, 2017 Accepted: August 06, 2017 Published: September 23, 2017 ABSTRACT Here, the immunomodulatory effects of water-soluble polysaccharide from Grifola frondosa on RAW264.7 macrophages and its molecular mechanisms were investigated. G. frondosa polysaccharide could obviously enhance immunostimulatory activity such as the release of nitric oxide and cytokine production. Western blotting results showed that G. frondosa polysaccharide elevated the TLR4, which might act as an upstream regulator of MyD88 induced G. frondosa polysaccharide. MyD88 promoted IKKβ in endochylema and translocate NF-κB p65 subunit into the nucleus which increased the NO production and cytokine/chemokines level. The results suggested that G. frondosa polysaccharide activated macrophages through TLR4-MyD88-IKKβ-NF-κBp65 signaling pathways.

Aluminum (Al) has been well known as an environmental factor that may affect several enzymes and other biomolecules related to Alzheimer's disease. The increasing use of Al in the preparation and storage of food currently represents the main form of Al exposure for the general public. The present study was aimed to develop a household procedure for the rapid test determination of Al in edible jellyfish. The method was developed based on the reaction of Chrome Azurol S with Al in acidic medium, forming a colored compound on the surface of filter paper. Experimental design methodologies were used to optimize the measurement conditions. The proposed method was applied successfully to the analysis of Al in edible jellyfish products in clinical laboratory and household settings.

Aluminum (Al) has been linked to the development of some cardiovascular diseases and mung bean is a functional food with the ability to detoxify. We aimed to evaluate the preventive effect and possible underlying mechanisms of the mung bean polyphenol extract (MPE) on Al-induced cardiotoxicity. Control, AlCl3 (171.8 mg Al per kg body weight), MPE + AlCl3 (Al-treatment plus 200 mg MPE per kg body weight), and a group of MPE per se were used. Al intake induced a significant increase of serum CK and LDH activity and the level of Na+, Ca2+, malondialdehyde and advanced oxidation protein products in the AlCl3-treated rats’ heart tissue. Administration of MPE significantly improved the integrity and normal ion levels of heart tissue, and attenuated oxidative damage and the accumulation of Al in Al-treated rats. MPE significantly inhibited Al-induced increase of myocardial p-JNK, cytoplasmic NF-κB, cytochrome C, and caspase-9 protein expressions. Therefore, these results showed that MPE has a cardiac protective effect against Al-induced biotoxicity through ROS-JNK and NF-κB-mediated caspase pathways. Furthermore, the stability constant for the vitexin-Al complex was analyzed (log K = log K1 + log K2 = 4.91 + 4.85 = 9.76). We found that MPE-mediated protection against Al-cardiotoxicity is connected both with MPE antioxidant and chelation properties.

In this paper, the supercritical carbon dioxide extraction of oleoresin from Dao-kou roasted chicken flavor spice blends was optimized under particular conditions of extraction temperature, pressure, and time by response surface methodology (RSM). On the basis of single-factor experiments, the Box-Behnken design with three factors and three levels was applied to obtain the optimum process parameters for good extraction ratio. The optimum extraction conditions are as follows: extraction temperature 48 °C, pressure 39 MPa, and time 134 min. Under such conditions, the oleoresin extraction ratio reached 3.68 % experimentally. The flavor effect of that extracted by supercritical fluid extraction (SFE) on chicken sausage base was compared with that extracted by steam distillation, alcohol, and water, and the composition of essential oils were analyzed by GC-MS/MS. The results showed that 65 compounds could be extracted by SFE; it was better than that extracted by traditional methods of 63, 46, and 34 compounds; and the flavor effect was nicer than that extracted by other three methods.

The fermentation performance of a genome-shuffled strain of Candida versatilis S3-5, isolated for improved tolerance to salt, and wild-type (WT) strain were analysed. The fermentation parameters, such as growth, reducing sugar, ethanol, organic acids and volatile compounds, were detected during soy sauce fermentation process. RESULTS The results showed that ethanol produced by the genome shuffled strain S3-5 was increasing at a faster rate and to a greater extent than WT. At the end of the fermentation, malic acid, citric acid and succinic acid formed in tricarboxylic acid cycle after S3-5 treatment elevated by 39.20%, 6.85% and 17.09% compared to WT, respectively. Moreover, flavour compounds such as phenethyl acetate, ethyl vanillate, ethyl acetate, isoamyl acetate, ethyl myristate, ethyl pentadecanoate, ethyl palmitate and phenylacetaldehyde produced by S3-5 were 2.26, 2.12, 2.87, 34.41, 6.32, 13.64, 2.23 and 78.85 times as compared to WT. CONCLUSIONS S3-5 exhibited enhanced metabolic ability as compared to the wild-type strain, improved conversion of sugars to ethanol, metabolism of organic acid and formation of volatile compounds, especially esters, Moreover, S3-5 might be an ester-flavour type salt-tolerant yeast. ? 2016 Society of Chemical Industry

Microcapsules are attractive core-shell configurations for studies of controlled release, biomolecular sensing, artificial microbial environments, and spherical film buckling. However, the production of microcapsules with ultra-thin shells remains a challenge. Here we develop a simple and practical osmolarity-controlled swelling method for the mass production of monodisperse microcapsules with ultra-thin shells via water-in-oil-in-water (W/O/W) double-emulsion drops templating. The size and shell thickness of the double-emulsion drops are precisely tuned by changing the osmotic pressure between the inner cores and the suspending medium, indicating the practicability and effectiveness of this swelling method in tuning the shell thickness of double-emulsion drops and the resultant microcapsules. This method enables the production of microcapsules even with an ultra-thin shell less than hundreds of nanometers, which overcomes the difficulty in producing ultra-thin-shell microcapsules using the classic microfluidic emulsion technologies. In addition, the ultra-thin-shell microcapsules can maintain their intact spherical shape for up to 1 year without rupturing in our long-term observation. We believe that the osmolarity-controlled swelling method will be useful in generating ultra-thin-shell polydimethylsiloxane (PDMS) microcapsules for long-term encapsulation, and for thin film folding, buckling and rupturing investigation.

In recent decades, a growing number of refractory organic pollutants with serious health risks are frequently detected in aquatic environment. Sulfate radical (SO4 −)-based advanced oxidation process (SR-AOP) under light irradiation (ultraviolet (UV) light, visible light or simulated solar light) is considered as a prospective method for refractory organic pollutants degradation. The satisfactory removal depends on the generation of active radicals SO4 − as well as OH from the activation of persulfate (PS) and peroxymonosulfate (PMS). In this paper, different methods of PS/PMS photo-activation, including catalyst-free, metal catalyst and metal-free catalyst activation, under different light sources as well as their activation mechanisms are reviewed comprehensively. The effects of key parameters are commendably assessed, such as PS/PMS concentration, catalyst dosage, initial contaminant concentration, pH, and coexisting organic or inorganic matters. This study will help to deeply understand the photo-activated SR-AOP for the refractory organic pollutants removal and present better perspectives for future researches.

In this study, a novel persulfate combined with iron-carbon microelectrolysis (PS-ICME) system was explored to pretreat the landfill leachate. In the static batch experiments, response surface methodology (RSM) was used to determine the relationship between three independent variables (pH, the ratio of iron to carbon (Fe-C ratio), persulfate dosage) and response values (Chemical oxygen demand (COD) removal efficiency). Experimental results showed that the COD removal efficiency reached to 62.91% under the optimal conditions: initial pH 7, Fe-C ratio 3, and persulfate dosage 85 mM. The dissolved organic matter (DOM) in landfill leachate was characterized by three-dimensional excitation-emission matrix spectroscopy (3D-EEMs). Combined with electron spin resonance (ESR) spectrum investigation, the enhanced mechanism for landfill leachate pretreated by PS-ICE in near-neutral pH was elucidated. In the column continuous flow experiment, it had been confirmed that dissolved oxygen plays an important role in the PS-ICME system. Based on the above conclusions, PS-ICME system has a satisfactory performance on pretreatment of landfill leachate.

The salt-tolerant yeast strain Candida versatilis is usually added to high-salt, liquid-state soy sauce fermentation. The genome of C. versatilis was sequenced in our previous study but the reason for its high-osmolarity ability was not clear. RESULTS The 9.7 Mbp genome of C. versatilis contained 4711 CDS. Candida versatilis was the closest to another yeast, Zygosaccharomyces rouxii, added to soy sauce fermentation. The protein sequence of the whole genome was divided into 4338 groups, accounting for 92.1% of all the predicted protein of C. versatilis using OrthoMCL. Mitogen-activated protein kinase (MAPK) signal pathways, including high osmolarity and cell integrity, were predicted and proved by investigating the expression changes of the key genes CvHOG1, CvGPD1, and CvFPS1 in a high osmotic environment and by testing the variations of intracellular glycerol and extracellular glycerol. CONCLUSION Candida versatilis exhibited strong osmotolerance because it could synthesize intracellular glycerol and absorb glycerol from the environment cooperated with the shut down of glycerol efflux channel in membrane. ? 2018 Society of Chemical Industry

A novel method was explored to improve the waste-activated sludge (WAS) dewaterability using Fe(II) combined with calcium hypochlorite, and the possible mechanisms were investigated simultaneously. Capillary suction time (CST), specific resistance to filtration (SRF) of sludge, and water content (WC) of dewatered sludge cake were selected as the factors to evaluate the sludge dewaterability. The maximum reduction of WC (30.76%) was achieved under the optimal conditions of Ca(ClO)2 0.04 g/g total suspended solids (TSS), FeSO4·7H2O 0.097 g/g TSS, and pH 7.3, while the reduction of CST and SRF reached 91.24 and 99.47%, respectively. Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy of extracellular polymeric substances (EPSs) showed that sludge dewaterability was mainly related to the degradation of tryptophan and tyrosine protein-like substances in sludge EPS. Economic analysis suggested Fe(II) combined with calcium hypochlorite treatment had greater potential on enhancing WAS dewaterability compared with the traditional sludge treatment.

Two novel HLA class II alleles, DRB4*03:01N and DQB1*03:276N, containing large deletions were identified during routine typing. Extraction of DNA encompassing the deletions was carried out with a panel of capture oligonucleotides followed by whole genome amplification. Next generation DNA sequencing was then used to characterize the sequences. DRB4*03:01N has a 16 kilobase pair deletion stretching upstream from intron 2 toward centromeric DRB8. DQB1*03:276N has two deletions separated by 844 nucleotides. The first deletion (3.7 kilobase pairs) is upstream of intron 1 and the second deletion removes 3.3 kilobase pairs further upstream towards centromeric DQA2.

Surfactin is primarily produced by Bacillus natto TK-1 and is one of the most powerful biosurfactants. It consists of a heptapeptide interlinked with a β-hydroxy fatty acid. Because of its special structure, surfactin shows broad biological effects, including anti-tumour, anti-microbial and anti-mycoplasma activities. It also has potential anti-inflammatory activity; however, the anti-inflammatory mechanism of surfactin has not been explored. In this study, we investigated the anti-inflammatory mechanism of surfactin in lipopolysaccharide (LPS)-stimulated macrophages. Surfactin exhibited an anti-inflammatory effect without cytotoxicity at certain concentrations, and the lipopolysaccharide (LPS)-stimulated cells appeared normal after surfactin treatment. Surfactin significantly inhibited the increased expression of IFN-γ, IL-6, iNOS and nitric oxide (NO). TLR4 is the critical receptor for LPS; therefore, the TLR4 signal transduction pathway is the primary pathway that mediates LPS-induced inflammation. The results show that surfactin downregulated the LPS-induced TLR4 protein expression of macrophages and indicated that the surfactin-mediated signal pathway was involved in with TLR4. The subsequent studies demonstrated that surfactin exhibited anti-inflammatory effects by attenuating the activation of nuclear factor-κB (NF-κB), which is involved in the nuclear factor-κB (NF-κB) cell signalling pathways. These results suggest that surfactin may be a new therapeutic agent for inflammation.

As one of the major microbes in the soy sauce fermentation, Candida versatilis enriches the flavor and improves the quality of soy sauce. In this study, a combination of five different GC-MS and LC-MS-based metabolome analytical approaches was used to analyze the intracellular, extracellular and whole metabolites of C. versatilis. Our results found out that a total of 132, 244 and 267 different metabolites were detectable from the intracellular, extracellular and whole part, respectively. When exposed to 0. 9 and 18 % salt, respectively, 114, 123 and 129 different intracellular metabolites, 184, 200 and 178 extracellular metabolites and 177, 188 and 186 whole metabolites were detected, respectively. Our data showed that salt enhances the metabolic capacity of C. versatilis, especially its amino acid and enhances the synthesis and secretion of some metabolites of C. versatilis, especially the aldehydes and phenols, such as vanillin, guaiacol and 5-hydroxymethylfurfural. Our data also showed that special attention has to be paid to the generation of biogenic amines when C. versatilis was treated with salt.

Grifola frondosa is an important fungal research resource. However, there was little report about hyperglycemic activity of Grifola frondosa polysaccharide on insulin resistance in vitro. In this study, the hypoglycemic activity of a polysaccharide obtained from Grifola frondosa (GFP) on HepG2 cell and hpyerglycemic mechanism were investigated. The purity of the isolated polysaccharides was examined by HPLC. In this research, it was found that GFP enhanced the absorption of glucose of HepG2 cells in a dose dependent manner at 24 h of 30 ugmL⁻¹. GC-MS and FT-IR spectroscopy analysis results showed that glucose and galactose were the dominant monosaccharides in GFP and the major component of GFP was β-pyranoside. Western-blotting results showed that the HepG2 cell model treated with GFP activated the insulin receptor protein (IRS) in the cell membrane and increased phosphorylated-AktSer473 expression, which had an inhibition of glycogen synthase kinase (GSK-3). The down-regulation of GSK-3 stimulated synthesis of intracellular glycogen. The results above suggested that the GFP increased the metabolism of glucose and stimulated synthesis of intracellular glycogen through the Akt/GSK-3 pathway.

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na+, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na+/H+-antiporter played a primary role in Na+ extrusion and H+-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na+, enhance H+-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.

Cells transport mass dynamically, crossing cell membranes to maintain metabolism and systemic homeostasis, through which biomolecules are also delivered to cells for gene editing, cell reprograming, therapy, and other purposes. Quantifying the translocation kinetics is fundamentally and clinically essential, but remains limited by fluorescence-based technologies, which are semi-quantitative and only provide kinetics information at cellular level or in discrete time. Herein, a real-time method of quantifying cell internalization kinetics is reported using functionalized firefly-luciferase nanocapsules as the probe. This quantitative assay will facilitate the rational design of delivery vectors and enable high-throughput screening of peptides and other functional molecules, constituting an effective tool for broad applications, including drug development and cancer therapy.

BACKGROUND This study aimed to enhance and improve the quality and safety of soy sauce. In the present work, the change of biogenic amines, such as histamine, tyramine, cadaverine, spermidine, was examined by the treatment of Candida versatilis and Zygosaccharomyces rouxii, and the influence of salt-tolerant yeast on biogenic amines was analysed during the whole fermentation process. RESULTS The results showed that the content of biogenic amines was elevated after yeast treatment and the content of biogenic amines was influenced by using yeast. The dominating biogenic amine in soy sauce was tyramine. At the end of fermentation, the concentrations of biogenic amines produced by Zygosaccharomyces rouxii and Candida versatilis in the soy mash were 122.71 mg kg−1 and 69.96 mg kg−1. CONCLUSIONS The changes of biogenic amines in high-salt liquid soy mash during fermentation process indicated that a variety of biogenic amines were increased in the fermentation ageing period, which may be due to amino acid decarboxylation to form biogenic amines by yeast decarboxylase. The fermentation period of soy sauce should be longer than 5 months because biogenic amines began to decline after this time period. © 2013 Society of Chemical Industry

Summary The salt-tolerant Torulopsis versatilis mutant strain, T3-5, is able to grow in YPD medium containing 22% salt, whereas the original strain, T, used for soy sauce fermentation was only able to grow in the presence of 20% or less. Biochemical analyses showed that strain T3-5 had a 22% higher intracellular glycerol level than T. Sequencing of the glycerol transporter gene FPS1 revealed a change from G to A at position 198 upstream of the start codon, changes from G to A at positions 93 and 1084, respectively, within the FPS1 gene, and insertions of T base at positions 285 and 310, respectively, downstream of the termination codon when comparing the corresponding sequences of strains T and T3-5. An inverse relationship between osmotic pressure and the FPS1 transcription level was observed for both T and T3-5 using qPCR. Therefore, it is possible that lower transcription levels of FPS1 are at least partly responsible for increased osmotolerance in strain T3-5. Overexpression of T and T3-5 FPS1 in Saccharomyces cerevisiae resulted in lower salt tolerance, especially in the presence of 12% salt. The data suggest that decreased expression levels of and/or alteration in the FPS1 protein contributed to increased salt tolerance of T3-5.

Although surfactin is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism responsible for this process remain elusive. In this study, the signaling network underlying the apoptosis of human hepatoma (HepG2) cells induced by surfactin was investigated. It is found that the reaction oxygen species (ROS) production and intracellular calcium ([Ca(2+)]i) accumulation are both induced HepG2 cells apoptosis. The [Ca(2+)]i exaltation was partly depended on the Ca(2+) release from inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry) receptors channels, which both triggered endoplasmic reticulum stress (ERS). The results showed that surfactin induced the ROS production and ROS production led to ERS. The occurrence of ERS increased the [Ca(2+)]i level and the processes associated with blocking extracellular signal-regulated kinase (ERK) pathway. According to a comprehensive review of all the evidence, it is concluded that surfactin induces apoptosis of HepG2 cells through a ROS-ERS-Ca(2+) mediated ERK pathway.

Trehalose can protect organisms against disadvantage conditions, such as high osmolarity, heat, oxidation, desiccation and freezing. In this work, the ability to produce trehalose of soy sauce yeasts was studied first and the results showed that trehalose could be accumulated gradually in logarithmic phase and achieve a concentration peak in stationary phase. Then the relevance of growth conditions and intracellular trehalose level of soy sauce yeasts was investigated. Mutant strains (S3-2, T3-5) which can grow in hypertonic conditions, showed different results from their original organisms Zygosaccharomyces rouxii (S) and Torulopsis versatilis (T). For S and T could increase trehalose level from 7.0% and 7.5% to 8.4% and 9.6% with 9% salt stimulation, respectively. While the mutants could accumulate more trehalose under a higher salt (about 12%) stimulation compared to 0% NaCl situation, which suggested a significant feature of the mutants. And the original strains could increase the trehalose level from 8.4% and 9.6% to 10.1% and 10.9% respectively under heat shock (40 °C, 30mins). Moreover, the mutant strains could also product more trehalose in the thermal stimulation condition, but less than the original ones.

Although Musca domestica larvae lectin (MLL) is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism(s) responsible for these processes remain elusive. In the current study, the signaling network underlying the MLL-induced apoptosis of human hepatoma BEL-7402 cell was investigated. Our data found out that MLL causes a sustained increase of the intracellular Ca(2+) and this process was prevented by the intracellular calcium chelator, BAPTA-AM, suggesting the involvement of intracellular Ca(2+) in MLL-induced cell apoptosis. MLL also causes the production of reactive oxygen species and elevates the phosphorylation status of JNK, processes associated with the increased cytoplasmic Ca(2+). The mitochondrial permeability transition pore (MPTP) opening study showed that MLL treatment of BEL-7402 cells results in the opening of MPTP and a reduction of mitochondrial transmembrane potential. In such condition, cytochrome-c was detected to be released from mitochondria to cytoplasm through the MPTP. This eventually activates caspase-3 and thus results in apoptosis of the tested BEL-7402 cells. According to a comprehensive review of all the evidence, it is concluded that MLL induces apoptosis of BEL-7402 cells through a Ca(2+)/JNK-mediated MPTP pathway.

Summary Soy sauce is considered the most important food condiment in China for its distinct fragrance and salty taste. Highly valued for its unique tastes and flavours, soy sauce exhibits many beneficial functions, such as 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone and 4-ethylguaiacol. Soy sauce has been widely used in Asian countries for cooking. With the rapidly growing popularity of these unique tastes and flavours, it is important to have a comprehensive reference of its functional benefits. Meanwhile, the genes encoding the major enzymes of metabolism exhibit specialised roles. This review provides a valuable source for the abundant metabolism genes of strains and current knowledge on antioxidant activity, antihypertensive effect, anticarcinogenic activity, fortification and their relationships with soy sauce tastes and flavours.

The possible health impact of the exposures to Al from environment would be inevitable for humans. Using chelating agents and natural antioxidants against Al-induced biotoxicity become a natural and modern way to prevent the adverse effects of Al in people. This study was undertaken to determine the effectiveness of chlorogenic acid (CGA, 5-O-caffeoylquinic acid) in preventing aluminum chloride (AlCl3) induced hepatotoxicity and hematotoxicity in mice. Control, Al-treated (a single injection of 25 mg Al3+/kg, i.p.), Al + CGA (2 h after, a single dose of 100 mg/kg, i.p.), CGA + Al (administered to mice daily for 5 days at 30 mg/kg before Al-treatment) and group of CGA per se (administered to mice daily for 5 days at 30 mg/kg) were used. The levels of Al in liver and blood, the activities of transaminases in serum and osmotic fragility were increased by comparison with the control, while the activities of superoxide dismutase and catalase decreased significantly in the Al-treated group. However, treating mice with CGA at either dosing regimens, post- or pre- Al administration alleviate Al oxidative damaging effects, stabilize cell membrane, prevent hepatocyte apoptosis. CGA supplementation may be favorable to avoid Al-induced hematotoxicity and hepatotoxicity for humans.

An excess of dietary salt is the most common factor that contributes to the pathogenesis of hypertension. Dietary management is deemed critical to prevent and treat hypertension. We aimed at evaluating the preventive effect of the buckwheat (BW)-enriched diet on high salt-induced elevation of blood pressure (BP) and renal injury in order to provide a new focus on the design of strategies to prevent hypertension. Control, high salt (HS)-treated (8%), HS + tartary buckwheat (TB) (8% HS + 15% TB), and a group of HS + common buckwheat (CB) (8% HS + 15% CB) rats were used for 7 weeks. BP was monitored periodically during the study by the tail cuff method. HS intake caused a significant increase of BP, the level of serum Na(+) compared to the control group. BW significantly prevented the increase of BP, attenuated oxidative damage, and improved Na(+)/K(+)-ATPase activity in HS treated rats. These results show that a diet supplemented with whole BW has beneficial effects on hypertension, by decreasing blood pressure values and oxidative stress.

A water-soluble polysaccharide (BP-1) was obtained from highland barley (Hordeum vulgare L.) by hot water extraction and purification of sepharose column chromatography. BP-1 had an average molecular weight of about 6.7×104Da and was composed of glucose (Glc), xylose (Xyl), arabinose (Ara) and rhamnose (Rha) with a relative molar ratio of 8.82:1.92:1.50:1.00. It was found that BP-1 inhibited proliferation of human colon cancer cells (HT-29) in a time- and dose-dependent manner with half maximal inhibitory concentration at 48h of 48.18μg/mL. Western blotting results showed that BP-1 enhanced the phosphorylation of c-Jun N-terminal kinase (JNK), processes associated with the reactive oxygen species (ROS) formation and inhibited nuclear factor-κB (NF-κB) translocation from cytoplasm into nucleus. Meanwhile, the BP-1-induced apoptosis was related to the regulation of apoptosis-associated proteins, such as B-cell lymphoma-2 (Bcl-2), release of cytochrome C from mitochondria to cytoplasm and activation of caspase-8 and caspase-9. These results suggest that BP-1-induced HT-29 apoptosis through ROS-JNK and NF-κB-mediated caspase pathways.

A novel thermal sensitive hydrogel was formulated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and alpha, beta-glycerophosphate (alpha, beta-GP). A serial of hydrogels containing different amount of GP and HTCC with diverse quarternize degree (QD, 41%, 59%, 79.5%, and 99%) were prepared and characterized by rheological method. The hydrogel was subsequently evaluated for intranasal vaccine delivery with adenovirus based Zaire Ebola virus glycoprotein antigen (Ad-GPZ). Results showed that moderate quarternized HTCC (60% and 79.5%) hydrogel/antigen formulations induced highest IgG, IgG1, and IgG2a antibody titers in serum, as well as mucosal IgA responses in lung wash, which may attributed to the prolonged antigen residence time due to the thermal-sensitivity of this hydrogel. Furthermore, CD8(+) splenocytes for IFN-gamma positive cell assay and the release profile of Th1/Th2 type cytokines (IFN-gamma, IL-2, IL-10, and IL-4) showed that hydrogel/Ad-GPZ generated an overwhelmingly enhanced Th1 biased cellular immune response. In addition, this hydrogel displayed low toxicity to nasal tissue and epithelial cells even by frequently intranasal dosing of hydrogel. All these results strongly supported this hydrogel as a safe and effective delivery system for nasal immunization. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

Whole‐cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as alcoholic strength, temperature, pH and concentration of solute. Here, we describe the use of genome shuffling to improve the ethanol tolerance of acetic acid bacterium, which is a poorly characterised industrial strain. The best strain A3‐11 was constructed and selected after three rounds of shuffling from the wild‐type strain of acetic acid bacterium. A3‐11 could grow in liquid medium with 13% ethanol. During the vinegar fermentation, the organic acid components produced were distinctly improved by A3‐11. It was found that the content of acetic acid was twice as much as the control. Furthermore, the concentration of tartaric acid was 4.04‐fold more than the control. Notably, A3‐11 increased in butanedioic acid production by up to 71.73% compared with the control.

Summary Because of secreting large amounts of enzymes, Aspergillus oryzae was widely used in the fermentation of soy sauce in many Asian countries. Here, N+ ion implantation mutagenesis was applied to improve the ability of A. oryzae to secrete acid protease. Several mutants were screened using three kinds of plates (Casein medium, Czapek’s medium and carboxymethyl cellulose medium agar plates). Compared with the other mutants, the mutant A100-8 could secrete the most protease. Acid protease activity of A100-8 was enhanced about 44.1% at 36 h by koji fermentation test. In addition, A100-8 was stable by periodically subculturing and maintaining on the agar slant. The mRNA expression levels of two kinds of acid protease (serine carboxypeptidase and aspartyl protease) were measured by RT-PCR at different periods. Serine carboxypeptidase and some kinds of aspartyl protease of A100-8 were significantly (P

Tremella fuciformis, a typical dimorphic fungus, displays two distinct morphological transitions: (i) yeast-pseudohyphae transition; (ii) yeast-hyphae transition. To understand the mechanism of its dimorphism, we initiated a study by investigating how environmental factors affect the yeast-to-pseudohypha transition, including pH value, temperature, carbon source, nitrogen source, cultural time, ethanol and inoculum size. Our studies showed that the optimal conditions for yeast-to-pseudohypha transition were as follows: incubating at 25 to 30°C for 3 to 7 days in media containing glucose as the carbon source, ammonium as the nitrogen source and 10 ml/L ethanol. Below a threshold of inoculum size (105 cells/L), the transition ratio fluctuated markedly, which made us propose that quorum sensing molecules contribute to the dimorphic behavior in T. fuciformis.

The present study reported that nitric oxide (NO) was up-regulated by the induction of lectin purified from Musca domestica pupa (MPL) in macrophages without cytotoxicity. The mRNA expression and protein secretion of inducible nitric oxide synthase (iNOS) were strongly induced by MPL treatments. Subsequent investigation revealed that the nuclear factor-κB (NF-κB) inhibitory κB (IκB) in endochylema was inhibited and NF-κB translocated into the nucleus after MPL treatment. Meanwhile, the IKKβ was strongly induced and the production of the toll-like receptor 4 (TLR4) was significantly up-regulated. Moreover, MPL increased NO production via inducing the expression of iNOS through the activation of NF-κB, which suggested that MPL probably acted as an activating agent of the NF-κB activation.

The aim of this study was mainly to determine anticoagulant activity of a chemically sulfated polysaccharide (S-GFB), which was derived from water-insoluble polysaccharide of Grifola frondosa mycelia. Through 14-day seed culture, maximum mycelial biomass and polysaccharides, which appeared at about the 90th hour fermentation, were 7.9 and 1.3 g/L, respectively. The sulfur content in S-GFB was 17.7% (w/w). The molecular structure images of S-GFB and GFB were seen with atomic force microscopy. S-GFB showed particle lumps at 1 µg/mL, which was different from the long polysaccharide chain of GFB at 1 µg/mL. Anticoagulant activities of the S-GFB were investigated by studying the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. APTT and TT were effectively prolonged, but no clotting inhibition was observed in PT assay at different concentrations. It proved that S-GFB had an important effect on decompounding fibrin polymerization through the fibrinolytic assay. PRACTICAL APPLICATIONS The potential practical applications of this paper exhibited two points as follows: First, it took less than 4 days to obtain the maximum mycelial biomass in Grifola frondosa mycelial fermentation. So this method, which shortened the period of fermentation, may be utilized on industrial production of G. frondosa mycelia. Second, the water-insoluble polysaccharide accounted for a considerable proportion of polysaccharide obtained from G. frondosa. In order to make full use of the water-insoluble polysaccharide, we transferred it to water-soluble polysaccharide with high biological activity by chlorosulfonic acid-hydrochloride method.

Fermentation properties of top-fermenting yeast are under the control of multiple genes difficult to manipulate directly by classical breeding, metabolic engineering, or other genetic methods with specific genes or pathways as target. Here, genome shuffling is introduced to improve fermentation performance (such as the viability of the yeast, flavor of beer, and the fermentation time) by improving wort and ethanol tolerance of top-fermenting yeast. The strategy was performed not based on polyethylene glycol (PEG)-mediated protoplast fusion but using yeast sexual and asexual reproduction by itself. The best performing strain W3-8 was selected on the selective plates after 3 rounds of genome shuffling. The fermentation time of W3-8 was not only markedly shortened, but also, most flavor compounds were distinctly improved. In particular, ethanol yield was increased by up to 67% after the 3rd pitching compared with the control. Furthermore, W3-8 promoted desired amounts of esters and higher alcohols, in accordance with specific consumer preferences. Significant improvement in the fermentation traits of the top-fermenting yeast was achieved using genome shuffling.

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3-2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3-2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high-salt liquid fermentation, S3-2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3-2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3-2 distinctly improved the formation of 4-hydroxy-2 (or 5) -ethyl-5 (or 2) -methyl-3 (2H) -furanone by up to 75%. Another important flavour component, 4-ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry

Fermentation properties under the control of multiple genes of industrial Saccharomyces cerevisiae strain are difficult to alter with traditional methods. Here, we describe efficient and reliable genome shuffling to increase ethanol production through the rapid improvement of stress resistance. The strategy is carried out using yeast sexual and asexual reproduction by itself instead of polyethylene glycol-mediated protoplast fusion. After three rounds of genome shuffling, the best performing strain S3-10 was obtained on the special plate containing a high ethanol concentration. It exhibits substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. The cycle of fermentation of S3-10 was not only shortened, but also, ethanol yield was increased by up to 10.96% compared with the control in very-high-gravity (VHG) fermentations. In total, S3-10 possesses optimized fermentation characteristics, which will be propitious to the development of bioethanol fermentation industry.

Amino acids comprise the majority of the flavor compounds in soy sauce. A portion of these amino acids are formed from the biosynthesis and metabolism of the fungusAspergillus oryzae; however, the metabolic pathways leading to the formation of these amino acids inA. oryzaeremain largely unknown. We sequenced the transcriptomes ofA. oryzae100-8 andA. oryzae3.042 under similar soy sauce fermentation conditions. 2D gel electrophoresis was also used to find some differences in protein expression. We found that many amino acid hydrolases (endopeptidases, aminopeptidases, and X-pro-dipeptidyl aminopeptidase) were expressed at much higher levels (mostly greater than double) inA. oryzae100-8 than inA. oryzae3.042. Our results indicated that glutamate dehydrogenase may activate the metabolism of amino acids. We also found that the expression levels of some genes changed simultaneously in the metabolic pathways of tyrosine and leucine and that these conserved genes may modulate the function of the metabolic pathway. Such variation in the metabolic pathways of amino acids is important as it can significantly alter the flavor of fermented soy sauce.

Eicosapentaenoic acid (EPA), a well-known dietary n-3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca(2+)]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca(2+)]c generation, moreover, generation of ROS, overload of mitochondrial [Ca(2+)]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS-Ca(2+)-JNK mitochondrial pathways.

Zygosaccharomyces rouxii was applied in high-salt liquid state fermentation of soy sauce. The gene FPS1 encoding Fps1p was analyzed in the Z. rouxii in order to investigate the effect of gene FPS1 on glycerol accumulation. The sequence result showed that FPS1 had an open reading frame of 2079 bp. It was found that the expression level of FPS1 in the Z. rouxii decreased with the increase of osmotic stress. Furthermore, overexpression of FPS1 could depress the salt-tolerance compared to the control.

Salmonella typhiis a pathogen that causes the human disease of typhoid fever. The aim of this study was to investigate the viable but nonculturable (VBNC) state ofS. typhi. Some samples were stimulated at 4°C or −20°C, while others were induced by different concentrations of CuSO4. Total cell counts remained constant throughout several days by acridine orange direct counting; however, plate counts declined to undetectable levels within 48 hours by plate counting at −20°C. The direct viable counts remained fairly constant at this level by direct viable counting. Carbon and nitrogen materials slowly decreased which indicated that a large population of cells existed in the VBNC state and entered the VBNC state in response to exposure to 0.01 or 0.015 mmol/L CuSO4for more than 14 or 12 days, respectively. Adding 3% Tween 20 or 1% catalase enabled cells to become culturable again, with resuscitation times of 48 h and 24 h, respectively. The atomic force microscope results showed that cells gradually changed in shape from short rods to coccoids, and decreased in size when they entered the VBNC state. Further animal experiments suggested that resuscitated cells might regain pathogenicity.

Aspergillus oryzae is the most important fungus for the traditional fermentation in China and is particularly important in soy sauce fermentation. We report the 36,547,279-bp draft genome sequence of A. oryzae 3.042 and compared it to the published genome sequence of A. oryzae RIB40.

An antitumor peptide from Musca domestica pupae (MATP) was seen to inhibit proliferation and induce apoptosis in cancer cells in our previous investigation. However, the molecular mechanisms involved in MATP-induced apoptosis are still uncharacterized in the human liver cancer cell line HepG2. The present study was undertaken to elucidate the signaling events in MATP-induced apoptosis of HepG2 cells. In this study, the sustained activation of phosphorylated c-Jun N-terminal kinase (JNK) and the obvious inactivation of phosphorylated Akt(Ser473), which prevented IκBα from degeneration, were induced by MATP. Simultaneously, the apoptosis induced by MATP was reversed by SP600125 (a JNK inhibitor) whereas it was aggravated by LY294002 (an Akt inhibitor). These results proved that JNK and Akt independently participated in the apoptosis of HepG2 cells, which were treated with MATP. Moreover, the activation of phosphorylated JNK together with the inactivation of phosphorylated Akt(Ser473) restrained nuclear factor-κB (NF-κB p65) from entering the nucleus. The apoptosis induced by MATP was increased by pyrrolidine dithiocarbamate (NF-κB p65 inhibitor) and the restriction of NF-κB p65 from entering the nucleus induced the decrease of Bcl-2. Simultaneously, MATP induced the increase of Bax, but this mechanism did not depend on the decrease of NF-κB p65 in the nucleus. The release of cytochrome c from the mitochondria, which intensified the expression of caspase-9 and caspase-3, was enhanced by the increase in Bax-to-Bcl-2 expression ratio. The apoptosis of HepG2 cells was induced ultimately by the increase in caspase-3. Taken together, these findings suggest that MATP-induced apoptosis through a JNK-mediated and Akt-mediated NF-κB pathway.

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.

DNA sequencing is a powerful technique for identifying allelic variation within the natural killer (NK) cell immunoglobulin-like receptor genes. Because of the relatively large size of the KIR genes, each locus is amplified in two or more overlapping segments. Sanger sequencing of each gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

Zygosaccharomyces rouxii was mostly used in high-salt liquid fermentation of soy sauce. To better understand the osmo-adaption mechanism, two key salt-tolerance genes GPD1 coding for glycerol-3-phosphate dehydrogenase and FPS1 coding for a putative glycerol transporter were evaluated in the wild-type Z. rouxii (S) and a higher salt-tolerant mutant strain Z. rouxii 3-2 (S3-2) previously constructed. It was found that several mutations occurred in ZrGPD1 and ZrFPS1 in S3-2 compared with the control strain S. The mutation of ZrGPD1 in S3-2 resulted in the increase of transcription level of ZrGPD1 compared with the control. At the same time, the mutation of ZrFPS1 resulted in the decrease of transcription level of ZrFPS1. In addition, overexpression of S3-2GPD1 and S3-2FPS1 in Saccharomyces cerevisiae could cause the stronger salt tolerance compared to SGPD1 and SFPS1, respectively. The results suggested the improvement of salt tolerance in S3-2 was due to the increase of glycerol contents, which was resulted from the increase of transcription level of ZrGPD1 and the decrease of transcription level of ZrFPS1.