Abstract:To obtain the recombinant expression of gene amyM for maltogenic amylases from Bacillus stearothermophilus in Bacillus subtilis，the target gene was PCR-amplified using plasmid opt-amyM/T as template，and ligated with the expression vector pHY300PLK，then transformed into the expression host Bacillus subtilis CCTCC M 2016536. The activity of maltogenic amylases reached 250.7 U/mL after cultivated in TB culture for 48 h. By optimizing the types of nitrogen source and the complex of it，the concentration of nitrogen source and carbon source etc. The optimal fermentation condition was determined as：25.0 g/L yeast extract and 5.0 g/L soy peptone of complex nitrogen source，5.0 g/L of glycerin，initial pH of medium 6.5 and the optimal temperature was 41℃. Under optimal conditions，the production of maltogenic amylases reached 396.7 U/mL，which was 1.6 times of that before optimization. Further experiments were determined the enzymatic properties of maltogenic amylase: the optimum temperature of maltogenic amylase was 60 ℃，the optimum pH was 5.5，the half-life was 325 h，respectively. The K_m was 0.95 g/L and the specific activity is 2 646 U/mg.

Abstract:In order to reduce the inclusion bodies of the linoleic acid isomerase（PAI）in Escherichia coli，a recombinant expression strain E.coli BL21（pCold-Mpai）was constructed by using maltose-binding protein（MBP）and low temperature induced expression system pColdV，and its induction conditions were optimized. SDS-PAGE showed that the fusion protein MBP-PAI was successfully expressed，and the best induction conditions were: induction temperature 15 ℃，IPTG 0.1 mmol/L and induction time 12 h. Under the best induction conditions，the soluble expression and enzyme activity of MBP-PAI was 18 times and 1.5 times that of PAI，respectively. After purification by MBPTrap HP affinity chromatography column，the enzyme activity of MBP-PAI was 1.58 U/mg，which could convert linoleic acid to trans10，cis12-conjugated linoleic acid.

Abstract:Patulin（PAT） is a mycotoxin mainly produced by Aspergillus and Penicillium. PAT contamination worldwide is considered to be a serious health concern. New strategies to prevent or reduce patulin contamination in fruits and derived products are meaningful. Recently，biological approach for PAT detoxification is being explored. In this study，the efficacy of patulin degradation by Sporidiobolus pararoseus and the degradation mechanisms involved were investigated. The findings revealed that S. pararoseus can significantly reduce the accumulation of PAT in apple wounds. PAT（5 μg/mL） was completely degraded at 18 h post incubation with S. pararoseus in vitro. Further research found that PAT degradation is indepeμndent of the absorption of yeast cells and cell walls. The intracellular enzymes obtained from the S. pararoseus cells showed they are more efficient to degrade PAT than the yeast cells. Our findings contributes to the understanding of the mechanisms of patulin degradation by S. pararoseus，which provided a foundation for the detoxification of patulin in fruits and derived products.

Abstract:Glutamate decarboxylase（GAD）is a key enzyme that can be used to biosynthesize γ-aminobutyric acid（GABA）. In our study，four antibiotic-free marker recombinant Bacillus subtilis strains were constructed and glutamate decarboxylase derived from Lactococcus lactis ssp. lactis IL 1403 was expressed in those strains. By comparing growth curves and GAD production curves of the four recombinant strains，the recombinant strain B.subtilis WB600/ pUB-P43-gadB（opt）-dal with highest enzyme activity was selected. After fermentation of the B.subtilis WB600/ pUB-P43-gadB（opt）-dal for 42 h in primary fermentation medium，the volumetric activity of the fermented broth reached 4.1 U/mL. By modifying of the composition of fermentation medium，volumetric activity of the fermented broth was increased by 79% and reached 7.4 U/mL，which was the highest activity of GAD for the reported recombinant Bacillus subtilis strains.

Abstract:Aflatoxin seriously polluted agricultural products and threatened the life and health of human and animal. Conidial germination is one of the most decisive factors in survival and invasion of A. flavus，and blocking germination could be one of the effective breakthrough points to keep contamination in the upstream controls. In this study，it was found that extracellular KCl could regulate the conidial germination in A. flavus. By using K+ specific fluorescent probe PBFI-AM，the results showed that a certain concentration of K+，which was suitable for spore germination，can stimulate A. flavus spores to produce higher potassium currents instantly，indicating that K⁺ response may be an early signal of spore germination. As a specific voltage potassium channels blocker，4-AP can obviously inhibit the spore germination. And KCNA is identified with K+-channel filter sequence by using bioinformatics prediction，and further analysis supports the potential of KCNA as voltage-gated potassium channel α subunit. In addition，the ion channel currents of KCNA expressed in Xenopus laevis oocyte were observed using two-electrode voltage clamp. Our studies indicated KCNA involving the regulation of conidial germination in A. flavus，which was one of voltage-gated potassium channels.

Abstract:Many of biopharmaceutical proteins are produced by mammalian cells because the proteins require correct folding and glycosylation. However，production of recombinant proteins in mammalian cells has some issues to be solved，including higher manufacturing cost and heterogeneity of glycans attached to the proteins. To overcome the issue，we here established a HEK293 cell lines disrupted Golgi alpha-1，2-mannosidase I. In double-KO cell line，which was knocked out both MAN1A1 and MAN1A2，the high mannose-type N-linked glycans were increased，whereas complex type glycans was greatly reduced. The glycans of an endogenous protein LAMP2 were sensitive to endoglycosidase-H treatment in double-KO cells，indicating that most of glycans on the proteins were high mannose-type glycans. Our results suggest that disruption of alpha-1，2-mannosidases in mammalian cells，especially double-KO cells，is useful for production of recombinant proteins with high mannose-type glycans.

Abstract:The research investigated the effects of hypocrellin production of agitation and amylase addition under solid state fermentation by Shiraia bambusicola. The optimized experimental conditions were as follows：the first agitation on the third day，intermittent agitation every 36 hours，bacterial mesophilic α-amylase（2.85 U/g） added at the start time and glucoamylase（29.78 U/g） added on the third day. After optimization，hypocrellin production reached 60.74 mg/g within 13 d，which was 3.66 folds than previous study. This is the first report that amylase addition is used in SSF and it would have significant effect on hypocrellin production.

Abstract:A new company strain of A8, which can promote 2-keto-L-gulonic acid（2-KGA） produced by K. vulgare in vitamin C（VC）mixed culture fermentation，was separated from the source of Longwei Lake silt，Dongling Shenyang. The ITS rDNA was amplified by PCR and sequenced to obtain a 610 bp ITS rDNA. Analysis was performed by homology comparison with the NCBI database to construct the phylogenetic tree basing on ITS rDNA sequences. And the strain of A8 was determined as Rhodotorula mucilaginosa based on the molecular identification combining with morphological identification. The R. mucilaginosa and K. vulgare composed of a new mixed culture of VC fermentation system and the production yield of 2-KGA was 52.56 g/L.

Abstract:CL-RBF algorithm was proposed to predict the protein subcellular localization，which is considered about cluster results within one label and between different labels of the ML-RBF method. Silhouette coefficient was introduced to get the optimal number of centroids on hidden layer. The previous approach only considered optimization of clustering algorithms within the same label. In this paper，larger distance between two centroids which were generated from two labels was taken into account，when there were less samples covering these two labels. Besides，gradient descent algorithm was used to adjust the parameters. The final adjustment was made by analyzing the distance between train samples，the hidden centers obtained by label L and the clustering centers not belonging to label L. Bag of words and AAC method were employed to extract the feature of protein sequence. Compared with the methods which have been introduced previously for bacterial protein subcellular localization prediction via 10-fold cross-validation test，the new predictor performed more powerful and flexible on four different multi-label datasets.The prediction server was available on https://njau.applinzi.com/homepage_final.jsp.

Abstract:Major types of typical Chinese brewing vinegars with different characteristics were evaluated by evaluation panels of Chinese vinegars. The flavor wheel of Chinese brewing vinegars was presented first in this study which included a total of 45 sensory descriptors in 16 categories. Sixteen kinds of the important and typical descriptors were chosen by variance analysis，principal component analyses and other methods including 6 kinds of the taste terms（acidity，sweetness，bitter，saltness，astringent and umami）. Ten kinds of olfactory terms（sour，sweet，burnt，sauced，fruity，bran incense，incense smoke，grain，alcoholic aroma and flowery）.Through the descriptive analysis of the selected Chinese brewing vinegars，the difference of the flavor characteristics was found.

Abstract:Bifidobacterium longum CCFM760 is screened from stools of a healthy two-year-old youngster in Binhu District，Wuxi City，Jiangsu Province，China. In this study，the ability to grow and produce acid，artificial simulated gastrointestinal fluid tolerance，antibacterial ability，oligosaccharide utilization ability and antibiotic tolerance of B. longum CCFM760 were assessed，as well as its total superoxide dismutase (SOD) activity. It was found that this bacterium had good growth acidogenicity and artificial simulated gastrointestinal fluid tolerance，and obviously inhibited Escherichia coli and Staphylococcus aureus. Meanwhile，it could significantly utilize seven oligosaccharides and was sensitive to 14 antibiotics. The total activity of SOD in the culture reached（99.14±9.91）U/mL. In addition，the genome sequence of the strain was sequenced to explain the ability of oligosaccharide metabolism，antibiotic sensitivity and anti-oxidation of the bacteria. These results demonstrated that B. longum CCFM760 is a safe and potential probiotic.

Abstract:The study aimed to investigate the growth prediction models of Staphylococcus aureus in coconut milk at different temperatures（20，25，30 and 36 ℃）. Growth of inoculated bacteria in coconut milk was determined and modified Gompertz modified Logistic and Baranyi models were fitted by using the Matlab software. The optimal primary model was selected by comparing the residual and goodness of fit，and the growth parameters were thus obtained. The secondary model was established by square root and quadratic polynomial equations and evaluated by the correlation coefficient，the accuracy factor and the bias factor. At between 20~36 ℃ temperatures，the Baranyi model was suitable as a primary prediction model for simulating the growth of Staphylococcus aureus in coconut milk. The quadratic polynomial equation could better express the relationship between temperature and the maximum specific growth rate and the lag phase compared with the square root model. Therefore，Baranyi and quadratic polynomial models were chosen to describe the growth of Staphylococcus aureus in coconut milk at different temperatures.

Abstract:In order to improve the yield of Monacolin K in lipid-lowering Monascus and reduce the production cost，seven strains of Monascus were screened for the efficient production of Monacolin K in this study. Among the tested strains，Monascus ruber 9901 was found to be the most suitable one for the purpose. Various kinds of rice were used as substrates for Monacolin K production by solid-state fermentation of Monascus ruber 9901，among which Guangxi indica rice was found as the best substrate. Afterwards，the appropriate fermentation time，the shaking frequency，the time of temperature adjustment，the amount of water addition and inoculation were systematically studied by single factor experiments. Based on these results，the response surface design was further used for the process optimization. The results showed that the optimal conditions were 45 mL water amount，10% inoculation amount，and incubated at 30 ℃ for 50 h followed with 25 ℃ until 20 d. Under the optimal conditions，the maximal yield of Monacolin K reached 10 453.07 mg/kg，corresponding to 48.97% higher than that of the control.

Abstract:Bacterial-derived FAD-conjugated glucose dehydrogenase is rare. Using lactose as an inducer，fed-batch fermentation of E. coli BL21（DE3） was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy. Enzyme activity and dry cell weight reached 1 341 U/L and 12.4 g/L respectively. By using Histrap HP，HiPre TM 26/10 Desalting and DEAE anion exchange chromatography，a purified enzyme was obtained with a specific enzyme activity of 109 U/mg.The molecular weight conformed to the theory about approximately 60 000 as determined by SDS-PAGE. The isoelectric point of the enzyme was 5.3. Absorption spectroscopy of full wavelength scanning by microplate reader showed that the enzyme was FAD conjugated. Compared with other substances，the Km of the enzyme was lowest of 2.56 mmol/L when glucose was used as substrate，and k_cat/K_m was 3 487.7 L/（mmol·s）. The enzyme kept relatively stable when the pH ranged from 5.5 to 8.0，with optimal pH of 7.0. When the pH is higher than 8.0， the enzyme activity decreases rapidly. The Tm of the enzyme measured by differential scanning calorimetry（DSC） was 77 ℃ showing high thermal stability. This experiment provides reference for the further industrial production and application of the enzyme.