Abstract:To evaluate the accuracy of genetic markers of horA and horC genesto rapidly detect the spoilage ability of beer spoilage bacteria，the genotypes and phenotypes of 53 spoilage bacteria strains which were isolated from 5 Chinese breweries were compared in this study. The physiological features studied included M13-PCR fingerprint，horA and horC genes，beer spoilage ability and hop resistance ability. The clustering analysisof M13-PCR fingerprint indicated that the similaritycoefficient was less than 70% between different species and the similarity between strains in the same specie also varied greatly. Hence，the genotypes of 53 strains were different from each other. Phenotype analysis showed that almost all strains with strong spoilage ability or hop resistance ability contained horA or horC genes，which confirmedthe good accuracy of these genetic markers. However，some non-spoilage or weak hop-resistant strains also contained horA or horC genes，leading to a highfalse positive rate of 78.6%. A few weak spoilage strains did not contain horA or horC genes，which led to a false negative rate of 28%. Two strong hop-resistant strains did not contain horA or horC genes and they were supposed to have other mechanism of hop resistance. It is interesting that horA and horC geneswere found to be conserved in L. plantarum species. These results indicated that horA or horC genesare not suitable to be used as genetic markers for rapidly detectionof the spoilage ability and hop resistance of all beerspoilage bacteria.

Abstract:In order to compare the differences in antimicrobial resistance between different sources and different pathogenic Vibrio parahaemolyticus，the antibiotic resistance of 44 V. parahaemolyticus strains was detected by modified K-B disk method，and antimicrobial resistance genes（ARGs） were detected by PCR.The results showed that there were no significant differences in antimicrobial resistance phenotype between seawater and freshwater shrimp isolates，and the antimicrobial resistant rates were 100% and 95.5% respectively；However，the multidrug resistance of pathogenic strains reached 73.7%，significantly higher than that of non-pathogenic strains（16%）.The type and number of ARGs between different sources and different pathogenic strains also has no significant differences. This study provides the basic data for the drug resistance diversity of V. parahaemolyticus，and provides relevant help for further study of antimicrobial resistant bacteria risk assessment.

Abstract:This study is aiming to establish a method for total DNA extraction from the frozen soil of the Glacier No.1 in the Tianshan Mountains. Four DNA extraction methods（SDS-CTAB，TENP，TEN and Zhou method）were used for DNA extraction from the frozen soil，and the yield，purity and integrity of the total DNA produced were compared. Among the four extraction methods tested，TEN method produced the highest purity with a A260/A280 and A260/A230 ratio of （1.82±0.40）and（0.97±0.38），respectively，and the yield of TEN method was（392±44） ng/g frozen soil. We further modified the TEN method by adding a pretreatment step. The A260/A280 and A260/A230 ratio of the extracted DNA reached（1.92±0.15）and（2.03±0.31），respectively，and the DNA yield of TEN4 method reached（449±61） ng/g frozen soil. 16S rDNA，26S rDNA PCR analysis，and restriction digestion analysis of the DNA extracted by TEN method further proved that the DNA was qualified for the construction of a meta-genomic DNA library. In summary，a modified TEN method suitable for DNA extraction from the frozen soil of the Glacier No.1 in the Tianshan Mountains was established，which could be used for meta-genomic DNA library construction.

Abstract:Using the genomic DNA from Lactobacillus casei CICIM B1192 as the templet，we amplified the coding gene Lcldh2 by PCR technique，which encodes an L. casei L-lactate dehydrogenase（L-LcLDH2）. Then，Lcldh2 was successfully expressed in E. coli BL21（DE3）mediated by an expression vector pET-28a（+）. Using PPA as the substrate，L-LcLDH2 displayed the enzymeactivity of 47 U/mg，by which the asymmetric reduction of the substrate PPA afforded L-PLA with enantiomeric excess（eep） of more than 99%. Bioinformatic prediction revealed that Lcldh2 is 906 bp in length，encoding 301 amino acids. Theoretical relative molecular mass and isoelectric point of L-LcLDH2 is 32 585 and 5.5，respectively. L-LcLDH2 is a stable cytoplasmic protein without signal peptide and has a highly conserved sequence GXGXXG. The acquisition of L-LcLDH2 has provided a new biocatalyst for the preparation ofhighly optically pureL-phenyllactic acid.

Abstract:To improve vitamin K2 production，1，4-dihydroxy-2-naphthoicacid polyprenyltransferase （MenA）which was a key enzyme for vitamin K₂ synthesis in Bacillus subtilis natto was overexpressed. The effects of pH on cell growth and vitamin K2 production was also investigated in a 5 L fermentator. It was shown that the vitamin K₂ production was enhanced by 51% when menA gene was overexpressed. The mono-control of pH could not balance the cell growth and vitamin K₂ production. A two-stage pH control strategy was employed，i.e. pH was controlled at 7.0 between 0~40 h and switched to 4.0 between 40~80 h. The biomass and vitamin K₂ production reached（11.46±0.67）g/L and（63.60±1.81） mg/L，which were 1.5 and 2.6 times of that of pH uncontrolled condition by original strain，respectively.

Abstract:Oral administration of GLP-1（Glucagon-like peptide-1）has attracted attention as treatment for diabetes. GLP-1 is a short peptide and secreted by L cells.GLP-1 can increase insulin release and reduce blood sugar .However，one of the major limitation of native GLP-1 is its rapid degradation by DPP-4，leading to its short half-life in vivo. In this study，we examine whether yeast spores were used as a carrier of GLP-1. Sporulation will happen when yeast cell face nutrient limitation，and previous work has shown that the yeast spores can hold secretory proteins in the periplasmic space of the spore wall because its outer layers work as a diffusion barrier. By being held in the spore wall，the proteins are protected from environmental stresses. Yeast spores were used as carrier to protect GLP-1 from being digested by gastric and intestine juice.The results showed that GLP-1 was successfully expressed in yeast spores. After pre-treated with simulated gastric and intestine juice，spores with GLP-1 obviously enhanced insulin secretion from MIN6 cells compared with GLP-1. Using yeast spores as carrier，the anti-protease degradation ability and insulinotropic activity of GLP-1 were significantly enhanced. Our study will lay a foundation for further development of oral anti-diabetic drugs.

Abstract:Blanching and drying are two essential processing steps of dehydrated vegetables. The conventional methods have many disadvantages of time-consuming，high energy consumption，low product quality，as well as heavy pollution to environment. This study investigated the infrared dry-blanching and dehydration on leafy vegetables. The characteristics of vegetables were determined via the measurement of peroxidase（POD） activity，the changes on Vitamin C content and leaf color. The study showed that the POD activities of five selected vegetables were reduced to about 20% of their initial activity with infrared dry-blanching. Meanwhile，infrared dry-blanching also can remove partial moisture from vegetables and retain the high quality. In addition，the optimal conditions of infrared dry-blanching on the five different vegetables were also obtained from this study.

Abstract:Lactobacillus bulgaricus is one of the commonly used strains in fermented dairy products，and it is very important for the breeding of the strains. The objective of this study was to evaluate the antibiotic resistance of five Lactobacillus bulgaricus strains isolated from traditional fermented dairy products in Mongolia. The antibiotic resistance was analyzed by broth dilution method，and the resistance genes were detected by polymerase chain reaction（PCR）. The results showed that all tested strains were sensitive to linezolid，ampicillin，clindamycin，rifampicin，vancomycin，trimethoprim，and quinupristin/dalfopristin，while variably resistant to tetracycline，kanamycin，chloramphenicol，gentamicin，streptomycin，ciprofloxacin，erythromycin，and neomycin. Based on the PCR，five different antibiotic resistance genes，namely erm（B），vanX，aac（6'）-aph（2''），parC，and rpoB，were detected in the tested strains；and each strain carried at least two resistance genes. These results serve as useful reference for strain selection for future studies.

Abstract:Citrate is one of the high-volume organic acids with the global production of 1.7 million tons annually mainly produced by microbial process using Aspergillus niger as natural producer. Glucose absorption was the first step of citrate fermentation and needed to be enhanced. The sugar transporters were analyzed based on transcriptome data，phylogenetic tree analysis and sequences alignment. The evm.model.unitig_0.1770 gene，whose relationship was the closest with KlHGT1，was predicted containing 11 transmembrane segments and named as HGT1. The growth test on glucose limited medium showed the diameter of HGT1 transformants were 50% to 150% larger than the diameter of H915-1. After addition of 30 g/L glucose into fermentation medium，the glucose consuming time of HGT1 transformants reduced for 12 h than that of H915-1. Finally，the citrate titer of HGT1 tranformant was increased for 14.7% comparing to H915-1，the fermentation time reduced for 6 h and the maximum specific citrate production rate increased for 29.5%.

Abstract:α--L-Rhamnosidase generates prunin and L-rhamnose by hydrolyzing naringin，which can be used to debittering citrus juices. In the present study，the α--L-rhamnosidase，previously identified from Aspergillus niger JMU-TS528，was docked with naringin and the binding energy was calculated by molecular dynamics simulation and MM-PBSA method. The results of binding energy indicate that var der Waalsforce is the main driving force for enzyme-substrate combination；whereas the electrostatic interaction and non-polar solvents contribute less. In addition，the critical amino acid residues for hydrophobic effect are Trp236，Ala340，Ile462，Phe461，Tyr516，Val522 and Trp528. Ser286，Phe465 and Pro520 are important amino acid residues which could form steady hydrogen bonds with naringin. The key amino acid residues identified in the study provides valuable targeting sites for remoulding α--L-rhamnosidase in protein engineering.

Abstract:N-acetylglucosamine（GlcNAc），derivative of glucosamine（glucosamine，GlcN），is widely used in the field of health food and pharmaceutical. Glucosamine-6-phosphate N-acetyltransferase（GNA） is one of the crucial enzymes in the GlcNAc synthesis pathway. On the basis of recombinant GlcNAc-producing Bacillus subtilis，we overexpressed glucosamine-6-phosphate N-acetyltransferase，originates from Caenorhabditis elegans（Cegna1），in the engineered B. subtilis，which enhanced GlcNAc titer to 7.31 g/L. Compared with the engineered B. subtilis with overexpression of glucosamine-6-phosphate N-acetyltransferase derived from Saccharomyces cerevisiae，the GlcNAc yield of the recombinant B. subtilis with Cegna1 increased by 24.51%. By fed-batch fermentation in 3 L bioreactor，GlcNAc titer further increased to 24.23 g/L，which is 24.69% higher than the reference strain. Further investigation results showed that the recombinant Cegna1 has higher affinity and catalytic efficiency for GlcN-6P and Ac-CoA. Finally，it is found that the substrate GlcN-6P has obvious inhibition on the the recombinant Cegna1. Identified substrate inhibition of Cegna1 provides future directions for improvement of GlcNAc production by directed evolution of Cegna1 for alleviating substrate inhibition.

Abstract:In this paper, the pearl industry by-product of Pinctada martensii was as the object of the study, based on the previous research results, the hydrophilic colloids, which was effectively enhancing the thermal stability of Water-Soluble Protein（WSP） of Pinctada martensii were screening. Meanwhile, the effect of the best hydrophilic colloid on Pinctada martensii muscle water-soluble proteins（WSP and F-D WSP） under different heat-treatment conditions was studied. The results showed that the thermal stability of WSP was significantly affected by carrageenan, pectin and guar colloid under the same heat treatment temperatures（P<0.05）.And in the range of heat treatment temperature（60～100 ℃），the effect of carrageenan was the best . Moreover, the effect of carrageenan on the thermal stability of WSP and F-D WSP was significantly different（P<0.05）. In the range of 60～100 ℃，the effect of carrageenan on the stability of WSP was observed with the increasing of heat treatment temperature and the stability of F-D WSP was enhanced in the initial stage and then weakened at high temperature stage.