Abstract:With the rapid development of food industry and the increase in consumer safety awareness, consumers are also paying more and more attention to food quality and safety. Frozen storage is the most common method for long-term storage of food, but there are still some deteriorations in food quality after freezing and thawing, such as the nutrient loss caused by the loss of thawed juice, the serious oxidation of lipids and proteins, and the damage to muscle fiber tissue. Therefore, it is particularly important to seek a new safe and efficient thawing technology. Magnetic nanoparticles assisted heating is a new type of food thawing method, which greatly avoids the conventional problems of traditional thawing and new thawing methods. Magnetic nanoparticle assisted heating is a new thawing method of food, which greatly improved the comment problems in the traditional and new thawing methods, such as serious oxidation and poor sensory performance. The present article reviews the quality deterioration issues in the thawing of food and the application of magnetic nanoparticle-assisted heating in the thawing of fish, in order to provide reference for the application of this technology in food defrosting.

Abstract:Functional membrane microdomains(FMMs) can be used as the endogenous spatial scaffolds for pathway enzyme assembly. Moderately increasing the proportion of FMMs in plasma membrane can significantly increase the synthesized product yield. However, the cell growth and product synthesis were both inhibited after further modification of FMMs. Based on the previous research of our group, the rational modification of plasma membrane was carried out to alleviate the effects of excessive FMMs modification on cells. First, the plasma membrane was rationally engineered by overexpression of PlsX, PlsY and PlsC. Then, taking the synthesis of N-acetylglucosamine(GlcNAc) by Bacillus subtilis as an example, it was found that GlcNAc titer in the plasma lemma-modified strains was （5.18±0.16） g/L, which was almost 41.9% higher than that of the control strain. In addition, cell membrane modification also reduced the adverse effect of FMMs over modification on the growth of the strain.

Abstract:Xanthine oxidase is a key enzyme in uric acid formation, which can effectively reduce the content of uric acid in vivo by inhibiting the activity of xanthine oxidase. To explore the inhibitory effects of Astragalus polysaccharide on xanthine oxidase, the variation of uric acid content of reaction system per unit time was determined by UV-Spectrophotometry, and the inhibition ratio of Astragalus polysaccharide to xanthine oxidase was calculated. The semi-inhibitory concentration IC₅₀ was also obtained. The inhibition type of Astragalus polysaccharide on xanthine oxidase was determined by the Lineweaver-Burk plot, and a Dixon plot was used to obtain the inhibition kinetic parameter(K_i). The inhibitory effects of Astragalus polysaccharide combining allopurinol were explored by the toxicological studies. Astragalus polysaccharide could inhibit the activity of xanthine oxidase, and the IC₅₀ was 1.37 mg/mL. The kinetic analysis showed that Astragalus polysaccharide had competitive and reversible inhibitory effect on xanthine oxidase, with an inhibition constant (K_i ) of 0.59 mg/mL. There was antagonism effect when Astragalus polysaccharide was combined with allopurinol. Astragalus polysaccharide could inhibit the activity of xanthine oxidase.

Abstract:Prodigiosin(PG), a secondary metabolite produced by Serratia marcescens, has attracted attention owing to its anti-microbial, anti-malaria, anti-tumor and immunosuppressive activities. However, there is still limited understanding of the regulatory mechanism for prodigiosin biosynthesis in S. marcescens. In this study, a Tn5G transposon insertion mutant library using the strain S. marcescens JNB5-1 as the parental strain was constructed, and BVG90_04085(PsrB), a DeoR family transcription regulator, was identified and elucidated to positively regulate prodigiosin production in strain JNB5-1. Further, the molecular mechanism behind PsrB in regulating prodigiosin production in this strain was investigated. Results showed that when fermentation was done in LB medium, the prodigiosin productivity of psrB mutants SK8-61 and ΔPsrB was only 0.22 and 0.26 times as high as that of the parental strain JNB5-1. The expression level of pig gene cluster analyzed by RT-qPCR showed that the psrB deletion mutant ΔPsrB comparing with the parental strain JNB5-1, and the expression level of a key gene in the prodigiosin pathway, pigABCDEFGHIJKLMN, was down-regulated by 5.81 to 22.93 times. Further, electrophoretic mobility shift assay (EMSA) and other experiments confirmed that regulator PsrB could directly bind to the promoter region of the pig gene cluster, suggesting that the molecular mechanism by which PsrB regulated prodigiosin production in S.marcescens was probably through direct binding to the pig gene cluster, and thus positively regulated the transcriptional level of pig gene cluster, affecting the synthesis of prodigiosin in S. marcescens. Finally, the psrB overexpressed strain JNB5-1/pXW1906 found during prodigiosin fermented in the fermentation medium could synthesize 8.61 g/L prodigiosin, which was 1.56 times of that produced by the parental strain JNB5-1 (5.53 g/L).

Abstract:The effect of heat combined with ethanol on the spore killing efficacy and permeability of inner membrane was studied in this paper. The survival rate, inner membrane fluidity, OD₆₀₀ value and inner membrane permeability of Bacillus subtilis spores treated by heat combined with ethanol were studied by plate counting, fluorescence polarization, spectrophotometry and flow cytometry. The results showed that heat combined ethanol could kill the spores, and the survival concentration of spore decreased by about one logarithm at 80 ℃ combined with 75% ethanol. The fluorescence polarization degree of spore suspension decreased by 0.31 at 60 ℃ and 80 ℃ combined with 75% ethanol, indicating that the fluidity of spore inner membrane increased greatly. The OD₆₀₀ value decreased the most after 80 ℃ and 75% ethanol treatment, and the permeability of spore inner membrane changed significantly. The results of flow cytometry showed that the positive area changed 93.69%. The results showed that the change of endomembrane permeability was one of the important reasons of spore death.

Abstract:The brewing process of yellow peach fruit wine was studied in this research using the concentrated yellow peach juice as raw material. The effects of yeast inoculation amount, fermentation temperature, initial sugar content, pH value and nitrogen content on the quality of yellow peach wine were analyzed by single factor experiment, and the optimal product type of yellow peach wine was further determined through sensory evaluation. The fermentation process of peach wine was then optimized by Box-Behnken response surface method. The results showed that the optimal fermentation conditions were 195 g/L initial sugar content, 200 mg/L added nitrogen content, 23 ℃ of main fermentation temperature, initial pH 3.4 and 9.3×106 CFU/mL inoculation amount of yeasts. The semi-sweet yellow peach wine produced under this condition had the highest sensory score. Comparing this yellow peach wine with three commercial products, the antioxidant content and antioxidant capacity of fruit wine produced in the laboratory were superior to those of products. According to SPME-GC-MS quantitative detection and odor activity value analysis, isoamyl alcohol, 1-propyl alcohol, ethyl acetate, ethyl lactate, isoamyl acetate, ethyl butyrate, ethyl isovalerate, decanoic acid, hexanoic acid, octanoic acid and gamma-decalactone were the main flavor compounds in yellow peach wine. Principal component analysis showed that the fruit wine fermentation in the laboratory had a strong correlation with a variety of alcohols and esters. This research could provide a theoretical basis for the development and quality improvement of yellow peach wine.

Abstract:This study aimed to examine the hepatoprotective effects of phloretin on acute liver damage induced by thioacetamide(TAA) in Kunming mice. Quercetin was used as the positive control drug. The hepatoprotective effect was studied by means of serological index, including the serum levels of alanine aminotransferas(ALT), aspartate aminotransferase(AST), γ--glutamyl transferase(γ-GT), alkaline phosphatase(ALP), and total bilirubin(TB), as well as the histopathological changes. In the groups with high and low doses of phloretin, the serum ALT activity was (149.14±21.55) U/L and (189.43±16.64) U/L, respectively, and the serum AST activity was (123.58±35.90) U/L and (168.64±33.32) U/L, respectively. The serum γ-GT activity was (103.19±20.93) U/L and (129.30±27.91) U/L, respectively, and the ALP activity was (19.67±3.32) King U/dL and (24.17±3.58) King U/dL, respectively. The total serum bilirubin contents were (198.14±29.64) mol/L and (227.04±34.20) mol/L, respectively. Compared with the model group, phloretin significantly prevented the increase in serum ALT, AST, γ-GT, ALP and TB in acute liver damage induced by TAA(n-=8, p-<0.05), and the decrease was dose-dependent. A significant reduction was produced in the histopathological hepatic lesions. The results showed that phloretin had good hepatoprotective activity on acute liver injury induced by thioacetamide in mice, and it could be considered as a candidate drug and food additive worthy of further study.

Abstract:Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase(hGnT-I) works in Golgi glycosylation pathway and adds a β-1,2 linked GlcNAc to Man5Gn2 structure on glycoproteins. The recombinant plasmid pET28a-MGAT1ΔTM was constructed and expressed in the Escherichia coli Rosetta strain to obtain the active hGnT-I in large quantity. After optimizing the induction conditions, the soluble recombinant protein His-hGnT-IΔTM was successfully obtained and then purified by Ni-NTA column. The in vitro activity assay and substrate specificity detected by high-performance liquid chromatography(HPLC). The results suggested that the recombinant protein hGnT-IΔTM with the molecular weight of 42 800 could be successfully expressed in the ROSETTA system of E. coli, showing the corresponding glycosyltransferase activity in vitro. Except for its natural substrate M5GN2, hGnT-IΔTM also showed comparable glycosyltransferase activity against the non-natural substrate M3GN2. The products were verified by enzymatic digestion with β-N-actyl-glucosaminidase, indicating that the protein catalyzed the addition of a β glycoside bond linked to N-acetyl-glucosamine in the reaction. The prokaryotic expression system developed in this study could be used to produce a large number of purified functional recombinant hGnT-IΔTM, which could be used to the further investigation of this protein and the in vitro chemo-enzymatic synthesis of N-glycans.

Abstract:Blakeslea trispora (-) strain is a very important industrial strain producing β-carotene and lycopene. The genome extraction of Blakeslea trispora (-) is time-consuming and laborious, which brings difficulties to the transformants selection and genome edition. Four different lawn pretreatment methods were compared, boiling, boiling with NaOH, enzymolysis with mixed enzymes(2 g/dL lysozyme, 3 g/dL cellulose and 3 g/dL snailase), boiling after enzymolysis with mixed enzymes. The result showed that NaOH was the most prominent factor in the genome releasing process. The optimization was carried out including the NaOH concentration, the boiling time and the genome source (the supernatant or the pretreated lawn).The results showed that if the concentration of NaOH was not lower than 20 mmol/L, the target gene could be well exemplified with the supernatant as the genome source, and the boiling time did not play an important role in the process of genome releasing. The stability test of genome under different NaOH concentration showed that the genome concentration (150~350 ng/μL) and the purity (OD₂₆₀/OD₂₈₀ 1.8~2.0) were both high during the whole testing period (up to 108 h), and there was no obvious decline with the time. There was no prominent difference between the PCR results with the genome extracted by the rapid method and the traditional method as the template. The subsequent sequencing and restriction enzyme digestion of PCR products showed that NaOH treatment had no effect on the following experiments. Finally, it was confirmed that the rapid genomic extraction method had certain universality from two aspects of amplifying different genes and different filamentous fungi ITS of the same strain.

Abstract:Dextranase can specifically hydrolyze the α-(1,6) glycosidic bond in dextran, and it has important applications in food industrial production and prevention of dental caries. It is mainly derived from microorganisms. The marine bacterium Catenovulum sp. DP03 was screened and the complete genome sequence was determined. The dextranase genes were cloned and heterologous expressed in Escherichia coli. Then the properties of recombinant dextranases were compared and analyzed. There were two dextranases genes found in DP03, i.e., GL002870 and GL002872 which were 2 511 bp and 2 805 bp, respectively. The 3D structure of Cadex2870 and Cadex2872 were similar to that of AoDex. The catalytic regions Q418-D440 of AoDex corresponded to Q431-D453 of Cadex2870 and Q425-D447 of Cadex2870, respectively. The enzyme activities of Cadex2870 and Cadex2872 were 16.2 U/mg and 4.0 U/mg, respectively. The optimum catalytic temperatures for Cadex2870 and Cadex2872 were 45 ℃ and 30 ℃ respectively and the optimum catalytic pH values were 7 and 8, respectively. The products of Cadex 2870 hydrolyzed dextran were maltoheptaose, maltopentaose, maltotetraose and a small amount of maltose. And the hydrolysates of Cadex2872 were maltoheptaose, maltopentose and maltotetraose. The marine bacterium Catenovulum sp. DP03 contained two genes encoding dextranase. The two dextranases were different in the protein structure and enzymatic properties.

Abstract:Fucoxanthin is a kind of carotenoids that participates in the photosynthesis of marine algae and has multiple functions such as anti-oxidation, anti-cancer, anti-obesity and anti-diabetes, showing important applications value as the biological medicine and functional food. Marine diatom Phaeodactylum tricornutum has high content of fucoxanthin, which is considered as a new source of algae-based fucoxanthin besides brown seaweed. Aiming to improve the fucoxanthin production, P. tricornutum CCMP2561 was applied in this study to systematically evaluate the effects of organic carbon, nitrogen sources and their concentrations on the cell growth and fucoxanthin production under mixotrophic (photo fermentation) conditions. The optimal combination of carbon and nitrogen source was 0.10 mol/L of glycerol, 0.02 mol/L of total nitrogen mixed by tryptone and urea (1∶1). Under this optimal combination, the maximum biomass concentration, fucoxanthin content and yield were achieved at 3.94 g/L, 20.83 mg/g and 81.97 mg/L in P. tricornutum respectively, which were 3.15, 11.64, and 36.59 times to those before optimization under 2 000 lx of light intensity, 20 ℃ of culture temperature and 150 r/min of shaking speed. The optimal medium and approach for mixotrophic cultivation developed in this study could provide a newly practical technology for significant enhancement of fucoxanthin production in P. tricornutum.

Abstract:To improve the control effect of electron beam irradiation on foodborne pathogens, the effects of electron beam irradiation on the growth of pathogenic bacteria were studied by analysis of D₁₀ value, growth curve, biofilm formation and dominant bacteria in mixed culture of four common foodborne pathogens, including Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Bacillus cereus. The results showed that electron beam irradiation had strong disinfecting influence on these four pathogens and theD₁₀ values ranged from 0.330 kGy to 0.648 kGy. In addition, multiple irradiation could decrease the D₁₀ values, leading to the lower tolerance of pathogenic bacteria to irradiation. Sublethal dose electron beam irradiation could inhibit bacterial growth, which shoD₁₀wed longer lag phase and lower colony quantity level of stationary phase than that of the control. And this effect was especially significant at lower temperature of 15 ℃. Among different pathogenic bacteria, Escherichia coli was the most sensitive to irradiation, with the lowest D₁₀ valueand the greater lag degree of lag phase than other pathogenic bacteria, while Bacillus cereus was the least sensitive to irradiation. When the four pathogens were mixed and co-cultured after irradiation, the growth of Bacillus cereus irradiated by electron beam was inhibited and the percentage decreased from 33.49% to 25.06%, while the percentage of other three pathogens increased. Sublethal dose electron beam irradiation could affect the biofilm formation of pathogenic bacteria. After irradiation, the ability of forming biofilm of Escherichia coli and Bacillus cereus was enhanced, while that of Salmonella typhimurium or Listeria monocytogenes was weakened.

Abstract:This work was aimed to develop a method for rapid identification of bacteria with high similarity of conserved sequences in DNA, and to identify a Bacillus strain collected from soil. The prokaryote strain was isolated and purified from the soil after diluting and coating the plate, which collected from the planting area with animal fertilizer in Tong'an National Agricultural Science and Technology Park, Xiamen. The 16S and gyrB gene of the strain were amplified by PCR and sequenced. The ATCC strains with similar sequences selected from Genbank were compared. After the 16S sequence and the gyrB sequence were linearly spliced, the phylogenetic tree was constructed with Paup*4.0, and the identification results were verified by physiological and biochemical methods. The 16S sequence and gyrB gene sequence were constructed separately, however, the strain was failed to be identified to specie level. After the joint construction, the Bacillus HX2016002 isolated from soil was identified as Bacillus cereus. This method obtained a high self-expanding phylogenetic tree, and the identification results were consistent with the physiological and biochemical experiments. The sequence analysis with the known Bacillus cereus in Genbank showed that the method was generally applicable in Bacillus cereus identification. The phylogenetic tree based on the combination of 16S and gyrB gene has the value of further research in the identification of genus strains with high conserved sequence similarity.

Abstract:To obtain an acid fragrance tobacco extract, the extraction technology was first optimized. The extracts were then fermented using microbe from Kombucha, and the tobacco extract with acid fragrance were obtained after concentration. The flavor components of prepared tobacco extract were analyzed by gas chromatography-mass spectrometry (GC-MS), and the application of tobacco extract was also estimated combined with sensory quality evaluation. The results showed that the extraction rate and sugar contents of tobacco extract reached 55.34% and 13.14 g/L after the extraction process optimization, respectively. Compared with the unfermented tobacco extract, the content and types of flavor components were both increased in the fermented tobacco extract, such as phenylacetic acid, lactic acid, 3-methylbutyric acid and other acidic flavor components. Several characteristic aroma components of tobacco were also increased, such as megastigmatrienone, β-damascone, β-ionone, and maltol. The cigarette sensory was effectively improved if the as-prepared fermented tobacco extract was applied, with the quality and quantity of aroma significantly enhanced. Meanwhile, the effect of fermented tobacco extract was better than the unfermented tobacco extract. A special acid fragrance tobacco extract was obtained through the optimization of extraction process and biological fermentation, which could be used as a reference in the preparation of tobacco extract with characteristic aroma.