Abstract:Cronobacter sakazakii is a kind of foodborne pathogenic bacteria, which can cause neonatal meningitis, septicemia and other diseases. Loop-mediated isothermal amplification technique (LAMP) is a new type of isothermal nucleic acid amplification technique with high sensitivity, short time-consuming, high specificity and low equipment requirements. It can be combined with a variety of detection methods for the detection of foodborne pathogenic microorganisms. This paper aims to elucidatethe basic principles of LAMP and summarize the progress of LAMP technology combined with different detection methods in the detection of C. sakazakii.

Abstract:Vine tea is classified as an Ampelopsis grossedentata of the Ampelopsis michx. in the the family of Vitacea. Vine tea is rich in flavonoids, among which dihydromyricetin (DMY) is the most important component. Scientific research has found that vine tea and its main bioactive components have various biological effects such as anti-cancer, anti-oxidation, lipid-lowering, anti-inflammation, anti-bacteria and anti-fatigue. Current studies have found that vine tea and its main bioactive components participate in different biological functions via targeting some important genes and regulating signal transduction pathways. However, the molecular mechanism of its effects is still unclear. In this paper, the biological activities, signal transduction pathways and key target genes of vine tea and its main bioactive components were systematically reviewed according to the updated domestic and international progress, expecting to provide theoretical reference for the in-depth study of vine tea and its bioactive components, and to be conducive to the further development and utilization of vine tea and its main bioactive components in the future.

Abstract:Hot water extraction was used to extract bee pollen crude polysaccharides to investigate the effect of different extraction methods on the antioxidant activities of bee pollen polysaccharides. The effect of temperature and ethanol volume fraction on the extraction rate of bee pollen polysaccharide from three kinds of pollenwere studied. The scavenging effect of bee pollen crude polysaccharides towards superoxide and hydroxyl radical was detected under pyrogallol autoxidation and fenton oxidation, respectively. At last, the effect of pollen polysaccharides on myofibrillar protein oxidation was explored by measuring the content of protein carbonyl group (molar concentration) in the flesh of acrolein-treated large yellow croaker (Pseudosciaenacrocea). The results indicated that the antioxidant effect of camellia crude polysaccharides extracted at 80℃ was most significant against superoxide radicals. The scavenging effect of ethanol volume fraction on superoxide free radicals of three kinds of pollen polysaccharides was significant, but the scavenging effect onhydroxyl radicals was insignificant. Pollen polysaccharides extracted at 80 ℃ and 95% ethanol concentration significantly reduced the carbonyl content and increased total sulfhydryl content in the large yellow croaker myofibrillar protein treated with acrolein. The effect of crude polysaccharides from wild chrysanthemum pollen was the best. It was concluded that the antioxidant activity of bee pollen was affected by extraction temperature and ethanol volume fraction, and bee pollen polysaccharides couldinhibit protein oxidation, among which wild chrysanthemum pollen polysaccharides showed the best antioxidant effect.

Abstract:Athrobacter luteu β-1,3-glucanase (βglⅠ) specifically acts on β-1,3-glycosidic bonds in β-glucan, which has outstanding advantages in yeast lytic activity. However, the expression level of βglⅠis low resulting in high cost of its production. This study achieved the secretory expression of βglⅠ by using seamless cloning technology in Bacillus subtilis, and improved the extracellular production of βglⅠ by optimizing expression elements such as Tat and Sec signal peptides, RBS and 5′UTR. The results showed that the signal peptide SPLipA increased the expression level of βglⅠ by 0.4 times. Then, RBS and 5′UTR were compared on the basis of this signal peptide, and the yield of βglⅠincreased by cry3A 5′UTR was 1.2 times higher than that of the parent strain. In addition, a Ni2+ affinity chromatography column was used to purify βglⅠ and its enzymatic properties were studied. The results showed that the optimum temperature and pH was 50 ℃ and pH 6.0, respectively. βglⅠwas more stable in the range of 0～45 ℃ and pH 4.0～9.0, and the specific activity of βglⅠ was 973 U/mg.

Abstract:To further improve the ability of Bacillus subtilis natto to synthesize vitamin K₂, the effect of rotational speed(0、200 r/min) on cell growth and product formation was investigated in 250 mL shake flasks. A two-stage rotational speed control strategy was proposed by analyzing the fermentation process curves and kinetic parameters under different conditions, as well as by observing the morphological changes between the bacteria at different time points after diluting and coating the fermentation broth with scanning electron microscopy. The conditions (temperature, speed, pH and time) affecting the control strategy were optimized by orthogonal design. When the rotate speed was maintained at 0 r/min, the highest yield of vitam in K₂ was obtained at 108 h and the morphology of bacteria was mostly folded at the later stage of fermentation. When the rotate speed was maintained at 200 r/min, the biomass was higher than that at 0 r/min. The bacteria were broken at the later stage of fermentation, and a few of them had a smooth surface. After optimization, the yield of vitamin K₂ reached （35.6±0.13） mg/L at 40 ℃ and pH 6.5 when the rotate speed was maintained at 200 r/min from 0 h to 36 h, and maintained at 0 r/min from 36 h to 132 h, which was 1.67 times and 9.674 times higher than that before optimization (0、200 r/min), respectively. The stage-by-stage control of rotational speed could effectively improve the ability of Bacillus subtilis natto to synthesize vitamin K₂, which is of great guiding significance and reference value for the large-scale production of vitamin K₂.

Abstract:Aspergillus oryzae often contains intact biosynthetic gene cluster for producing cyclopiazonic acid (CPA) toxins. As a result, CPAs has been recognized as a potential threat to food safety. Since CPAs may present in different forms of derivatives, we designed a strategy for incorporating exogenous fluorinated precursor into the CPA biosynthesis pathway. The results showed that after 5-F-L-Trp was used as the substrate and a molecular probe, it was accepted as the substrate of the first biosynthetic enzyme CpaS, producing 5-F-cAATrp, the very first biosynthetic intermediate. Meanwhile，5-F-cAATrp seemed an unsuitable substrate for the next catalytic enzyme CpaD, resulting abolishment of downstream tailoring steps and significant accumulation of this compound. The enrichment effect of CPA metabolites by the molecular probe therefore provides a new opportunity for more convenient and specific detection of CPA toxins targeting only one analyte. These findings are of significance for develop new method for detect and control the CPA toxins in Aspergillus-associated food products.

Abstract:The mechanism of Guiqi polysaccharides(GQP) in delaying the aging of rat brain tissue was investigated. 60 SD rats were randomly divided into the control group(Control), model group (Model), GQP low-dose group(LP), GQP medium-dose group(MP), GQP high-dose group(HP), and positive drug resveratrol group(Res).The aging model of rats was established by subcutaneous injection of D-galactose(D-gal). The experimental group and the positive drug group were orally administered GQP and resveratrol, respectively. The activities of total superoxide dismutase(T-SOD) and glutathione(GSH) were determined and the level of malondialdehyde(MDA) and ATP of ratbrain tissues were measured. The pathological changes of brain tissue were observed by hematoxylin-eosin (HE) staining.The change of mitochondrial membrane potential and the degree of swelling of mitochondrial membrane were detected. In addition,the expression levels of Bax and Bcl-2 were tested by immunohistochemical method. The results showed that D-gal significantly reduced the levels ofT-SOD, GSH and ATP in brain tissues of rats, and significantly increased the content of MDA(P<0.05). However, GQP and Res reversed the above indicators(P<0.05). There were insignificant pathological changes of brain tissue in normal rats, but the nuclei in model group were significantly enlarged and deep-stained, and GQP significantly alleviated the above-mentioned lesions. Compared with the control group, the mitochondrial membrane potential, ATP content and Bcl-2 expression were significantly decreased(P<0.01), while the mitochondrial membrane was significantly swollen and the expression of Bax was increased in model group(P<0.01). Meanwhile, GQP and Res increased the mitochondrial membrane potential, ATP content and Bcl-2 expression (P<0.05), but decreased the swelling of mitochondrial membrane and the expression of Bax(P<0.05).Consequently, it was suggested that GQP might play a role in delaying aging by reducing the oxidative damage and changing the mitochondrial function.

Abstract:Due to the mechanical harvesting, about 10%~20% of the harvested tomatoes which did not reach the required maturity of product processing such as tomato paste would be screened out and discarded, resulting in a waste of resources. In order to make rational use of these resources, their quality characteristics were systematically studied. Four processing cultivars, i.e., IVF3535, 8210, Jinfan-11 and 3311, were selected to investigate the quality changes related to flavor and nutrition during four ripening stages, including mature green stage, turning stage, firm ripe stage and ripe stage. The results showed that the dry matter of four processed tomatoes decreased at first and then increased during ripening, while the soluble solid and soluble sugar contents showed insignificant changes. And among them, IVF3535 was superior to the other three cultivars in dry matter, soluble solid and soluble sugar contents. The contents of lycopene, phytofluene, phytoene and β-carotene increased significantly during ripening and have accumulated at the turning stage, while the titratable acid and tomatine decreased significantly, and there was insignificant difference between the mass fraction of tomatine after the turning stage and that at the ripening stage (P>0.05). Therefore, the discarded tomatoes in turning and post-turning stages were qualified to be reused. This study could provide a reference for the efficient utilization of immature tomatoes in the tomato processing industry.

Abstract:The anti-fatigue effect of ginsenoside Rg1 on fatigue model mice and its influence on mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) gene expression in skeletal muscle were investigated. Seventy male BALB/c mice were randomly divided into seven groups. Besides the blank group, mice were injected intraperitoneally with cyclophosphamide to create an immunodeficiency model. And then the mice were subjected to the full-load swimming exhaustion experiment. Changes in fatigue-related indexes in liver and skeletal muscle of mice were determined by ELISA. The expression levels of TFAM and NRF-1 genes in the skeletal muscle of mice were detected by RT-PCR. The results showed that the duration of weight-bearing swimming was the longest in the middle dose group of ginsenoside Rg1, which increased by 271.4% compared with that of the blank group. The content of liver glycogen was significantly increased. The level of MDA in liver of mice in each dose group decreased, while the level of LDH increased. Medium dose of ginsenoside Rg1 could significantly increase the expression of TFAM mRNA and NRF-1 mRNA levels in the skeletal muscles of mice. Ginsenoside Rg1 could effectively adjust the fatigue-related biochemical indicators in skeletal muscle of fatigue mice, exhibiting better anti-fatigue effect.

Abstract:The quick detection of the effect of sodium stearyl lactate(SSL) on the quality of pre-proofed frozen dough and its fried doughnuts was carried out.SSL was added into the homemade fermented dough and the characterization wasdone after 4 freeze-thaw cyclesstorage.The effect of SSL on the quality of freeze-thaw fermented dough and its deep-fried doughnut was determined by the extension and rheological properties, freezable water content of the dough, and the specific volume, texture characteristics, crust color, internal texture structure, oil absorption capability,moisture loss rate, and sensory score of its fried donuts. Compared with the blank group, the results showed that with SSLdose increased, the extension properties and elastic modulus of pre-proofed doughincreased at first and then decreased, while its loss tangent (tanδ)and freezable water contentdecreased atfirst and then increased.The hardness, viscosity and chewiness offried donutsdecreased at first and then increased, while the elasticity, specific volume and sensory scores first increased and then decreased, and the oil uptake and moisture loss rate of donuts first decreased and then increased. The optimal amount of SSL was 0.2%, which could reduce the freezable water content of dough by 8.16%, increase the specific volume of donut by 22.73%, and reduce the oil uptake by 5.39%.

Abstract:This study aimed to investigate the secretory expression of the pullulanase gene from Bacillus subtilis and the application of the recombinant enzyme. The BsP gene encoding pullulanase in B. subtilis strain 168 was expressed in Escherichia coli and B. subtilis, respectively. The recombinant enzymes expressed in both hosts could be secreted to the outside of cell via its own structure. The enzymatic properties of the expression products secreted to the culture medium significantly differed from those accumulated in the cytoplasm of Escherichia coli. The specific enzyme activity of recombinant enzyme BsBsP expressed in B. subtilis and secreted to the culture medium was 153.7 U/mg, which was 5.5 times higher than that of the products accumulated in the cytoplasm of E. coli. The optimum pH and temperature of BsBsP were 6.0 and 45 ℃, respectively. The recombinant enzyme BsBsP could not hydrolyze amylose, and it was considered as a type I pullulanase. Hydrolysis of starch paste with the recombinant enzyme BsBsP significantlyreduced the broken rate of vermicelli in the processing of potato starch vermicelli. B. subtilis pullulanase could use its own structure to achieve high-efficiency secretory expression, and the recombinant enzyme BsBsP is suitable for starch-based vermicelli production.

Abstract:Fatty acid ethyl ester (FAEE) is the most potential new energy source in the new century. Compared with the current common chemical synthesis methods, the production of FAEE in microbial cell factories has many advantages, such as low environmental pollution and low production cost. Saccharomyces cerevisiae has the talent to produce ethanol and fatty acyl-CoA. The production of fatty acyl-CoA, i.e., the precursor of FAEE, was thus improved by modifying the metabolic pathways of S.cerevisiae. By introducing codon-optimized heterogeneous gene WS2(wax ester synthase), a metabolic pathway was constructed and it could use glucose as a substrate to generate FAEE through a series of reactions. To enrich the fatty acyl-CoA, it was necessary to further reform its branches, such as blocking sterol ester way(ΔARE1,ΔARE2), triacylglycerol pathway (ΔDGA1, ΔLRO1) and β-oxidation pathway(ΔPXA2) to reduce fatty acyl-CoAutilization. The results showed that the knockout of three pathways greatly increased the yield of FAEE. Strain BYW2 expressing WS2 gene had a 9-fold increase in FAEE yield compared to the original strain BY4741, and the shake flask yield reached 11.72 mg/L. Due to the low yield of the target product FAEE, the fermentation was optimized in the shaking flask. Three factors including the addition of ethanol and rapeseed oil addition, fermentation time and ethanol addition mode were optimized. The results showed that the addition of ethanol and rapeseed oil significantly increased the yield of FAEE. The optimal fermentation time was 60 h, and the ethanol addition mode was to add 2% of the fermentation volume every 6 h after 20 h.Under the optimal conditions, the highest yield of FAEE reached144.4 mg/L, which was 12.3 times higher than that before optimization, and 480 times higher than that in the original strain BY4741. In order to further increase the yield of FAEE, the difference of FAEE yield between uracil deficient strain BYD5W2 and uracil non-deficient strain BYD5W2* was compared by fermentation tank. The results showed that the highest FAEE yield of uracil deficient strain BYD5W2 was 0.618 g/L, and that of uracil non-deficient strain BYD5W2* was 1.35 g/L, which was the highest reported FAEE yield by S. cerevisiae currently.

Abstract:This study investigated the yellow-brown anion exchange resin formed by ferric ion pollution in the purification process. The pigment is produced and aggravated under alkaline conditions, and cannot be removed by conventional methods such as high salt and surfactants. Phosphoric acid, acetic acid, citric acid and hydrochloric acid were used for testing in this study. It was found that concentrated phosphoric acid could remove resin pigments more effectively than citric acid, while concentrated acetic acid and hydrochloric acid could not. Based on the above conclusions, the effect of phosphoric acid on the pigment in the resin under different concentrations and treatment times was further studied. The results showed that the ferric ion concentration and resin capacity increased with the increase of phosphoric acid concentration, however, prolonging incubation time led to the increase ofresin capacity, but gradually decreased ferric ion concentration. Hence, it was inferred that during the treatment ofresin pigment with phosphoric acid, there was secondary adsorption of iron ions to the porous structure of resin lumen with the increase of incubation time, resulting in the gradual decrease of ferric ions in the solution. Through data analysis in combination with color contrast, it was determined that incubating anion exchange resins with 0.5 mol/L phosphoric acid for 8.75~11.35 minutes could significantly remove the stubborn pigment problems caused by ferric ions, and restored the resin capacity recover, while avoiding related impurities to pollute the final protein product. The new method for removing impurities in chromatography resin studied in this paper could provide a theoretical reference for the cost reduction and efficiency enhancement of related biological products in the downstream industrialization.