Abstract:The decarboxylations of the intermediates involved in the industrial production of furanone starting from diethyl α-methyldiglycolate and diethyl oxalate have been examined. Furanone is obtained through two successive Claisen condensations, hydrolysis of esters, methylation, and decarboxylation in this process route. During the reaction, both ester groups of diethyl α-methyldiglycolate are removed during the process, but the approaches of two decarboxylations are obviously different. After the Claisen ester condensation, the first ester group is removed in the form of a carbonate by an addition-elimination mechanism in the presence of CH3ONa; the second ester group is removed in the form of CO2 through a sodium carboxylate intermediate under basic conditions following the methylation. In order to verify the generality of decarboxylation of the intermediate: the decarboxylation of β-carbonyl ester occurred without hydrolysis under basic conditions. The alkylation of α-ethyl acetoacetate was carried out to observe whether the product (α-ethyl-α-butyl acetoacetate) would undergo decarboxylation. However, the expected decarboxylation product of α-ethyl-α-butyl acetoacetate (3-ethyl-2-heptanone) was not observed. Therefore, the results indicated that common β-carbonyl esters could not undergo decarboxylation attacked by a base through an addition-elimination mechanism. The driving force for the two abnormal decarboxylation in the preparation of flavor furanone is due to the formation of stable aromatic conjugated structures after decarboxylation. This research has clarified the reaction mechanism of the preparation process of the flavor furanone for the first time, which will be helpful for the improvement and optimization of the technical route.

Abstract:In order to study the optimal enzymatic hydrolysis process for preparing thermal reaction bovine bone flavoring, bovine bone extract (BBE) was used as raw material, and compound enzyme was selected. The effects of enzymatic hydrolysis temperature, enzymatic hydrolysis time, compound enzyme addition amount and compound enzyme addition ratio on the degree of hydrolysis of enzymatic hydrolysate and the sensory score of thermal reaction bovine bone flavoring prepared by Maillard reaction of enzymatic hydrolysate with sugar and amino acid were investigated by single factor and response surface test. The mass ratio of free amino acid was determined by high performance liquid chromatography (HPLC), and the mass ratio of key odor compounds of thermal reaction bovine bone flavoring prepared before and after enzymatic hydrolysis of BBE was analyzed by comprehensive two-dimensional gas chromatography-olfactometry-mass spectrometry (GC×GC-O-MS). The results showed that the better enzymatic hydrolysis process of BBE was enzymatic hydrolysis temperature of 50°C, enzymatic hydrolysis time of 4 h, enzyme addition amount of 1250 U/g, and the ratio of compound flavor protease to compound protease was 1: 1. Under this condition, the sensory score was 8.24. Compared with BBE, the mass ratio of 15 free amino acids in the enzymatic hydrolysate increased significantly. The odor profile and taste profile of BBE and enzymatic hydrolysate were measured by electronic nose and electronic tongue. It was found that the nitrogen oxide odor characteristics of enzymatic hydrolysate were enhanced, and the umami, richness and salty taste characteristics of enzymatic hydrolysate were more obvious. The key odor compounds of thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were analyzed by solid phase microextraction (SPME) combined with GC×GC-O-MS. A total of 164 volatile odor compounds were identified, including 27 aldehydes, 30 ketones, 12 alcohols, 16 acids, 7 sulfur-containing compounds, 10 esters, 18 pyrazines, 10 furans, 6 thiazoles, 4 thiophenes and 24 other compounds. Through gas chromatography-olfactometry (GC-O) analysis, 74 odor-active compounds were detected. There were 22 key odor compounds with odor activity value (OAV) greater than 1. The most important key odor compounds in the thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were bis(2-methyl-3-furanyl) disulfide (OAV was 13078 and 8052, respectively). Compared with the thermal reaction bovine bone flavoring prepared by BBE, 5 new key odor compounds (E,E)-2,4-decadienal, 2-methyl-3-furanthiol, methyl furfuryl disulfide, S-methyl thiohexanoate and β-caryophyllene appeared in the thermal reaction bovine bone flavoring prepared by enzymatic hydrolysate. The purpose of this study was to provide a theoretical basis for the enzymatic hydrolysis process in the industrial production of thermal reaction bovine bone flavoring.

Abstract:D-Allulose, an important rare sugar, has a wide range of application value in food, cosmetics and pharmaceutical industry. At present, biological methods are mainly used for D-allulose production in industry. Due to the disadvantages of the traditional enzymatic methods, such as complicated steps, high cost and difficult product separation and purification, it has been difficult to meet the needs of industrial production. In recent years, whole-cell synthesis systems have attracted much attention because of their low cost, convenient operation and easy separation. The study is aimed at heterologously expressing D-allulose-3-epimease (DAEase) from Caballeronia insecticola in Bacillus subtilis WB800 to catalyze D-fructose to D-allulose. Recombinant strains with different constitutive promoters were firstly designed and constructed to improve the expression of DAEase in B. subtilis. Then, the conditions of the whole cell reaction system (temperature, pH, metal ions, cell concentration) were optimized, and the conversion efficiency of D-fructose under different substrate concentrations was explored. The results showed that the promoter PylbP could maximally express DAEase in B. subtilis WB800, and the optimal temperature, pH and metal ions of recombinant DAEase were 65℃, pH 9.5 and 5 mmol/L Mg2+, respectively. The whole cell reaction almost reached to equilibrium in 4 h with a conversion rate of 30.06% when 500 g/L D-fructose was added as the substrate. This research provided experimental and theoretical basis for producing D-allulose industrially.

Abstract:The purpose of this study was to establish polysaccharide gums treatments for clarifying and improving the color of restored juice of Docynia delavayi Schneid. fruit. By using spectrophotometry, HPLC, and UPLC-MS-MS, the gum effects on the basic characters (color, turbidity, browning value, pH value, soluble solid content, and vitamin C content), total phenolic content, chlorogenic acid content, polyphenolic composition, in vitro antioxidant activity, and non-enzymatic browning kinetic parameters of the restored juice were elucidated. The results indicate that the addition of weight percentage 0.4% apple high-methoxy pectin (AHP), 0.1% tamarind seed polysaccharide (TSP), or 0.2% chitosan (CTS) endowed Docynia restored juice of clear golden color. The clarifying effectiveness from high to low was in the order of CTS, TSP, AHP. Cold precipitation combining with gums treatments resulted in an improved clarifying effect and reduced the initial non-enzymatic browning rate constant k0 of the juice from 0.150 d-1 to 0.071~0.078 d-1 at 55 oC. The changes in color parameters, turbidity, browning value, and k0 were closely, linearly related to total phenolic content. Conclusive considerations on clarity, inhibition against non-enzymatic browning (reducing k0), DPPH-scavenging ability, and dietary fiber concern suggest that, in general, weight percentage 0.1% TSP treatment gave relatively the best restored juice among treatments. The polyphenolic compositions of the restored juice contained mainly chlorogenic acid and its derivatives, flavonoids (including quercetin-O-glucoside, kaempferol-O-glucoside and dixylosides, phloridzin, and chrysin), and isoflavonoids (including daidzin, pisumionoside). TSP treatment lightly reduced the compositions of chlorogenic acid and its derivatives, flavonoids, and isoflavonoids contents. Thermal treatments resulted in a great loss in the above polyphenolic compositions, accompanying with an increased content of hydroxyl chlorogenic acid derivatives, revealing that chlorogenic acid and hydroxyl chlorogenic acid derivatives were the key compositions for non-enzymatic browning reaction. These results are expected to provide a theoretical and technical support for clarifying treatments on restored Docynia delavayi juice.

Abstract:As a common food component, fat could interact with cow’s milk allergens, and thereby affecting the extraction effect and detection accuracy of allergens. However, research on degreasing methods for allergens in high-fat complex matrices was rarely reported. Chocolates containing cow’s milk allergens were used to simulate the high-fat complex matrix and decreased by n-hexane, isopropanol, ethyl acetate and lipase. The degreasing rate and protein loss rate were determined, the changes of protein structure, particle size, fat distribution and microstructure were compared, and the concentration of major milk allergens extracted from simulated matrix before and after degreasing were determined by indirect ELISA. The results showed that the sample treated with n-hexane had the highest degreasing rate (87.87%), and the lowest protein loss rate (6.86%). Degreasing would change the spatial structure of proteins, with the overall structure changing from compact to loose, and there is no obvious change in protein in the n-hexane and ethyl acetate treatment groups. The particle size of decreased slightly after degreasing, but the isopropanol treatment caused the protein to aggregate into larger particles, and the particle size increased from 297.9 nm to 445.1 nm. After degreasing by n-hexane and ethyl acetate, the fat distribution in simulated matrix was more uniform and the fat droplets became smaller. The microstructure of the simulated matrix changed from a dense and smooth clump to an incompact and porous structure, while the isopropanol and lipase groups still showed partial agglomeration. The indirect ELISA results showed that the extraction rate of casein in the ethyl acetate group increased by 61.53%, and the concentrations of α-lactalbumin and β-lactoglobulin were highest in the n-hexane group, which were 0.73 mg·mL-1 and 1.89 mg·mL-1, respectively. N-hexane degreasing treatments can significantly improve the extraction effect of cow’s milk allergens in high-fat complex matrices, hoping that the results can provide reference for the accurate detection of allergens in high-fat complex matrix foods.

Abstract:Volatile sulfur-containing compounds (VSCs) have the characteristics of strong aromas and low thresholds, which have important impacts on the flavor of baijiu, wine and other foods. In order to fully understand the distribution differences of VSCs in baijiu, solid phase microextraction (SPME) combined with comprehensive two-dimensional gas chromatography-sulfur chemiluminescence detection was used to analyze the VSCs in the six samples both qualitatively and quantitatively. The differences of VSCs in 6 kinds of Wuling soy sauce aroma-type baijiu samples were investigated using principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA). The results demonstrated that there were significant differences in the VSCs contents of the six kinds of soy sauce aroma-type baijiu. Excellent separation of the six samples could be achieved by OPLS-DA. Based on the variable projection importance greater than 1, 14 VSCs were screened out as the different components from the analysis results to distinguish the flavor characteristics of different Wuling samples. The results of HCA showed that the six kinds of soy sauce aroma-type samples could be divided into two categories according to the type and content of VSCs. This study aims to enrich the research on the flavor of baijiu and provide a suggestion for the classification of baijiu grades.

Abstract:Whole egg is rich in protein, fat, and many vitamins and minerals. It is a natural source of nutritional supplements, but high levels of phosphorus limit its use in the diet of patients with kidney disease. Activated carbon, hydrotalcite and diatomite were selected to dephosphorize the whole egg liquid, and the dephosphorization rate, protein loss rate and color difference were taken as the indexes, the optimum amount of adsorbent, adsorption time and adsorption temperature were optimized, and then, the adsorption isotherms, adsorption kinetics and adsorption thermodynamics of the three adsorbents in the whole egg liquid were fitted. The results showed that the optimum dosage of activated carbon was 8 g/L, adsorption time was 120 min and adsorption temperature was 35℃, while the optimum dosage of hydrotalcite was 8 g/L, adsorption time was 150 min and adsorption temperature was 35℃, and the optimum dosage of diatomite was 8 g/L, the adsorption time was 90 min and the adsorption temperature was 35℃. In the process of adsorption, hydrotalcite had the least loss of protein and the highest adsorption specificity for phosphoric acid groups. Activated carbon had the greatest effect on a*, diatomite had the greatest effect on b*, and all three adsorbents increased L*. The study of adsorption mechanism showed that hydrotalcite was adsorbed in the form of monolayer. Freundlich adsorption isotherm n of three adsorbents were all greater than 1. With the increase of adsorption temperature, the KF of activated carbon, hydrotalcite and diatomite increased gradually. The quasi-first-order kinetic model R2 of three adsorbents were lower, and fluctuated with the increase of temperature. The quasi-second-order kinetic model R2 of diatomite was higher, and the chemical adsorption degree of diatomite was higher. The ΔG of activated carbon, hydrotalcite and diatomite were all negative and the ΔH were greater than 0, which meant that the reaction was spontaneous and endothermic.

Abstract:As one of the key hydrolytic enzymes for starch digestion, the hydrolysis of α-amylase enables rapid starch digestion, leading to a rapid rise in postprandial blood glucose levels. Persistent postprandial blood glucose increases are positively correlated with metabolic diseases such as diabetes, hyperglycemia and hyperlipidemia. Using natural compounds to inhibit the activity of α-amylase is of great significance in reducing the incidence of these metabolic diseases. Using chlorogenic acid and α-amylase as the research object, the in vitro simulated digestion experiment was carried out. When the substrate concentration was 10 mg/mL, the activity of α-amylase was 2 U/mL. when the hydrolysis time was 20 min, the inhibition rate was 67.3%, and the IC50 was 4.2 mg/mL, which confirmed that chlorogenic acid inhibited the activity of α-amylase. Based on enzyme reaction kinetics, the results of Lineweaver Burk double reciprocal mapping method found that the inhibition type of the enzyme was mixed inhibition. Molecular docking technique was used to reveal the intermolecular interaction between chlorogenic acid and α-amylase. It was predicted that the inhibition mechanism of chlorogenic acid on α-amylase was a mixed inhibition, which was consistent with the results of enzyme reaction kinetics. Molecular docking simulation results showed that chlorogenic acid could not only form hydrogen bonds with Glu261, Asp328 and Tyr193 at the catalytic active site of α-amylase to achieve competitive inhibition with starch, but also form hydrogen bonds with His327 and Ala232 amino acid residues of α-amylase to achieve non-competitive inhibition combined with α-amylase substrate complex. Based on the inhibitory effect of chlorogenic acid on α-amylase, it might illuminate an approach towards the strategic inhibition of digestive enzymes by chlorogenic acid and provide some new insights into the utilization of chlorogenic acid for slowing starch digestion. It might provide theoretical basis and beneficial reference for the designing foods for type II diabetic patients with chlorogenic acid.

Abstract:Pyruvate is an important organic acid, which plays an important role in the energy metabolism of organisms. As a precursor, pyruvate can participate in the biosynthesis of many organic compounds. Therefore, the metabolic engineering strategy was used to modify corynebacterium glutamate to produce pyruvate. First, five key genes related to pyruvate metabolism tributaries of Corynebacterium glutamicum were successfully knocked out by homologous recombination method: pyruvate quinone oxidoreductase gene pqo, pyruvate carboxylase gene pyc, aminotransferase gene alaT, valine-pyruvate aminotransferase gene avtA, pyruvate dehydrogenase gene aceE. The yield of pyruvate reached 14.64 g/L after 72 h of shaking flask fermentation. In order to further increase the metabolic flux of pyruvate pathway, transketolase gene tkt, transaldolase gene tal, phosphoenolpyruvate carboxykinase gene pck were overexpressed to increase the supply of precursors for pyruvate synthesis. Finally, the yield of pyruvate reached 15.39 g/L after 72 h shaker fermentation, which was 28 times higher than that of the wild-type strain, which is helpful to provide theoretical basis and reference for the future study on the production of pyruvate by microbial fermentation.

Abstract:Ficus carica Linn, a tender and juicy fruit, is susceptible to damage and infection caused by microorganisms. In this work, pathogenic fungi were isolated from infected Ficus carica fruit and leaves during post-harvest storage. Based on morphological, internal transcribed spacer (ITS) sequence analysis of fungal isolates enabled species delimitation and their position on the phylogenetic combined tree, four pathogenic fungi were isolated from infected fruit and leaf. All isolates may have caused similar symptoms of the disease in healthy Ficus carica fruit. Four strains were identified as Alternaria alternata (179#), Fusarium odoratissimum (180#), as Colletotrichum endophyticum (181#), and Mucor racemosus (182#), among which Fusarium odoratissimum , Colletotrichum endophyticum and Mucor racemosus were the first report of causing disease of Ficus carica fruit. According to biolog microbial identification system, the carbon metabolic fingerprinting showed that four strains had similar metabolic characteristics to 60 carbon sources, including 56 available carbon sources such as L-arabinose, D-fibrinose and α -cyclodextrin, and 4 unavailable carbon sources such as N-acetyl-D-galactosamine, N-acetyl-β -D-mannosamine, L-trehalose and D-methyl lactate. This study provides a preliminary reference for biological features and post-harvest control measures for Ficus carica fruit diseases.