Table of Content

25 May 2021, Volume 42 Issue 10
Food Chemistry
Effect of Maltose on Gelatinization and Rheological Properties of Waxy Cereal Starches
LIN Nan, XIAO Yu, YANG Xinbiao, ZHENG Mingzhu, PAN Chu, QIN Zhixin, LI Xuezhen, ZHANG Luyue, LIU Jingsheng
2021, 42(10):  1-7.  doi:10.7506/spkx1002-6630-20200514-165
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This paper reports on the effect of adding different amounts (2%, 6% and 10%) of maltose to proso millet starch, waxy rice starch and waxy corn starch on their gelatinization and rheological properties as determined using a rapid viscosity analyzer (RVA), a rheometer, differential scanning calorimetry (DSC) and low-field strength nuclear magnetic resonance (LF-NMR). The results showed that maltose could increase the pasting temperature of the three waxy cereal starches and reduce the peak viscosity, final viscosity and setback value. The shear stress and consistency of starch paste decrease with maltose addition, but the pseudoplasticity remained unchanged. Addition of 10% maltose had a greater influence on waxy corn starch than on proso millet starch and waxy rice starch, decreasing the consistency coefficient K value from 32.546 to 4.801 Pa·sn and giving rise to a more obvious shear thinning phenomenon. Thermodynamic studies showed that addition of maltose could increase the gelatinization temperature and gelatinization enthalpy value of the three waxy cereal starches in a concentration-dependent manner. Low-field nuclear magnetic resonance revealed that addition of maltose increased the content of bound water and immobile water in the entire system, and reduced the content of free water, which further explains its ability to reduce the viscosity of the system and increase starch gelatinization temperature. The results of this study can guide the application of maltose in waxy cereal foods.
Effect of Acid-Coagulation pH on Physicochemical Properties, Protein Structure and Microstructure of Rushan Cheese Curd
WEI Guangqiang, WANG Yutan, MAO Xingju, LIAO Ziyu, YANG Jing, HUANG Aixiang
2021, 42(10):  8-13.  doi:10.7506/spkx1002-6630-20200324-361
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In this work, the effect of acid-coagulation pH on the physicochemical properties, protein structure and microstructure of Rushan cheese curd was studied along with its effect on the stretch molding of Rushan cheese. The results showed that with the increase in acid-coagulation pH, κ-casein was degraded; the particle size of Rushan cheese curd rose initially and then declined, reaching an average of (26.74 ± 0.11) μm at pH 4.8, and so did all tested components, reaching the highest value at pH 4.8. The firmness of Rushan cheese curd increased significantly (P < 0.05). At an acid-coagulation pH of 4.8, the secondary structure of casein exhibited the smallest α-helix to β-sheet ratio and β-turn content, which were 0.398 and 20.55%, respectively. The flexibility and stability of the curd were excellent. The microstructure observation showed that the fat globules in the agglomerates were distributed in a uniform and orderly manner. It formed a dense milk gel with protein and water. In summary, the acid-coagulation pH affects the degradation and structural change of casein and consequently the physicochemical properties and structure of Rushan cheese curd. An acid-coagulation pH of 4.8 can impart a dense structure together with strong water-holding capacity and plasticity to Rushan cheese curd and make Rushan cheese easy to mold by stretching it. This study is expected to provide a scientific basis for the selection of processing parameters of Rushan cheese.
Interaction between Apple Polyphenols and Pectin and Its Effect on the Physicochemical Properties and Stability of Turbid Juices
REN Jiaqi, LIU Xin, LEI Lin, ZHAO Jichun, ZENG Kaifang, MING Jian
2021, 42(10):  14-22.  doi:10.7506/spkx1002-6630-20200506-058
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In order to further understand the effect of polyphenols commonly present in cloudy apple juice on the structure of pectin and the stability of composite systems containing them, the three major polyphenols (phlorizin, chlorogenic acid, epicatechin) in apples were separately mixed with 5 mg/mL apple pectin in water at different concentration gradients (0.03–0.70 mmol/L) determined according to their contents in apples from different varieties and producing area and of different maturities. The structure of apple pectin were investigated by ultraviolet spectroscopy, infrared spectroscopy and scanning electron microscopy. The physicochemical properties and stability of composite systems were analyzed by rheology, thermogravimetry, particle size and turbidity retention rate. The results showed that the polyphenols interacted noncovalently with pectin mainly through hydrogen bonds, and intramolecular and intermolecular hydrogen bonds, C–H, and C=O bonds all participated in the reaction. The surface of the composite systems was smooth, showing a sheet-like structure. The composite systems had higher thermal stability, and reduced particle size and turbidity than did pectin. Chlorogenic acid at 0.08 mmol/L could increase the apparent viscosity of the system and enhance the pseudoplasticity. Phlorizin at 0.03 mmol/L could improve the turbidity retention rate of the composite system. The research results provide a theoretical reference for improving the processing technology for cloudy apple juice products.
Enzymatic Modification of Dough Added with Grifola frondosa Flour and Quality Evaluation of Noodles Made from It
FANG Donglu, MA Xiaohui, ZHAO Mingwen, ZHENG Huihua, CHEN Hui, HU Qiuhui, ZHAO Liyan
2021, 42(10):  23-31.  doi:10.7506/spkx1002-6630-20200620-272
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In order to mitigate the negative impact of Grifola frondosa flour on noodle quality, glucose oxidase (GOX) and transglutaminase (TG) were applied and the color and texture properties of enzymatically modified noodles were analyzed to determine the optimal enzymatic modification process. Meanwhile, its nutritional properties and flavor were evaluated as well. The results showed that the color and texture were significantly improved by using the two enzymes, TG being more effective than GOX. Treatment with 0.9% TG increased the maximum shear force by 108.77% as compared to the control, gave the highest hardness, cohesiveness, gumminess, chewiness and resilience together with a breaking rate of noodles during cooking of 0%. Further, the quality of TG-modified noodles added with G. frondosa was compared with that of commercially available noodles with Lentinus edodes and traditional noodles. It turned out that the contents of protein, fat and total dietary fiber in noodles added with G. frondosa were 13.86%, 0.47% and 7.53%, which were significantly higher than those of the control group. In addition, the contents of K, Zn and essential amino acids were also increased significantly. The flavor of the different noodles was clearly distinguished through E-nose and E-tongue. In general, the color, texture and cooking properties of noodles added with G. frondosa were improved by TG treatment effectively. The addition of G. frondosa flour could remedy the limitation of traditional noodles in nutrition, flavor and health protection. This study provides a theoretical reference for the development of functional staple foods derived from mushroom.
Effect of Chia Seed Coat Polysaccharide on the Emulsion Stability and Quality of Ice Cream
LIU Tingting, ZHANG Shanshan, ZHAO Wenting, CHEN Yuetong, ZHANG Yanrong
2021, 42(10):  32-37.  doi:10.7506/spkx1002-6630-20200729-361
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To investigate the applicability of chia seed coat polysaccharides at different concentrations (0.5, 1.0, 1.5 and 2.0 mg/mL) as an emulsion stabilizer in ice cream products, the effects of different concentrations of chia seed coat polysaccharides on the quality of ice cream were studied by determining the stability of ice cream slurry and the overrun, melting rate, texture and the bubble size distribution of ice cream. The results showed that with the increase of chia seed coat polysaccharide concentration, the stability of ice cream slurry and the overrun and texture properties (elasticity, adhesiveness and chewiness) of ice cream increased, and the melting rate and hardness decreased. The bubble size distribution was more even, and the number of bubbles, uniform in diameter, increased. Addition of chia seed coat polysaccharide could increase the apparent viscosity of ice cream slurry, making it a pseudoplastic non-Newtonian fluid. Ice cream with 0.5 mg/mL concentration of chia seed coat polysaccharide exhibited slightly improved properties compared with the blank control group. However, upon addition of chia seed coat polysaccharide at a level greater than 1.5 mg/mL, the melting resistance and stability of ice cream were significantly improved, together with reduced hardness, improved microstructure, and smoother texture. In conclusion, addition of Chia seed coat polysaccharides could improve the quality, microstructure and stability of ice cream remarkably, which provides a scientific basis for the application of chia seed coat polysaccharides in cold drinks.
Preparation, Characterization and Sustained Release Property of Black Rice Anthocyanin-Loaded Chitsan γ-Polyglutamic Acid Nanoparticles
PAN Fei, ZHAO Lei, CHEN Yanlin, HAO Shuai, ZHANG Huimin
2021, 42(10):  38-44.  doi:10.7506/spkx1002-6630-20200410-139
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In order to improve the stability and sustained release property of black rice anthocyanin (BRA), chitosan was used to encapsulate BRA by ionic coacervation method with γ-polyglutamic acid (PGA) under pH-driven conditions in this study. Using combination of one-factor-at-a-time method and response surface methodology with particle size and encapsulation efficiency as response variables, the optimal preparation conditions were obtained as follows: chitosan concentration 0.8 mg/mL, PGA-to-chitosan ratio 7:20, BRA concentration 1.98 mg/mL, pH 5.0 and stirring time 30 min. The encapsulation efficiency and loading efficiency of the BRA nanoparticles prepared using the optimized conditions were 50.90% and 4.77%, respectively, with particle size of (352.95 ± 6.42) nm, PDI of 0.23 ± 0.02, and zeta potential of (29.56 ± 1.09) mV, suggesting small particle size and good dispersion stability. Observation by scanning electron microscope (SEM) demonstrated that the nanoparticles of BRA were spherical in shape. Compared with free BRA, the release rates of the BRA nanoparticles in simulated gastric and intestinal fluids were reduced by 52.65% and 36.69%, respectively, indicating that the prepared BRA nanoparticles have good sustained release property and thus have good application prospects.
Effect of Quercetin on the Structure and Gel Properties of Pork Myofibrillar Protein under Oxidative Conditions
JIA Na, SUN Jia, LIU Dan, JIN Boyang, LIU Dengyong
2021, 42(10):  45-51.  doi:10.7506/spkx1002-6630-20191103-021
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In order to study the effect of quercetin on the structure and gel properties of pork myofibrillar protein under oxidative conditions, an oxidation system (40 mg/mL protein, 10 μmol/L FeCl3, 100 μmol/L VC, and 1 mmol/L H2O2) was established and quercetin at different concentrations (10, 50, 100, 150 μmol/g) was added to the system. Sulfhydryl content, surface hydrophobicity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern, gel strength, water-holding capacity, microstructure, rheological properties and hydration characteristics were determined after 12 h of incubation in the system. The results showed that quercetin significantly reduced the total sulfhydryl content of myofibrillar protein (P < 0.05). Quercetin at 10 μmol/g decreased the surface hydrophobicity and increased it slightly at higher concentrations. Quercetin weakened the intensity of the band of the myosin heavy chain (MHC) and it also weakened the intensity of the band of actin at concentrations of 100 and 150 μmol/g. Both the MHC and actin participated in the formation of macromolecular protein aggregates and the aggregates could be reduced. Quercetin enhanced the gel strength and water-holding capacity and made the gel microstructure more compact. It resulted in partial conversion of free water into immobile water and enhanced the water-binding capacity as well as the G’ and G” of the protein. Therefore, quercetin improved the gel properties of myofibrillar protein by covalently cross-linking with its sulfhydryl group and properly enhancing its surface hydrophobicity; moreover, quercetin could concentration-dependently enhance the gel-forming capacity of myofibrillar protein.
Extraction, Purification and Structure Analysis of Soluble Acidic Polysaccharides from Soybean Dregs
WANG Siqi, HU Yanbo, ZHAI Liyuan, SHI Zenghui, ZHAO Jun
2021, 42(10):  52-57.  doi:10.7506/spkx1002-6630-20210118-187
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The crude polysaccharide extracted from soybean dregs was purified and fractionated into two soluble acid polysaccharide fractions with uniform charge, SSPS-A1 and SSPS-A2. SSPS-A1 was further subjected to gel filtration chromatography, yielding a homogenous polysaccharide with uniform charge, SSPS-A1-c. Its molecular mass and monosaccharide composition were measured, and its structure was analyzed by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy. The results showed that SSPS-A1-c had a molecular mass of 31.6 kDa, mainly composed of galacturonic acid (GalA), galactose (Gal) and arabinose (Ara) with a molar ratio of 17.3:44.9:19.1 together with lesser amounts of rhamnose (Rha), glucose (Glc) and xylose (Xyl). IR spectroscopy analysis showed that SSPS-A1-c had the characteristic absorption peaks of acidic polysaccharides such as O–H, C–H and C=O and it may be methylated or acetylated to a certain extent. NMR results showed that SSPS-A1-c had chemical shifts of α-1,5-Araf, α-t-Araf and β-t-Galp, which was speculated to contain arabinogalactan (AG)-type pectin domain. Besides, it also exhibited chemical shift of α-1,4-GalpA, which was inferred to contain the homogalacturonan (HG)-type pectin domain. The NMR results were consistent with the monosaccharide composition and infrared analysis results. The research results will provide a theoretical basis for the application of soluble acidic polysaccharides from soybean dregs, and will be of great significance to the comprehensive development and utilization of soybean dregs.
Analysis of Butyric Acid in Sufu and Fermentation Characteristics of Butyric Acid-Producing Bacteria Isolated from Sufu
MA Yanli, DUAN Zhe, LIANG Jingjing, LI Suping, DING Yufeng
2021, 42(10):  58-64.  doi:10.7506/spkx1002-6630-20200210-083
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This study investigated bgutyric acid contents in different types of sufu and the pattern of change in butyric acid content during grey sufu fermentation, and it also evaluated fermentation characteristics of butyric acid-producing bacteria isolated and identified from grey sufu. The results showed that the butyric acid content of grey sufu was 25.4 mg/g dry sample, much higher than that of other kinds. Butyric acid was mainly produced during the first 30 days of post-fermentation, which was closely related to specific microorganisms in the post-fermentation process. The lactic acid content of grey sufu was low and gradually decreased during post-fermentation, which could be converted into butyric acid by specific microorganisms. Clostridium butyricum BP01 quickly entered the logarithmic growth stage and then the stationary phase after 18 hours of cultivation. The pH of the final product was 5.8, and the strain could utilize lactic acid as a carbon source, and the butyric acid content of the final product increased significantly when glucose and lactic acid were used as a mixed carbon source. The strain had the strongest ability to inhibit Escherichia coli followed by Staphylococcus aureus and Bacillus cereus. It could tolerate a certain concentration of NaCl, and had certain temperature adaptability, indicating its potential to be applied in grey sufu fermentation. These results showed that grey sufu was rich in butyric acid, which was much higher than any other food resource reported so far.
Effect of Stepwise Fermentation Modes on Microbial Population and Quality of Pixian Broad Bean Paste Mash during Fermentation
LI Xiongbo, DENG Weiqin, LI Heng, FAN Zhiyi, LI Jiezhi, CHEN Gong
2021, 42(10):  65-72.  doi:10.7506/spkx1002-6630-20200108-104
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Two stepwise fermentation modes of low salt and low temperature followed by high salt and high temperature for the production of Pixian broad bean paste were established in this study. In mode 1, salinity, temperature and fermentation time were set as 6%, 12 ℃ and 12 days for the early stage; 6%, 37 ℃ and 4 days for the middle stage; 15%, 37 ℃ and 14 days for the late stage, respectively. The conditions for mode 2 were the same as those for mode 1 except that the salinity was 9% at the early and middle stages of fermentation. The traditional high temperature fermentation was set as the control group. The changes of mold count, total bacterial count and physicochemical indexes were detected during the fermentation process. Meanwhile, the biogenic amines and volatile components of broad bean paste mash samples at the end of fermentation were analyzed. The results showed the mold count and total bacterial count kept stable at first, and then decreased rapidly in the two stepwise fermentation modes. In the control group, the mold count kept decreasing during fermentation, while the total number of bacteria decreased at the beginning and then increased slowly to reach a plateau. At the end of fermentation, the contents of total acid, amino acid nitrogen and biogenic amine in the mode 1, mode 2 and control groups were 0.96%, 0.92% and 0.87%; 0.76%, 0.83% and 0.66%; and 122.93, 126.50 and 176.12 mg/kg, respectively. In addition, the types and contents of volatile compounds in modes 1 and 2 were higher than those in the control group. The total content of volatile compounds especially esters was the highest in mode 1. Sensory evaluation showed that the sensory quality of mode 1 was the best, followed by mode 2, while the control group was the worst. The comprehensive analysis showed that the quality of broad bean paste mashes produced by the stepwise fermentation modes was better than that of the control group, especially mode 1.
Artificial Neural Network-Genetic Algorithm-Based Optimization of High Cell Density Cultivation of Recombinant Escherichia coli for Producing Pullulanase
CHI Lei, WANG Jingyu, HOU Junchao, WEI Jiajia, WEI Tao, HU Xiaolong, HE Peixin
2021, 42(10):  73-78.  doi:10.7506/spkx1002-6630-20200101-006
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In this study, the high cell density cultivation of recombinant Escherichia coli BL 21 for the production of a novel thermostable pullulanase was optimized using artificial neural network and genetic algorithm. The effects of culture temperature, medium pH, and carbon-to-nitrogen (C/N) molar ratio were tested in a 5 L bioreactor. The results suggested that the optimal culture conditions before the induction phase were as follows: temperature 34.4 ℃, pH 6.87 and C/N ratio 6.1, and the optimal culture conditions after induction were 32.5 ℃, pH 6.69 and 5.3 C/N ratio. The maximum biomass, protein concentration and pullulanase activity obtained under these conditions were 56.5 g/L, 3.21 g/L and 268.3 U/mL, respectively.
Gene Mining, Expression and Identification of Novel Glutamate Decarboxylases Based on Genome Mining
LI Xiang, XIE Yuli, JIA Yuanyuan, ZHANG Shan, WANG Hongyan, LIU Xianli, LUO Xiangyan, TANG Cunduo
2021, 42(10):  79-85.  doi:10.7506/spkx1002-6630-20200102-012
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In this study, three hypothesized glutamate decarboxylases (GAD) genes from Lactococcus lactis, Lactobacillus senmaizukei and Enterococcus sulfureus, respectively named as LlGAD, LsGAD and EsGAD, were excavated using genome mining technology with the high-activity GAD gene (LbGAD) from Lactobacillus brevis as the probe. By means of pET28a plasmid, these four genes were expressed in E. coli BL21. The expressed products of LsGAD and LlGAD had a good solubility, exhibiting GAD activity of 34.17 and 38.91 U/mL, respectively in the fermentation broths. The specific activity, temperature characteristics, pH characteristics and Kcat/Km value of LsGAD were significantly superior to those of the other GAD enzymes. In addition, the biosynthesis of γ-aminobutyric acid (GABA) from L-glutamic acid by whole-cell catalysis was studied. When L-glutamic acid at 6 g/L was transformed for 24 h, the maximum yield of GABA of 58% was obtained. In conclusion, this study has realized a leap from genomic data to real enzymes by obtaining GAD with excellent performance as well as the biosynthesis of GABA, which will lay a solid foundation for the low-cost and large-scale biosynthesis of GABA.
Spatial Heterogeneity of Prokaryotic Microbial Community Diversity in Pit Mud from Degraded Cellar for the Production of Strong-flavor Baijiu Revealed by High Throughput Sequencing
HU Xiaolong, YU Miao, CAO Zhenhua, WANG Kangli, TIAN Ruijie, HAN Suna, LI Hua, LI Jianmin, ZENG Tian, LI Hong
2021, 42(10):  86-93.  doi:10.7506/spkx1002-6630-20200101-004
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In this study, high-throughput sequencing technology combined with network analysis and multivariate statistical analysis was used to analyze the prokaryotic microbial community diversity and microbial community networks, as well as the physicochemical factors affecting the microbial community in pit muds from the upper (PW-T), middle (PW-M) and lower (PW-U) layers of the wall, and from the bottom (PB) of a degraded fermentation pit used for the production of strong-flavor Baijiu. The results showed that the α and β diversity of the pit mud microbial community had obvious spatial heterogeneity in the degraded pit, and the process of degradation could proceed from top to bottom. As the depth of the pit was increased, the α diversity index of the pit mud microbial community showed a significant upward trend on the whole. Specifically, the Shannon index of microbial diversity followed the decreasing order of PW-U (3.38) > PB (2.51) > PW-M (1.09) > PW-T (0.33) (P < 0.05), and there was only one dominant phylum (Firmicutes, about 99%) in PW-T and PW-M, compared to six including Firmicutes, Bacteroidetes, and Euryarchaeota in PW-U and four in PB. All the pit muds had low water content (with an average of 32.7%), low pH (with an average of 4.42), and low available phosphorus content (with an average of 259.27 mg/kg), which had a great impact on the microbial community structure as determined by redundancy analysis (RDA). According to the co-occurrence network analysis, the genera in different classes (interclass) had a high co-occurrence incidence (accounting for 78.74% of the total correlation). The total content of the nine hubs in the co-occurrence network was very low (0%–2.84%), and it had a significantly positive correlation with the pH and water content of pit mud (P < 0.05 or P < 0.01); all hubs except Syntrophomonas showed a significantly negative correlation with Lactobacillus (P < 0.01). The pH of pit mud had a significantly positive or negative correlation with various physicochemical factors (P < 0.05) and a significantly positive correlation with Simpson index, which indicates that pH could be used as an indicator to evaluate pit mud quality. This study will provide theoretical support for further uncovering the degradation mechanism of pit mud, preventing the degradation of pit mud, and making high-quality artificial pit mud.
Conversion of Linoleic Acid to Conjugated Linoleic Acid by Lactobacillus plantarum p-8
ZHAO Wei, ZHANG Feng, ZHANG Heping, ZHAO Guofen
2021, 42(10):  94-103.  doi:10.7506/spkx1002-6630-20200225-273
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In this study, the ability of whole and disrupted cells of Lactobacillus plantarum p-8 and recombinant linoleate isomerase family from this strain to convert linoleic acid (LA) to conjugated linoleic acid (CLA) were evaluated and the underlying mechanism was explored. The results showed that whole and disrupted cells of L. plantarum p-8 cultured in MRS medium could inefficiently catalyze the conversion of LA to c9,t11-CLA, t10,c12-CLA and t9,t11-CLA, and only a small amount of t10,c12-CLA was observed in the cells. The results of real-time PCR exhibited that lower expression levels of linoleate isomerase family may be responsible for lower CLA production. Members of the independently expressed recombinant linoleate isomerase family, flavin denine dinucleotide (FAD) and nicotinamide adenine dinucleotide were found essential for the conversion of LA to c9,t11-CLA, t10,c12-CLA and t9,t11-CLA through a pathway consistent with that in L. plantarum AUK1009. Homologous modeling of linoleic acid hydrase presented three domains. The substrate-binding site and the FAD site were located in the hydrophobic cavity at the junction of the three domains. M76 and Y180 were essential residues.
Hydrolysis Site of Casein by Milk-Clotting Protease from Moringa oleifera Lam. and Preparation and Evaluation of Casein Phosphopeptide and Casein Glycomacropeptide
SHI Yanan, ZHANG Jiayan, HUANG Aixiang
2021, 42(10):  104-110.  doi:10.7506/spkx1002-6630-20200322-322
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In this study, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to explore the cleavage site and hydrolysis characteristics of casein by a milk-clotting protease from Moringa oleifera Lam. Then, casein phosphopeptide and casein glycomacropeptide in the resulting hydrolysate were analyzed. The results showed that the protease could specifically cleave κ-casein at the Arg93-His94 site leading to milk clotting, which is distinct from other reported chymosins. The Michaelis constant (Km) and maximum reaction rate (Vm) of the milk-clotting protease were 0.49 mg/ml and 45.2 U/min, respectively, and it could promote the aggregation and coagulation of casein micelles. The enzyme could hydrolyze α, β and κ-casein to different extents and exhibit the fastest hydrolysis rate toward α-casein, indicating that it was suitable for the processing of soft cheese. Casein phosphopeptide (CPP) was obtained by barium-ethanol precipitation method with casein as the substrate, with a yield of 15.87%. CPP could delay the precipitation of calcium by 23 min, which was beneficial to the intestinal absorption of calcium. When buffalo milk was used as the substrate, the concentration of casein glycomacropeptide (CGMP) was 6.61 mg/mL in whey, and the degree of glycosylation was 477.527 μg/mg. This study provides an important theoretical basis for the development and application of new plant chymosins in cheese and casein-derived bioactive peptides.
Metatranscriptomic Analysis of Microbial Metabolic Process in Chilled Tan Sheep Meat in Different Chilled Storage Periods
ZHAO Junyi, YANG Bo, LUO Ruiming, ZHANG Heyu, SU Chunxia, HU Qianqian, ZHAO Xiaoce
2021, 42(10):  111-119.  doi:10.7506/spkx1002-6630-20200410-146
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In this study, metatranscriptomics was used to analyze the relationship of differential genomic transcription and metabolic pathways in microbes on the surface of chilled Tan sheep meat sealed in trays and stored for 0, 4 and 8 days with microbial community succession over the 8-day storage period. The results showed that the number of differentially expressed bacterial genes at 0, 4 and 8 days of storage were 429, 15, and 1 529, respectively. Through KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of these DEGs, the differentially expressed genes involved in nucleotide metabolism, lipid metabolism, energy metabolism, carbohydrate metabolism and amino acid metabolism were significantly enriched, indicating that these metabolic pathways played important roles in the succession of microbial community on chilled Tan sheep meat. At the three storage periods, Proteobacteria was the dominant microorganism. Pseudomonas and Enterobacteriaceae, belonging to the Proteobacteria phylum could rapidly grow and reproduce to dominance by utilizing oxygen in the storage environment and glucose in the mutton as the carbon source. As storage proceeded, oxygen was depleted, and the pH of Tan sheep meat was reduced so that lactic acid bacteria quickly grew to be the third most microorganism after Pseudomonas and Enterobacteriaceae. The results of this study can provide a theoretical reference for predicting the microbial community and quality of Tan sheep meat and for controlling the microbial load on it during chilled storage.
Preparation and Activity Analysis of DPP-IV Inhibitory Peptides from Pacific Oyster (Crassostrea gigas)
CHEN Hong, ZHANG Qian, CHEN Yulei, WENG Ling, ZHANG Lingjing, XIE Shaohao, LIU Guangming, CAO Minjie
2021, 42(10):  120-126.  doi:10.7506/spkx1002-6630-20200226-294
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To prepare dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides, Pacific oyster flesh (Crassostrea gigas) was hydrolyzed with five different proteases, including papain, alcalase, pancreatin, neutrase and protromex. The pancreatin hydrolysate was found to have higher DPP-IV inhibitory activity as compared with the other four hydrolysates. The optimal hydrolysis conditions with pancreatin were determined to be 0.8%, 8.0, 37 ℃ and 90 min for enzyme dosage, pH, temperature, and hydrolysis duration, respectively. The hydrolysate obtained using the optimized conditions was purified and fractionated sequentially by ultrafiltration, Sephadex G-15 column chromatography and high performance liquid chromatography (HPLC) into different peptide fractions. Two DPP-IV inhibitory peptides were obtained and identified as Glu-Ile-Thr-Ala-Leu-Ala-Pro-Ser-Thr-Met-Lys (EITALAPSTMK) and Ile-Leu-Ala-Pro-Pro-Glu-Arg (ILAPPER) by mass spectrometry (MS). Their digestibility characteristics in gastrointestinal fluid were analyzed using online simulation with BIOPEP. Two peptides APSTM and ILAPPER were synthesized by solid-phase synthesis for DPP-IV inhibitory activity evaluation. The results showed that the half-maximal inhibitory concentration (IC50) values of the two peptides were 354.81 and 16.98 mmol/L, respectively. Molecular docking results indicated that inhibitory peptides interacted with the active site of DPP-IV through hydrogen bonds, van der Waals forces and p bonds. The results of this study provide a theoretical basis for the development of functional foods from oyster in the future.
Screening for and Identification of Lactic Acid Bacteria with Antioxidant Activity from the Intestinal Tract of Fish
DING Lili, LÜ Xinran, GAO Yongyue, CUI Xiaoling, WANG Xiaomi, BAI Fengling, YI Shumin, GUO Xiaohua
2021, 42(10):  127-132.  doi:10.7506/spkx1002-6630-20200328-414
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In this study, lactic acid bacteria (LAB) with antioxidant activity were isolated from the intestinal tract of seawater and freshwater fish for use in the prevention and attenuation of oxidative deterioration during the processing of aquatic products. The results were showed that five LAB strains with strong antioxidant activity were screened from the 48 LAB strains, among which strain CY1-2 from the intestinal tract of grass carps exhibited the strongest antioxidant activity. The antioxidant activity of the cells of this strain was determined to be (67.29 ± 0.42)%, (60.67 ± 1.44)% and (29.87 ± 1.14)% in the scavenging of 1,1-diphenyl-2-picryl-hydrazyl-hydrate (DPPH), hydroxyl and superoxide anion radicals and (40.77 ± 0.50)% in the inhibition of lipid peroxidation, respectively, and the percentages of hydroxyl and superoxide anion radical scavenging effect and anti-lipid peroxidation effect of the cell-free extract were (64.27 ± 1.26)%, (21.97 ± 1.47)%, and (51.03 ± 0.40)%, respectively. In addition, strain CY1-2 exhibited antibacterial activity against both gram-positive bacteria and gram-negative bacteria, including Bacillus licheniformis, Staphylococcus aureus, B. cereus, Shigella sp., Escherichia coli, Pseudomonas aeruginosa and Pseudomonas fluorescens. It was identified as Lactobacillus plantarum by physiological and biochemical characterization and 16S rDNA sequence analysis. Owing to its strong broad-spectrum antibacterial activity L. plantarum CY1-2 has the potential to be developed as a novel natural microorganism-derived antioxidant from aquatic products.
Improvement of the Product Specificity of Bacillus sp. Y112 Cyclodextrin Glucosyltransferase by Site-Directed Mutagenesis of Arginine 81
LI Xiaohan, GUO Jiaomei, SONG Kai, SUN Jingjing, WANG Wei, HAO Jianhua,
2021, 42(10):  133-138.  doi:10.7506/spkx1002-6630-20200205-031
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The cyclodextrin glucosyltransferase (CGTase) from the marine Bacillus sp. Y112 was analyzed by homologous modeling and amino acid sequence alignment; it was found that the arginine residue at position 81 from the N-terminus may affect the product specificity. In this study, when this residue was mutated to threonine by site-directed mutagenesis, the yield of α-CD was decreased, and the percentage of β-CD in the cyclization products was increased from 64% to 71%, suggesting the improved product specificity. The underlying reason may be related to the size of the substituted amino acid residue and change in hydrogen bonding interaction. At the same time, the enzymatic properties of the mutant retained the thermophilicity, thermostability, and alkali resistance of the wild-type enzyme.
Metagenomic Comparison of Microbial Communities on Eggs from Different Areas of China
LI Xia, WEN Jiaqi, WANG Xiujuan, SONG Yuqi, DAI Weichang, WANG Yuhua
2021, 42(10):  139-145.  doi:10.7506/spkx1002-6630-20200402-026
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In this study, metagenomics of high-throughput sequencing were used to systematically analyze the composition and diversity of microbial communities on eggs from four major egg-producing areas of China, Jilin, Anhui, Beijing and Hebei. The results showed that the bacterial community composition on the eggshell surface consisted mainly of Actinobacteria, Firmicutes and Proteobacteria. The bacterial community composition on egg samples from Jilin consisted of Actinobacteria, Firmicutes and Proteobacteria, with relative abundance of 49.12%, 31.93% and 8.41%, respectively. The bacterial community compositions on egg samples from Anhui, Beijing and Hebei were similar, consisting of Proteobacteria, Firmicutes and Proteobacteria, with Proteobacteria being the dominant phylum, and the relative abundance of Proteobacteria in the three samples was over 50%, 77.03%, 57.93% and 84.28%, respectively. In addition, the composition of microbial community at the genus level on eggs was distinct among different producing areas. The most abundant bacterial genera on eggs from Jilin were Kocuria and Brachybacterium, with relative abundance of 10.1% and 9.83%, respectively. The distribution of bacterial genera on eggs from Anhui, Beijing and Hebei was similar, with Acinetobacter being dominant, and pathogenic bacteria were found, including Staphylococcus with relative abundance of 1.20% for eggs from Anhui, Staphylococcus with relative abundance of 3.53% and Escherichia-Shigella with relative abundance of 3.15% for eggs from Beijng, and Pseudomonas with relative abundance of 16.28% for eggs from Hebei. It can be concluded that there was some difference in the composition of microbial communities at the phylum and genus level on eggs from different regions, especially in the relative abundance. A significant compositional difference in the microbial community on eggs was found between the north region and the central south region (P < 0.01), and the north region was more suitable for egg production. In order to ensure the safety of liquid egg products, egg product enterprises should select a suitable egg disinfection method according to the microbial community composition on its surface, which is of great significance to the liquid egg processing industry.
Effect of Fermentation with High-Yield α-Galactosidase-Producing Lactic Acid Bacteria on Biochemical Properties and Breadmaking Characteristics of Chickpea Sourdough
WU Meng, CAO Weichao, CHENG Xin, MA Zilin, LUO Kun, XU Yan, HUANG Weining, LI Ning, GAO Tiecheng
2021, 42(10):  146-153.  doi:10.7506/spkx1002-6630-20200407-083
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In this study, Lactobacillus fermentum C2-8, which is able to produce high α-galactosidase activity, was used as a starter culture in chickpea sourdough fermentation. The effect of the starter culture on the contents of organic acid, raffinose, stachyose, reducing sugar, and free amino acid in chickpea sourdough as well as on its breadmaking characteristics was evaluated using solid phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS), a texture analyzer, an electronic tongue, and sensory evaluation. The results showed that chickpea sourdough fermented for 24 h contained 62.67 and 15.53 mmol/kg lactic acid and acetic acid, respectively; raffinose and stachyose in it were completely degraded, and the content of reducing sugar was 11.50%. The content of total free amino acid increased 1.26 times when compared to unfermented dough. Compared with chickpea bread (CB), the specific volume of chickpea sourdough bread (CSB) increased (P < 0.05) by 4.60%, while the hardness, and chewiness decreased by 18.49%, and 15.64%, respectively. The peak areas of volatile flavor compounds including alcohols, acids, esters and ketones in CSB markedly increased compared to CB. Interestingly, the peak area of phenylethanol was 2.04 times higher in CSB than CB. Tastes scores of sweetness, sourness, umami, and saltiness and overall flavor characteristics were improved in CSB than CB. Scores for all quality characteristics except color were higher in CSB than CB, with the former having a score for overall acceptability of 7.2 points.
Effect of Bacterial Community Succession on Lipid Hydrolysis and Oxidation during the Traditional Processing of Longxi Bacon, a Traditional Chinese Fermented Meat Product
LI Yanhu, YUN Jianmin, ZHAO Fengyun, ZHANG Wenwei, AI Duiyuan
2021, 42(10):  154-161.  doi:10.7506/spkx1002-6630-20200219-198
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In order to investigate the effect of bacterial community succession on lipid metabolism during the traditional production of Longxi bacon, Illumina MiSeq sequencing was used to analyze the bacterial community structure and succession at the genome level, and gas chromatography-mass spectrometry (GC-MS) was used to detect changes in fatty acid composition and contents during the production process. Additionally, changes in acid value (AV) and peroxide value (POV) were determined. The results showed that the sequencing depth covered most bacterial species in the samples. After sorting out and filtering the sequencing results, 186 866 effective sequences were obtained. After clustering, 580 operational taxonomic units (OTUs) were annotated to 414 genera in 15 phyla. During the whole production process, Proteobacteria and Firmicutes were the dominant bacterial phyla. Brochothrix, Carnobacterium, Latobacillus, Pseudoalteromonas, Psychrobacter and Cupriavidus were the dominant genera. Through function prediction with PICRUSt software, it was found that the abundance of the genes related to fatty acid metabolism was relatively high in the microflora. Fatty acid composition analysis revealed that palmitic acid (C16:0), stearic acid (C18:0), palmitic acid (C16:1), oleic acid (C18:1), and linoleic acid (C18:2n6c) were the major components, accounting for 83.84%–92.43% of the total fatty acids. According to correlation analysis, it was found that the bacteria related to lipid metabolism during the production of Longxi bacon mainly included Streptococcus, Staphylococcus, Lactobacillus, Cupriavidus, Carnobacterium and Brochothrix. Streptococcus and Lactobacillus were related to changes in saturated fatty acids and polyunsaturated fatty acids, while Carnobacterium, Staphylococcus, Brochothrix and Cupriavidus were mainly involved in the metabolism of unsaturated fatty acids, closely related to changes in palmitoleic acid, oleic acid and linoleic acid.
Analysis of Microbial Community Structure in Traditional Sourdough and Flavor Components in Steamed Bread Made with It
LI Xiaomin, HAN Wei, LI Qi, WANG Qing, TAN Xinyue, ZOU Qiulong, ZHANG Xiaolin
2021, 42(10):  162-170.  doi:10.7506/spkx1002-6630-20200109-107
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This study is aimed to figure out the structure of the microbial community in the traditional sourdough and to determine the flavor components of steamed bread made with it. The pH, titratable acidity and total bacterial colony counts of sourdough samples collected from Shanxi (SX), Hebei (HB), Qinghai (QH), Gansu (GS) and Henan (HN) provinces were measured, and the composition of the microbial communities in the five samples were determined by high-throughput sequencing. Besides, solid phase micro-extraction coupled to gas chromatography-mass spectrometry (SPME-GC-MS) was used to determine the flavor compounds in steamed bread. The results showed that the pH of these sourdoughs was between 3.69 and 3.83, the total titratable acidity (TTA) ranged from 3.73 to 4.30 mL, as determined by titration with 0.1 mol/L NaOH, and the number of lactic acid bacteria and yeasts ranged from 4.89 to 8.31 (lg(CFU/g)) and 4.75 to 6.08 (lg(CFU/g)), respectively. Lactobacillus, Lactococcus and Weissella were the dominant bacteria in the sourdoughs, and the dominant fungi were Saccharomyces, Pichia and Candida. Besides, the relative abundance of the dominant bacterial and fungal genera was different among the different samples. In total, 42 volatile compounds were identified in all samples, including alcohols, aldehydes, esters, aromatics and hydrocarbons, 30 of which were found in sample HB, 26 in samples SX, QH and GS, respectively, and 25 in sample HN. The total contents of flavor compounds in steamed breads made with the five sourdoughs were ranked in decreasing order as follows: SX > HN > GS > QH > HB.
Characterization of Novel Broad-Spectrum Bacteriocin Produced by Lactobacillus plantarum from Pickles
GAO Zhaojian, HUANG Lianghao, DING Feihong, ZHAO Yifeng, CHEN Teng
2021, 42(10):  171-177.  doi:10.7506/spkx1002-6630-20200213-135
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This research aimed to screen for Lactobacillus strains with the capability of producing bacteriocin that can specifically inhibit foodborne pathogens and spoilage bacteria and to study the characteristics of the bacteriocin, so as to lay a foundation for its application in food preservation. Out of 112 isolates from pickles, one, coded Hlh32, was found able to produce bacteriocin (BacH32) and identified as Lactobacillus plantarum. The maximum production of BacH32 (anti-Staphylococcus aureus activity of 1 850 AU/mL) was achieved when the culture reached the stationary phase (between 18 and 24 h) in MRS broth at 35 ℃. The bacteriocin was purified sequentially by ammonium sulfate salting-out, DEAE Sepharose Fast Flow anion exchange chromatography, Sephadex G-15 gel filtration chromatography and reversed-phase HPLC. As determined by Trinice SDS-PAGE, the molecular mass of bacteriocin Hlh32 was 6.7 kDa, which was significantly different from that previously reported. It was sensitive to proteolytic enzymes but to lipase, α-amylase not β-amylase, indicating its protein nature. Its antimicrobial activity was maintained well after incubation for 4 h at pH 2–9, 30 min at 37, 60, 80 and 100 ℃, or 15 min at 121 ℃. The bacteriocin was found to have board-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria together with fungi. BacH32 completely inhibited the growth of S. aureus cells at the early-log phase. These results indicate the potential of the bacteriocin for use as a food biopreservative.
Affinity Chromatographic Purification and Enzymatic Characterization of Trypsin from Grass Carp (Ctenopharyngodon idellus)
LI Chen, GAO Liufang, CUI Xiaodong, HAN Yuhang, ZHU Danxu, LI Jiao
2021, 42(10):  178-183.  doi:10.7506/spkx1002-6630-20200304-062
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Trypsin was extracted from the hepatopancreas of grass carps in this study. After homogenization and ammonium sulfate precipitation, crude enzyme solution was prepared by dialysis. Then, trypsin of electrophoretic purity was obtained by BTI-Sepharose affinity chromatography in only one step. The results showed that the molecular mass of the trypsin was about 27 kDa, and the Km was 3.4 × 10-5 mol/L. The optimum reaction temperature of the enzyme was 60 ℃ and it remained stabile at temperatures lower than 60 ℃. Its optimal reaction pH was 9.5, and the pH tolerance range was 6.0–12.0. Ba2+, Mg2+ and Fe2+ all could activate the enzyme in the concentration range of 0–10 mmol/L. EDTA and K+ inhibited the enzyme in the concentration range of 0–10 mmol/L, the former being more effective than the latter.
Effect of Protease Preparation on Physicochemical Properties and Microbiota of Shrimp Paste during Fermentation
LI Wenya, LIU Yang, LI Ning, ZHENG Xiaowei, SANG Yaxin, SUN Jilu
2021, 42(10):  184-192.  doi:10.7506/spkx1002-6630-20200301-014
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In order to improve the production process of shrimp paste, the effect of a commercial protease preparation on the physicochemical properties and microbial community of shrimp paste during fermentation was systematically studied in this work. The pH, amino acid nitrogen (AAN), malondialdehyde (MDA), total volatile basic nitrogen (TVB-N) and biogenic amine of shrimp paste were measured. The composition of the bacterial community was analyzed by high-throughput sequencing technology. The correlation between the bacterial community and physicochemical properties during shrimp paste fermentation was analyzed by Spearman’s test. The results showed that the contents of AAN and TVB-N in shrimp paste with the addition of the protease preparation increased more significantly during fermentation compared with the blank control group, shortening the fermentation time but simultaneously leading to a decline in the quality of shrimp paste. However, its addition had no apparent effect on the biogenic amine content. A total of 313 864 valid sequences clustered into 1 279 operational taxonomic units (OTUs) were obtained from shrimp paste samples. The addition of the enzyme preparation significantly affected the bacterial community composition and reduced the bacterial richness. At the genus level, Flavobacterium was the dominant bacterium in the control group, while Bacillus became dominant upon the addition of the enzyme preparation, together with increased relative abundance of Bacillus and reduced relative abundance of Lactococcus. The dominant flora was Bacillus, Flavobacterium and Lactococcus, which affected the quality and physicochemical properties of shrimp paste. In conclusion, the addition of the protease preparation had an important effect on the bacterial community composition, MDA, TVB-N and AAN contents in shrimp paste during fermentation, which would provide a theoretical basis for improving the production process of shrimp paste.
Metabolomic and Transcriptomic Analysis of Response Mechanism of Baker’s Yeast to Freezing Stress
MENG Lu, LIU Hancheng, LIU Yahan, LIN Xue, LIU Sixin, LI Congfa
2021, 42(10):  193-200.  doi:10.7506/spkx1002-6630-20200204-027
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In order to study the mechanism underlying the response of baker’s yeast (Saccharomyces cerevisiae) to freezing stress, intracellular metabolomic and transcriptomic analysis was carried out on the fermentation broth of baker’s yeast before and after treatment at ?20 ℃ for 7 days. In this experiment, the survival rate of baker’s yeast undergoing environmental stress was 43% after simulated dough fermentation without added sugar, and the fermentation capability was decreased by 42% as compared to the untreated control. The changes of 24 intracellular metabolites and the differential expression of 494 genes in S. cerevisiae were related to the response to freezing stress. Through the analysis of differential metabolic pathways, we found that the lack of intracellular amino acids and plasma membrane stiffness may be the major factors affecting cell growth and fermentation performance under freezing stress. However, the increase in the relative content of intracellular unsaturated fatty acids and the accumulation of trehalose could not eliminate the cellular damage caused by low temperatures. These results can improve our understanding of the response mechanism of yeast to freezing stress, which will provide new ideas for future studies of the resistance regulatory mechanism in yeast, and will be of great significance for the optimization and technical development of frozen dough.
Effect of Truncation of N-Terminal Structural Modules on Enzymatic Properties of Thermoacidiphilic Type III Pullulan Hydrolase TK-PUL
ZENG Jing, HE Chukuo, GUO Jianjun, YUAN Lin
2021, 42(10):  201-208.  doi:10.7506/spkx1002-6630-20200408-099
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In this study, the enzymatic properties of TK-PUL, a thermoacidiphilic type III pullulan hydrolase produced by the extremely thermophilic archaea Thermococcus kodakarensis KOD1 were compared with those of its N-terminal truncated mutants, in order to determine the effect of the truncation of N-terminal structural modules on the enzymatic properties of TK-PUL. The truncation of the structural module N1 improved the catalytic characteristics of TK-PUL, resulting in a 1.11-fold increase in α-amylase specific activity, a 1.12-fold increase in pululanase specific activity and a 1.15-fold increase in half-life at 100 ℃. The truncation of the structural module N2 enhanced the thermal stability of the enzyme, and extended the half-life at 100 ℃ by 1.25 times, whilst it decreased the pH stability, substrate binding capacity and specific activity of TK-PUL. Moreover, the truncation of the structural module N2 changed the substrate specificity of TK-PUL, resulting in an increase in the ratio of α-amylase to pullulanase activity from 0.49 to 0.60. The results showed that neither the structural module N1 nor N2 of TK-PUL was the essential structural regions for enzymatic catalysis, but both of them had important effects on the substrate binding capacity, catalytic activity and stability of the enzyme.
Optimization of the Production of Ethyl Hexanoate and Ethyl Butyrate by Cofermentation of Saccharomyces cerevisiae and Esterifying Bacteria from Pit Mud of Chinese Baijiu Using Response Surface Methodology
GUO Yan, DENG Jie, REN Zhiqiang, HUANG Zhiguo, WEI Chunhui, HUANG Mingcai
2021, 42(10):  209-217.  doi:10.7506/spkx1002-6630-20200408-105
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In order to increase the yield of ethyl butyrate and ethyl hexanoate in the fermentation process of Luzhou-flavor Baijiu, solid-state fermentation conditions for mixed culture fermentation of Saccharomyces cerevisiae and three strains of esterifying bacteria from Baijiu cellar pit mud were optimized using one-factor-at-a-time method and Box-Behnken design response surface methodology. The results showed that the water content and acidity of sorghum-based medium and inoculum quantity had significant effects on the production of ethyl hexanoate and ethyl butyrate (P < 0.01), while fermentation temperature had no significant effect (P > 0.05). The optimum fermentation conditions were as follows: temperature 32 ℃, water content 78.00%, acidity 2.60 mmol/100 g, and inoculum quantity 4.60%. Experiments carried out under these conditions led to yields of 9.55 mg/100 g and 1.87 mg/100 g for ethyl hexanoate and ethyl butyrate, respectively, which were 99.07% and 100% as compared to the predicted values, respectively. The optimal process was validated to be reasonable and feasible, which provides a theoretical basis for further improving the quality of Luzhou-flavor Baijiu and the application of functional microorganisms from Baijiu cellar pit mud in actual production.
Screening of Bacillus from Daqu by Enzymatic Characteristics to Enhance the Fermentation of Chinese Baijiu
HUANG Xiaoning, LIU Jingjing, HAN Beizhong, CHEN Jingyu
2021, 42(10):  218-224.  doi:10.7506/spkx1002-6630-20200407-073
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The α-amylase and protease activities of 105 Bacillus strains isolated from light-flavor, strong-flavor and sauce-flavor Daqu were evaluated. Out of them, B. licheniformis (B.L-1) and B. subtilis (B.S-1) were selected for application in enhanced fermentation of light-flavor Baijiu. The volatile compounds of fermented grains were determined after completion of the fermentation. A total of 38 volatile compounds were identified by headspace solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS). By multivariate statistical analysis, 21 and 11 significantly differential metabolites were identified in the samples fermented with B.L-1 and B.S-1, respectively (VIP > 1; P < 0.05). 4-Ethyl-2-methoxy-phenol, ethyl octoate, 3-methyl butanoic acid ethyl ester, and tetramethyl-pyrazine were the differential metabolites shared by both enhanced fermentation groups, and their contents significantly increased compared to the control group. The results indicated that enhanced fermentation with the selected Bacillus isolates could remarkably increase the contents of volatile flavor substances in the fermented grains. This study has provided a good way to select potential candidates for the construction of synthetic microbiota in Daqu.
Component Analysis
Preparation of Compound Seasoning from Lamb Meat: Analysis of Characteristic Flavor Compounds by SPME-GC-MS and Electronic Tongue
ZHANG Ting, QI Lulu, PAN Daodong, YANG Zhengcang, DANG Yali
2021, 42(10):  225-234.  doi:10.7506/spkx1002-6630-20200822-296
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In the preparation of a compound seasoning from lamb meat, we analyzed the volatile flavor compounds in lamb foreleg and hind leg from Inner Mongolia by solid-phase micro-extraction combined with gas chromatography-mass spectrometry (SPME-GC-MS). The hind leg meat was sequentially hydrolyzed with two proteases and simultaneously deodorized with β-cyclodextrin or ginger, and Maillard reaction was carried out on the enzymatic hydrolysate. The characteristic flavor compounds of the hydrolysate and the Maillard reaction products from it were analyzed by SPME-GC-MS, electronic tongue and sensory evaluation. Finally, the formulation of compound seasoning with the Maillard reaction products as the flavor base was optimized. Results showed that the contents of low-flavor threshold aldehydes, unsaturated alcohols and important acids were higher, and the contents of amines responsible for the fishy smell and 1-butanol responsible for the pungent odor in the hind leg were lower than those in the foreleg, indicating the former was more suitable for preparing seasonings than the latter. The odor was more effectively removed by adding 3% ginger rather than 5% β-cyclodextrin. The Maillard reaction could effectively enhance the overall flavor of compound seasonings, and the major characteristic flavor substances in the Maillard reaction products were 4-methyl-5-(β-hydroxyethyl) thiazole, 2-methyl-3-furan mercaptan and thiophene-2-mercaptan. This study can provide a theoretical reference for the development of high-quality meat seasonings.
Analysis of Volatile Flavor Compounds Dairy Products by Gas Chromatography-Ion Mobility Spectrometry
LI Juan, REN Fang, ZHEN Dawei, ZHANG Hao, XIE Jianchun
2021, 42(10):  235-240.  doi:10.7506/spkx1002-6630-20200503-020
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Gas chromatography-ion mobility spectrometry (GC-IMS) was used to analyze the volatile flavor compounds different dairy products, including two brands of pasteurized milk, five brands of ultra-high-temperature sterilized milk and one brand of reconstituted milk. A total of 29 compounds were identified, mainly aldehydes, ketones, alcohols, esters, and others. Methyl hexanoate, ethyl butanoate, 3-methylbutyl acetate, ethyl acetate, ethyl pentanoate, 1-pentanol, and limonene constituted the characteristic peak region of pasteurized milk; furfural, benzaldehyde, methyl isobutyl ketone, 2-octanone, 1-butanol, and isopropyl acetate constituted the characteristic peak region of reconstituted milk; and new volatile compounds generated by sterilization constituted the characteristic peak region of ultra-high-temperature sterilized milk. By means of principal component analysis (PCA), the different types or brands of dairy products were clearly classified. This study indicates the usefulness of GC-IMS to discriminate different dairy products.
Variation of Bioactive Components in Red Raspberry Juice during Fermentation for Deacidification
CHEN Sirui, TANG Ying, DONG Dan, JIANG Ying, HE Hongying, WANG Jinling
2021, 42(10):  241-248.  doi:10.7506/spkx1002-6630-20200304-065
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In order to explore the effect of fermentation with Issatchenkia terricola WJL-G4 on bioactive components in red raspberry juice, the contents of total phenolics, total flavonoids and anthocyanins in inoculated and uninoculated juices were analyzed by high performance liquid chromatography (HPLC). Correlation analysis and principal component analysis (PCA) were used to discuss the correlation between the fermentation process and the bioactive components. The results showed that the contents of total phenolics and anthocyanins in the inoculated and non-inoculated groups were decreased during fermentation, while total flavonoids were significantly increased in the inoculated group after 5 days of fermentation. After 8 days of fermentation, ten phenolic acids, seven flavonoids and one aromatic compound were?identified in the juice. The contents of cryptochlorogenic acid, p-coumaric acid, raspberry ketone, rutin and quercetin were significantly increased. Among the nine components which were significantly decreased during fermentation, the contents of arbutin, gallic acid, neochlorogenic acid and caffeic acid were higher than those in the control group. The PCA results showed that the first three principal components together explained 93.098% of the total variation, which could well describe the information of bioactive components in the fermented juice. The samples fermented for 1 to 4 days located in the positive interval of the plot for PC1 had higher synthetic scores, indicating fermentation for 1 to 4 days to be optimal for acid degradation in red raspberry juice. This study provides a theoretical support for research on the deacidification properties and comprehensive utilization of I. terricola WJL-G4.
Analysis of the Contents of 13 Intrinsic Free Sugars in Different Lily Samples
KONG Ying, WANG Huan, LANG Lixin, DOU Xiaoying, BAI Jinrong
2021, 42(10):  249-254.  doi:10.7506/spkx1002-6630-20200508-080
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In this study, the contents of intrinsic free monosaccharides and disaccharides in 15 lily samples collected from different producing regions of China were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the major free sugars in all lily samples were sucrose, glucose and fructose. In most lily samples, sucrose accounted for more than 86% of total free sugar contents. Compared with the bulbs from the same regions, the bulbils of Lilium lancifolium were richer in many free sugars. Compared with the fresh bulbs, dried lily scales contained decreased contents of total free sugars and increased types of free sugars. Among these edible and medicinal lilies, the bulbs of L. pumilum and L. davidii var. willmottiae had the highest sweetness, followed by the bulbs of L. lancifolium and L. brownii var. viridulum and the dried scales of L. davidii var. willmottiae, and the sweetness of the dried scales of L. brownii var. viridulum was the lowest. Hierarchical cluster analysis showed that most bulb samples belonging to the same Lilium species were clustered together, and principal component analysis showed that L. pumilum, L. davidii var. willmottiae and L. regale were richer in most types of free sugars. Besides, the purple bulbs of L. regale and the bulbils of L. lancifolium had higher contents of glucose and fructose, which may be related to the accumulation of anthocyanins. This study provides a theoretical basis for product development from lily germplasms.
Chemical Characterization of the Aroma of Tieguanyin Oolong Tea and Black Tea
GUO Li, DU Zhenghua, YAO Lihong, CHEN Xiaobing, ZHANG Yi, LIN Zhi, GUO Yaling, CHEN Mingjie
2021, 42(10):  255-261.  doi:10.7506/spkx1002-6630-20200428-361
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In this study, the aroma quality of oolong tea, black tea and green tea manufactured from the fresh leaves of Camellia sinensis cv. Tieguanyin was determined by sensory evaluation, and their volatile compounds were determined by solvent-assisted flavor evaporation combined with gas chromatography-mass spectrometry/flame ionization detector. We found that all the Tieguanyin tea samples scored 90 points for aroma with a flowery note. In total, 52 volatile components were detected in the tea samples, with linalool and fatty acid esters being the major ones. The characteristic aroma components of Tieguanyin tea included oxidized linaool II, oxidized linaool IV, linalool, trans-nerolidol, hexanoic acid 3-hexenyl ester, hexanoic acid 2-hexenyl ester, benzoate 3-hexen-1-ol, benzoic acid hexyl ester and neophytadiene. Black tea processing could promote the formation of linalool and its oxides, butanoic acid 3-methyl-, 2-hexenyl ester, hexanoic acid 3-hexenyl ester, hexanoic acid 2-hexenyl ester, neophytadiene, dihydroactinidiolide, beta-ionone, methyl salicylate. Oolong tea processing was conducive to the formation of hotrienol, 2,6-dimethyl-3,7-octadiene-2,6-diol, trans-nerolidol, octanoic acid 3-hexenyl ester, and benzoic acid hexyl ester. In conclusion, the aroma characteristics of Tieguanyin tea were closely related to its species-specific components and the manufacturing technology used; which will provide an important technical reference for the development of new Tieguanyin tea.
Simultaneous Determination of Phenolic Components in Dendrobium officinale Stems, Leaves and Flowers by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
HUANG Biao, HE Wei, WU Jianhong, WANG Hongmei, LI Wei
2021, 42(10):  262-268.  doi:10.7506/spkx1002-6630-20200529-363
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The study aimed to compare the phenolic components in Dendrobium officinale stems, leaves and flowers. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to determine the phenolic components. The samples were extracted with methanol in water, and the extract was separated on a Wates T3 C18 column by gradient elution. Then, the target compounds were analyzed by positive and negative electrospray ionization under the multiple reaction monitoring (MRM) mode. By employing 18 standard phenolic compounds as references, 15 phenolic compounds were detected in D. officinale samples, including 3,5-dihydroxybenzoic acid, tangeretin, chlorogenic acid, quercetin, rutinum, p-hydroxybenzoic acid, caffeic acid, syringate, kaempferol, vanillic acid, coumalic acid, sinapic acid, ferulic acid, 3-hydrocycinnamic acid, and salicylic acid. The contents of phenolic compounds in different parts of D. officinale were in the descending order of flowers > leaves > stems. Rutin was identified as the most dominant phenolic compound in the three parts There were differences in the major phenolic compounds in them. The major phenolic compounds in D. officinale flowers were rutin, caffeic acid, ferulic acid and p-hydroxybenzoic acid. The contents of rutin and caffeic acid in the leaves were higher than those of other phenolic compounds. The major phenolic compounds in the stems were rutinum, ferulic acid and vanillic acid. The results of this study may provide a scientific reference for the exploitation of phenolic substances in different parts of D. officinale.
Flavor Composition and Characteristic Esters in Mechanized Sauce-Flavor Baijiu Base Liquors from Fermentation Rounds
HE Dongmei, MA Yu, HUANG Yongguang, YOU Xiaolong, CHENG Pingyan, HU Feng
2021, 42(10):  269-275.  doi:10.7506/spkx1002-6630-20200515-173
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In this study, liquid-liquid extraction combined with gas chromatography-mass spectrometry (LLE-GC-MS) was used to qualitatively and quantitatively analyze the flavor compounds in mechanized sauce-flavor Baijiu base liquors from different fermentation rounds, and their odor activity values (OAVs) were calculated. There were significant differences in the flavor composition of the tested samples, especially esters, alcohols, acids, aldehydes and ketones. Esters played a major role in the flavor composition. The results showed that 12 esters including ethyl lactate, ethyl acetate, ethyl hexanoate, ethyl hexadecate, ethyl oleate, 9,12-octadecadienoic acid, ethyl tetradecanoate, 9-hexadecenoic acid, ethyl stearate, 13-methyltetradecanoic acid, ethyl phenylacetate and phenylethyl acetate were the ‘skeleton esters’ of mechanized sauce-flavor Baijiu base liquor. Ethyl laurate and ethyl tetradecanoate contributed the most to the floral and sweet flavor in the base liquors from the 1st and 2nd rounds of fermentation; ethyl 3-phenylpropionate contributed the most to the honey-like and sweet flavor in the first-round base liquor; ethyl valerate, amyl acetate and ethyl octanoate contributed the most to the fruity, flowery and sweet flavor in the third-, fourth- and fifth-round base liquors, which were the characteristic compounds responsible for harmonizing the aroma of the base liquors. All esters except ethyl oleate and ethyl hexadecate had little contribution to the sixth- and seventh-round base liquors. The flavor composition of mechanized base liquor was similar to that of the traditional one, and the characteristic esters were also similar between them. This study will provide a reference for promoting the mechanization of sauce-flavor Baijiu.
Analysis and Evaluation of Fruit Nutritional Quality and Aroma Components of Four Major Japanese Apricot (Prunus mume Sieb. et Zucc.) Cultivars in Fujian Province
JIANG Cuicui, YE Xinfu, LIN Yanjuan, FANG Zhizhen, ZHOU Danrong
2021, 42(10):  276-283.  doi:10.7506/spkx1002-6630-20200511-111
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Four major Japanese apricot cultivars named ‘Qingzhumei’, ‘Longyanmei’, ‘Hangmei’ and ‘Baifenmei’ in Fujian province were measured for fruit traits such as individual fruit mass and fruit dimension as well as nutrient components such as vitamin C (VC), total phenolics, flavonoids, soluble sugars, organic acids, aroma compounds and mineral elements. The results indicated that a significant difference in the contents of VC, total phenolics, flavonoids, soluble sugars, organic acids, aroma components and mineral elements existed among the four cultivars. Sucrose followed by glucose and fructose was the predominant sugar in Japanese apricot fruit. The major organic acid was citric acid, followed by malic acid, succinic acid, acetic acid and quinic acid. In total, 110 aroma components were detected in the four cultivars, including 30 esters, 26 alcohols, 17 aldehydes, 15 ketones, 7 acids, and 15 other substances, 26 of which were common to all cultivars. 2-Heptenal, hexanal, hexanol and trans-ocimenol were the major aroma substances in these cultivars. K, Ca and Se were abundant in all cultivars. The results of principal component analysis (PCA) showed that ‘Qingzhumei’ had the highest comprehensive score, which exhibited the highest total concentration of aroma compounds and the best overall quality. ‘Qingzhumei’ was suggested to be a good breeding material. These findings provide a theoretical foundation for determining the nutritional value of Japanese apricot fruit, breeding superior cultivars and rational processing and utilization of Japanese apricot fruit.
Safety Detection
Nondestructive Classification of Defects in Potatoes Based on Lightweight Convolutional Neural Network
YANG Sen, FENG Quan, ZHANG Jianhua, WANG Guanping, ZHANG Peng, YAN Hongqaing
2021, 42(10):  284-289.  doi:10.7506/spkx1002-6630-20200412-155
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At present, the detection of external defects in potatoes mainly depends on manual feature extraction and is consequently inaccurate. A classification method for fast and accurate online grading of potatoes based on the lightweight convolutional neural network is proposed in this paper. First, the Xception network model was trained using ImageNet dataset to establish a pre-trained network model. Then, based on the trained Xception network, this method replaced the Softmax classifier in the original Xception network with category 5 tags, and the potato defect dataset was trained in the Xception framework with transfer learning.Finally, based on the trained external defect recognition model, the classification performance of 5 tags defects was tested. The experimental results showed that when the learning rate was 0.000 01, the overall performance of the network model was optimal, the training accuracy rate was 98.88%, and the loss value was 0.034 9. Compared with nine other neural networks with different depths, the proposed lightweight network model had better recognition performance with average recognition accuracy of 96.04% under the same sample conditions. The model’s processing time was shorter than that of the ResNet152 network, with better recognition effect, and the recognition rate of the network model was 6.4 frames/s. This study could provide theoretical support for online classification of potatoes.
Mineral Enrichment Capacity and Geographical Origin Identification of Boletus edulis
CHEN Fengxia, YANG Tianwei, LI Jieqing, LIU Honggao, FAN Maopan, WANG Yuanzhong
2021, 42(10):  290-296.  doi:10.7506/spkx1002-6630-20191104-039
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The objectives of this research were to 1) determine the mineral element contents and enrichment characteristics of Boletus edulis and 2) establish a model using partial least squares-discrimination analysis (PLS-DA) combined with spectral data fusion for identifying different geographical origins of B. eduils. The mineral elements of B. edulis and soil samples from six producing areas in Yunnan province were determined and the near-infrared and ultraviolet spectral information was collected. The accumulation characteristics and enrichment capacity of mineral elements by B. edulis were analyzed based on mineral element contents and enrichment coefficient. Savitzky-Golay (SG), second derivatives (2D), standard normal variables (SNV) and multiple-scattering correction (MSC) were used to pre-process spectral data. A monospectral data model was established using PLS-DA. The results showed that B. edulis was rich in K and P, and all tested minerals varied with the geographical origin with the highest Na level being 20.70 times as great as the lowest level. The content of Fe in soils from the six producing areas was the largest, which may be related to the acidity of the soils. The enrichment capacity of P, K and Zn in B. edulis was strong, with an enrichment factor of 4.63 for P. The optimal pretreatment methods for near-infrared and ultraviolet spectra were SG + 2D and SG + MSC, giving an accuracy for the prediction set of 88.46% and 96.15%, respectively. The intermediate fusion model had the best prediction performance. The Holtelling T2 test revealed that all samples fell within the 95% confidence interval, and the accuracy for the training and prediction sets was 100.00% and 92.31%, respectively. The results obtained in this study will be useful for the rational utilization of wild edible fungi resources in Yunnan.
Rapid Detection of Shiga Toxin-producing Escherichia coli by Recombinase Polymerase Amplification Combined with Centrifugal Compact Disc Microfluidic Chip
Fan Yiling, WANG Shujuan, LI Qiongqiong, HU Ying, SONG Minghui, Qin Feng, Liu Hao, YANG Meicheng
2021, 42(10):  297-304.  doi:10.7506/spkx1002-6630-20200323-349
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Recombinase polymerase amplification (RPA) combined with a centrifugal compact disc microfluidic chip system was used to develop a method for the detection of Shiga toxin-producing Escherichia coli (STEC). Specific primers and probes targeting the stx1 and stx2 genes were designed and evaluated. The efficiency of RPA was verified using 19 STEC strains including 9 stx gene subtypes and 21 non-STEC strains. The fluorescence RPA-integrating microfluidic chip was evaluated using artificially contaminated beef samples. The inclusivity and exclusivity of fluorescence RPA were 100% when compared to the real-time polymerase chain reaction (real-time PCR) from the Microbiology Laboratory Guidebook of USDA, namely the United States Department of Agriculture. The fluorescence RPA microfluidic chip method could conduct 32 reactions simultaneously within 20 min with a sensitivity of 9.5 × 103 CFU/mL for STEC strains. The STEC strains could be detected in artificially contaminated beef samples at an inoculation level of 1 CFU/25 g after enrichment culture by the GB 4789.6-2016 Food Microbiological Examination-Diarrheagenic Escherichia coli. The relative trueness and the relative detection level of the method were both 100%. The fluorescence RPA microfluidic chip method can detect 9 stx gene subtypes of STEC with simple operation and fast response, and it is suitable for rapid and high-throughput detection of STEC.
Determination of Five Antiviral Drug Residues in Chicken Meat and Eggs Using On-line Solid Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry
MA Junmei, HE Liangna, SUN Lei, WANG Dong, ZHANG Leilei, ZHANG Yan, KANG Wenyi
2021, 42(10):  305-310.  doi:10.7506/spkx1002-6630-20200820-269
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In this research, we developed an on-line solid phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of arbidol, amantadine, rimantadine, memantine and oseltamivir in chicken meat and eggs. Samples were extracted with 1.0% formic acid in acetonitrile, and the extract was purified using an MCX on-line solid phase extraction cartridge. The target substances were separated using an XBridge C18 column, detected by electrospray ionization in the positive ion mode, and quantitated by the internal standard method. The results showed that the calibration curves of all analytes had a good linear relationship in the concentration range of 0.05–100 ng/mL. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.05–0.10 μg/kg and 0.10–0.20 μg/kg, respectively. The recoveries of the developed method were in the range of 80.03%–93.96%, with relative standard deviations (RSDs) of lower than 10%. The method was proven to be simple and sensitive, and could be used for the qualification and quantification of the five antiviral drugs in chicken meat and eggs.
Determination of Chlorpheniramine Residues in Foods of Animal and Plant Origin by GC-MS/MS
ZHOU Ruohao, LÜ Daizhu, MA Chen, LIANG Shuilian, XIANG Tantan, WANG Mingyue
2021, 42(10):  311-315.  doi:10.7506/spkx1002-6630-20191104-036
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In this study, an analytical method for the determination of chlorpheniramine residues in foods of animal and plant origin by gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed. The samples were pretreated with a modified quick, easy, cheap, effective, rugged, safe (QuEChERS) method and solid phase extraction, and the analytes were separated on HP-5MS GC column and quantitatively determined by an external standard method. The experimental results showed that the method exhibited a good linear relationship in the range of 0.01–0.5 and 0.03–1 μg/mL with correlation coefficient R2 higher than 0.999 for banana and an animal food sample, respectively. The spiked recoveries for three matrixes were measured to be 80.6%–107.8%, with relative standard deviation (RSD) of 2.3%–8.5%. This method was characterized by simple operation and good reproducibility. It was suitable for batch sample detection, providing a technical basis for evaluating the risk of dietary exposure to chlorpheniramine.
Simultaneous Determination of Typical Advanced Glycation End Products in Thermally Processed Foods by Ultra-high Performance Liquid Chromatography-Quadrupole-Tandem Mass Spectrometry
CHENG Weiwei, WANG Xia, ZHANG Zhongfei, LIU Guoqin, CHEN Feng, CHENG Kawing
2021, 42(10):  316-321.  doi:10.7506/spkx1002-6630-20200527-332
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A rapid and effective method was developed for the simultaneous determination of Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) in thermally processed foods by ultra-high performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UPLC-QqQ-MS/MS). After the defatted samples were reduced by sodium borohydride for 8 h, the protein was precipitated with a mixed solvent of chloroform and methanol (2:1, V/V) and then hydrolyzed with hydrochloric acid. Subsequently, CML and CEL were concentrated and purified on an Oasis MCX SPE column, and separated on ACQUITY UPLC HSS T3-C18 analytical column with gradient elution using a mobile phase consisting of 1 mmol/L aqueous ammonium acetate containing 0.1% formic acid and acetonitrile. The qualitative and quantitative analysis was performed in the positive ionization mode with multiple reaction monitoring (MRM). The results showed that the developed method had a good linear relationship in the concentration of 0.25–500 ng/mL with a correlation coefficient of over 0.999. The limit of detection (LOD) was 8 and 10 ng/g , and the limit of quantitation (LOQ) was 36 and 40 ng/g for CML and CEL, respectively. The average recoveries of CML and CEL at three spiked concentration levels were 96%–103% and 94%–107%, respectively, and the precision, expressed by relative standard deviations (RSD), was 1.48%–2.43% and 1.23%–1.84%, respectively. CML and CEL contents in 13 commercially available thermally processed foods were detected by this method. It was found that the contents of CML and CEL in infant meat floss and biscuits were significantly higher than those in baked and fried foods (P < 0.05). The results indicate that the method established in this study is rapid, precise, and effective for the simultaneous determination of CML and CEL in thermally processed foods.
Simultaneous Determination of Nine Phenolic Antioxidants in Vegetable Oil by Pass-through Solid Phase Extraction Cleanup and Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
ZHANG Yanxia, ZHAO Huinan, SUN Shanshan, XU Xiangjun, ZHENG Wenjing, XUE Xia, WANG Mingdong, LIU Yanming, ZHU Jianhua
2021, 42(10):  322-329.  doi:10.7506/spkx1002-6630-20200416-207
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A method for the determination of nine phenolic antioxidants in vegetable oil by pass-through solid phase extraction (SPE) cleanup combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The antioxidants in samples were extracted with 0.5% formic acid-acetonitrile (V/V), purified by a pass-through SPE procedure on an Oasis? PRiME HLB column. The chromatographic separation was carried out using a C18 column with gradient elution using a mobile phase consisting of acetonitrile and water. The target compounds were detected by triple quadrupole mass spectrometry with electrospray ionization (ESI) under the multiple reaction monitoring (MRM) mode. The calibration curves of the nine antioxidants showed good linearity in their own ranges with correlation coefficients of higher than 0.994. The average recoveries ranged from 82.2% to 115.2% at three spiking levels with relative standard deviation (RSD) of less than 9.3%. The limits of detection (LODs) of the analytes were in the range of 0.003-0.02 mg/kg. The limits of quantitation (LOQs) of the analytes were in the range of 0.01–0.05 mg/kg. This rapid and sensitive method is suitable for the detection of antioxidants in vegetable oil.