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Table of Content

25 September 2020, Volume 41 Issue 18
Food Chemistry
The Interaction of Whey Protein Isolate with Tannic Acid Improves the Stability of Rice Bran Oil Pickering Emulsion
LI Wanrong, LIU Pei, YU Jingyi, QIN Xinguang, LIU Gang
2020, 41(18):  1-7.  doi:10.7506/spkx1002-6630-20190906-077
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In this paper, whey protein isolate-tannic acid (WPI-TA) nanoparticles were prepared by alkaline method. The effect of the mass ratio of WPI to TA on the formation of WPI-TA nanoparticles was investigated by measuring the particle size and potential. Then, the optimal WPI-TA nanoparticle was selected as the emulsifier to prepare rice bran oil Pickering emulsion by a simple shear-induced emulsification technique. The impact of the interaction between WPI and TA was investigated on the thermal, ion and oxidative stability of rice bran oil Pickering emulsion. At the same time, the stability mechanism of the Pickering emulsion was explored. The results implied that WPI-to-TA ratio affected the formation and stability of WPI-TA nanoparticles. Besides, Fourier transform infrared spectroscopy showed that the interaction between WPI and TA caused a change in the secondary structure of the protein. The introduction of TA significantly increased the emulsification activity of WPI-TA nanoparticles, improved the stability of the Pickering emulsion to temperature and ionic strength while inhibiting the formation of primary and secondary oxides during storage. Therefore, WPI-TA nanoparticles are expected to be a potential advantageous stabilizer for Pickering emulsion.
Construction and In Vitro Digestion Properties of Pseudosciaena crocea Roe Protein Isolate-Stabilized Emulsion
WANG Xiaohan, JIANG Hui, WU Haitao, ZHANG Zhihui, CUI Haozhe, YU Jiahui, YANG He, TANG Yue
2020, 41(18):  8-13.  doi:10.7506/spkx1002-6630-20190914-167
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In order to improve the solubility and bioavailability of β-carotene, a stable β-carotene-loaded oil-in-water (O/W) emulsion system was constructed using Pseudosciaena crocea roe protein isolate (PRPI) as an emulsifier. The stability of the emulsion system with different storage times was investigated. The digestion process and absorption capacity were determined by in vitro simulated digestion. The results showed that the average particle size and appearance of the emulsion system did not change significantly after 14 days of storage. Simulated oral digestion had little effects on the microstructure of the emulsion system. However, during simulated gastric digestion, the emulsion system was hydrolyzed and the droplets were aggregated, the average particle size increasing from (255.46 ± 7.41) nm to (9 749.25 ± 406.66) nm (P < 0.05). After simulated intestinal digestion, the degree of droplet aggregation decreased significantly, the average particle size decreasing to (3 654.36 ± 527.91) nm (P < 0.05), but the retention rate and bioavailability of β-carotene remained at high levels of (81.64 ± 2.72)% and (93.13 ± 3.59)%, respectively. Therefore, PRPI could be used as an emulsifier to construct an emulsion delivery system with improved stability for improved β-carotene bioavailability.
Impact and Mechanism of Mg2+ in Brewing Water on the Taste Quality of Black Tea Infusions
LIU Yanyan, XU Yongquan, CHEN Jianxin, WANG Fang, CHEN Gensheng, YIN Junfeng, LIU Zhengquan
2020, 41(18):  14-20.  doi:10.7506/spkx1002-6630-20190723-294
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The effect of Mg2+ in brewing water on the taste quality and chemical ingredients of black tea infusion was investigated. The underlying mechanism was inferred from the interaction among the main taste substances of black tea infusion in the presence of Mg2+. The results showed that the quality of tea infusion with high Mg2+ concentration (≥ 16 mg/L) was fairly poor. As the concentration of Mg2+ increased, the umami taste, sweetness and bitterness of tea infusion decreased, while the astringency significantly increased. The contents of tea polyphenols, catechins, amino acids and caffeine, as well as Al3+, B3+, K+, Na+ and S6+ in tea infusion showed a downward trend, partially accounting for the decrease in the bitterness and umami taste of tea infusion. Furthermore, we found that Mg2+ not only enhanced the astringency of epiggallocatechin gallate (EGCG), caffeine and sodium glutamate solutions, but also weakened the bitterness of EGCG and caffeine solutions, the umami taste of sodium glutamate solution and the sweetness of sucrose solution, indicating a significant effect of Mg2+ on the taste components. With increasing concentration of Mg2+ in brewing water, the turbidity of EGCG-protein mixture solution increased significantly, which confirmed the conclusion that Mg2+ can strengthen the astringency intensity of tea infusion. The results of this study suggested that water that contain relatively low concentration of Mg2+ (≤ 4 mg/L) be used for brewing of black tea in daily life.
VE Loss and Its Effect on the Quality and Stability of Palm Oil during Frying
YU Mengdan, ZHU Tingting, SU Pei, BI Yanlan, LIU Wei, YUAN Fangbo
2020, 41(18):  21-26.  doi:10.7506/spkx1002-6630-20190723-311
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The effects of VE addition on the loss of tocopherol and tocotrienol and the quality of palm oil being used for frying French fries intermittently at (180 ± 5) ℃ were studied. The results showed that the higher the amount of VE added, the greater the absolute loss of tocopherol, but there was no linear relationship between them. VE had a significant protective effect on tocotrienol in palm oil, the higher the amount of VE added, the less the loss of tocotrienol and the more potent the effect in inhibiting the increase of acid value. However, VE had no significant effect in inhibiting the increase of polar compounds, and promoted the increase of carbonyl value and color darkening. Adding 1 000 mg/kg VE could prolong the induction period and reduce slightly the frying life of palm oil, while adding 4 000 and 8 000 mg/kg VE could shorten the induction period and extend the frying life of palm oil.
Effects of Heat Treatment on Interaction between Gliadin and Rutin and Rheological Properties of Emulsion Stabilized with Their Complex
LI Chunyi, WANG Qiming, TANG Yuwan, ZHANG Yuhao, ZHAO Jichun, MING Jian
2020, 41(18):  27-34.  doi:10.7506/spkx1002-6630-20191212-133
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The present study aimed to investigate the impact of heat treatment on the interaction between gliadin and rutin by intrinsic fluorescence spectroscopy, Fourier transform infrared spectroscopy, surface hydrophobicity measurement and scanning electron microscopy. For this purpose, gliadin-rutin complex was heat treated at 25, 60, 80 or 100 ℃ for 30 min. Gliadin was regarded as the control. Further, soybean oil emulsion stabilized with the treated complex was prepared and evaluated for their rheological properties. After the formation of the emulsion, the changes in potential, viscosity and microstructure were observed and frequency scanning and amplitude scanning were carried out. The results indicated that appropriate temperature treatment had significant effects on the surface hydrophobicity and microstructure of the protein, improved the viscosity of the emulsion, and enhanced the stability of the emulsion. The potential of the emulsion stabilized with gliadin-rutin complex treated at 80 ℃ was the highest among all temperatures tested, up to + 16.58 mV, indicating the highest emulsion stability. This provides a theoretical basis for the application of gliadin in dairy foods.
Effects of NaHCO3 Immersion on the Quality of Channel Catfish Subjected to Repeated Freeze-thaw Cycles
ZHANG Wei, JIAO Chunhai, XIONG Guangquan, WANG Lan, SHI Liu, LI Xin, DING Anzi, WU Wenjin
2020, 41(18):  35-41.  doi:10.7506/spkx1002-6630-20190716-221
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The objective of this research was to investigate effect of NaHCO3 immersion on the sensory quality, fat oxidation and protein oxidation of channel catfish subjected to repeated freeze-thaw cycles. The results showed that the pH value and whiteness of fish fillets decreased significantly, while the thawing loss rate and the degree of fat and protein oxidation increased significantly with increasing number of freeze-thaw cycles. After 4 freeze-thaw cycles, the 2-thiobarbituric acid (TBA) value reached 15 μg/g, indicating that the fish fillets showed deteriorated quality and basically could not be consumed. Compared to the control (without NaHCO3 immersion), NaHCO3 immersion treatment led to an increase in the pH value, and a decrease in the thawing loss rate. It was found that the degree of fat and protein oxidation was highly significantly linearly related to the number of freeze-thaw cycles (R2 > 0.9), and the oxidation rates of fat and protein were greatly reduced with increasing NaHCO3 concentration to 1.0 g/100 mL. Hence, NaHCO3 immersion could to a certain extent inhibit lipid oxidation and protein denaturation in channel catfish during repeated freeze-thaw, maintain water-holding capacity and retard quality deterioration, and this effect was better at 1.0 g/100 mL NaHCO3 concentration than at 0.5 g/100 mL.
Effect of Alfalfa Ice Structuring Proteins on Quality and Structure of Frozen Wet Gluten
QU Min, WU Zheng, DU Tingting, ZHU Xiuqing, CHEN Fenglian, LI Lingli, LU Manman
2020, 41(18):  42-50.  doi:10.7506/spkx1002-6630-20200225-272
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In this paper, ice-structuring proteins (AISPs) from alfalfa were extracted using ice spheres combined with phosphate buffer, and their amino acid composition and the properties and structures of the purified AISP fractions were identified. In order to study the effect of AISPs on the quality and structure of frozen wet gluten, AISPs was mixed with dry gluten powder in water for freezing treatment. The results showed that AISPs contained 17 amino acids with a ratio of hydrophilic amino acids to hydrophobic amino acids of 7:4. Two protein components with molecular masses of 34 and 52 kDa were obtained from the fractionation of AISPs, and their thermal hysteresis values were both 0.41 ℃. Schiff staining showed that the former was glycoprotein and the latter was non-glycoprotein, and the proportions of secondary structure in them were α-helix:β-sheet:β-turn:random coil = 13.19:60.21:12.21:19.16, and 16.87:39.41:26.69:17.03, respectively. The protective effect of AISPs on frozen wet gluten samples at ?40 ℃ was more significant than at ?18 ℃. With the increase in freezing time and the number of freezing-thawing cycles, the water-holding capacity of wet gluten protein decreased while the content of freezable water increased, and ω-gliadin was partially degraded. High-molecular-weight glutenin subunit (HMW-GS) was completely degraded. The B and C subunits of low-molecular-weight glutenin subunit (LMW-GS) in frozen samples were stable, while the D subunit increased. The frozen-thawed samples showed an increase in subunits B, C and D, especially for subunits B and C. After adding AISPs, the degradation of gluten protein components was alleviated to some extent. Scanning electronic microscopy (SEM) results showed that freezing treatment resulted in rough texture and enlarged pores in the wet gluten network, while the addition of AISPs made the gluten network clearer and more continuous.
Effect of pH on the Structure, Solubility and Surface Hydrophobicity of Albumin and Globulin from Rice Bran
YANG Jian, FU Tianxin, ZHANG Shu, FENG Yuchao, WANG Changyuan
2020, 41(18):  51-57.  doi:10.7506/spkx1002-6630-20190716-222
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The purpose of this study was to solve the problem of low extraction rate of rice bran protein and poor product functions and properties in order to increase the utilization rate of rice bran protein. The effects of pH on the structure, solubility and surface hydrophobicity of rice bran albumin and globulin were investigated based on the theory of protein chemistry and spectroscopic analysis. The results showed that with the increase in pH, the hydrodynamic diameter distribution of rice bran albumin and globulin followed a declining trend while the zeta potential absolute value exhibited an increasing trend. The content of α-helix and random coil structure in rice bran albumin increased gradually, while the content of β-fold structure decreased gradually. Tryptophan residues of rice bran albumin and globulin tended to be “exposed”. In conclusion, under alkaline conditions, rice bran globulin remained most of its secondary structures and subunit dissociation induced the tertiary structure of the protein, which was the main reason for the increase in the surface hydrophobicity. As for rice bran albumin, the transformation of secondary structural units from an ordered to a disordered state and subunit dissociation-induced protein unfolding were the reason for the increase in the surface hydrophobicity. The increase in the solubility of rice bran albumin and globulin was accounted for by the fact that the subunits were dissociate into small particles and the protein particles were charged under alkaline conditions.
Effect of Gluten on Quality Properties and Water Migration of Heat-Induced Gel from Pork
JI Hongfang, LI Shasha, ZHANG Lingwen, WU Shenglong, HAN Xiping, CHEN Fusheng, MA Hanjun
2020, 41(18):  58-63.  doi:10.7506/spkx1002-6630-20190911-140
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The cooking loss, water-holding capacity, texture, chemical forces, dynamic rheology, microstructure, water distribution and migration of pork batter gels with added gluten were measured to explore the effect of gluten on quality properties and water migration of heat-induced gels from pork. Results indicated that with the increase in gluten addition, the cooking loss decreased continuously (P < 0.05). The transverse relaxation time T2 always moved in the direction of short relaxation time, and the proportion of immobilized water rose significantly. The addition of gluten raised the water-holding?capacity, but the effect was not significant when the level of gluten addition was above 6% (P > 0.05). Both the hardness and chewiness increased remarkably. The springiness first increased until reaching the maximum at an addition level of 6%, and then decreased. The resilience increased to 0.329 with increasing addition of gluten up to 6%, and then did not significantly change at addition levels of 8% and 10% (P > 0.05). Hydrogen bonding and hydrophobic interaction were the main forces in the formation of gluten-pork batter gels. Additionally, G’ increased with elevated addition of gluten. Gluten could improve the gel formation ability of pork. The pork gel with 6% added gluten had a compact and uniform structure, smooth?appearance, and the best quality.
Preparation, Structure Identification and In Vitro Biological Activity of Polysaccharide from Perccottus glenii Head
GAO Yangyang, YAN Xiaohui, ZHU Chang, ZHANG Jialin, LIU Xuejun
2020, 41(18):  64-70.  doi:10.7506/spkx1002-6630-20190911-143
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The preparation of polysaccharide (PGP) and deproteinized polysaccharide (PGP-D) from Perccottus glenii head was investigated, and their monosaccharide compositions, structures, and bioactivities in vitro were studied. The results showed that the highest deproteinization efficiency was obtained by pepsin treatment with protein removal rate, polysaccharide loss rate, and half-maximum hydroxyl radical scavenging rate of 86.0%, 3.7%, and 47.6%, respectively. Scanning electron microscopy showed that the microscopic morphology of PGP after deproteinization changed from rough sphere-like to smooth flake-like. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that PGP-D was composed of arabinose, rhamnose, fructose, galactose, galacturonic acid, and glucuronic acid. UV-visible spectroscopy identification showed that PGP-D did not contain nucleic acid but contained trace amounts of protein. Fourier transform infrared spectroscopy analysis showed that PGP-D had the characteristic absorption peak of polysaccharide, which was an acidic polysaccharide containing β-glycosidic bonds. Both PGP and PGP-D had strong antioxidant and ACE inhibitory activities. PGP exhibited an inhibitory effect on α-glucosidase activity.
Bioengineering
Heterologous Expression, Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae
CHEN Ying, LI Chixia, CHEN Yujuan, TIAN Yuanyuan, ZHANG Meng, HAN Yingyi, WANG Yousheng
2020, 41(18):  71-76.  doi:10.7506/spkx1002-6630-20190711-161
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In order to prepare high-purity cyclic nucleotide phosphodiesterase 1 (PDE1) from Saccharomyces cerevisiae, the pGM-T-S. cerevisiae was used as a template to amplify the target gene by PCR, and an expression plasmid carrying this gene was constructed, and then transformed into Escherichia coli BL21 cells for expression. High-pressure broken bacteria extract was purified consecutively by nickel affinity column chromatography, Q-Sepharose ion exchange column chromatography and Sephacryl S200 column chromatography. The purified enzyme activity remained 92% of the original activity. This study may provide a basis for future crystallographic analysis of the protein and its metabolic regulation in S. cerevisiae.
Characteristics of Antibiotic Resistance and Plasmid Conjugative Transfer of IncI1 and IncN Plasmid Positive Salmonella
SHENG Huanjing, LI Yilan, WANG Zewei, NIU Qinya, MENG Lingyuan, CAO Chenyang, LI Wei, LIAN Luxin, YANG Baowei
2020, 41(18):  77-84.  doi:10.7506/spkx1002-6630-20190802-026
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Objective: In this study, the prevalence, antimicrobial susceptibility, antibiotic resistance genes and plasmid horizontal transfer of IncI1 and IncN plasmid positive Salmonella isolated from retail foods, clinical patients and food animals in the provincial-level regions of Beijing, Shanghai, Henan, Sichuan, Guangdong, Guangxi, Shaanxi and Xinjiang were investigated. Methods: The incompatible plasmids IncI1 and IncN in Salmonella were determined by a polymerase chain reaction (PCR)-based replicon typing method, the antimicrobial susceptibility was tested by the agar dilution method, and the antibiotic resistance genes were detected using PCR. The horizontal transfer of IncI1 and IncN plasmids was conducted via conjugation. Results: Forty-two IncI1 positive isolates (4.39%) and 3 IncN positive isolates (0.31%) were identified among the 956 strains of Salmonella. Of these, 26 representatives of IncI1 and/or IncN plasmid positive Salmonella showed the highest prevalence of antibiotic resistance to ceftiofur (100.0%), nalidixic acid (92.3%), ampicillin (92.3%), cefoperazone (88.5%), tetracycline (84.6%), ceftriaxone (80.8%), and sulfamethoxazole/trimethoprim (76.9%), streptomycin (76.9%) and chloramphenicol (61.5%) followed by kanamycin (26.9%), gentamicin (23.1%), polymyxin B (23.1%), ciprofloxacin (19.2%), amoxicillin-clavulanic acid (15.4%), cefoxitin (11.5%) and amikacin (3.8%). The IncI1 plasmid positive Salmonella exhibited broader antibiotic-resistance spectra and higher resistance to cephalosporins than the IncN plasmid positive one, whereas the IncN plasmid positive isolates were more resistant to aminoglycoside antibiotics. blaTEM and blaCTX-M were more frequently detected from the IncI1 positive Salmonella, while qnrA, qnrB and qnrS were more prevalent in the IncN positive isolates. The IncI1 and/or IncN plasmid in the donor strains could be transferred to the recipients Salmonella and Escherichia coli at conjugation frequencies ranging from 3.2 × 10-5 to 2.0 × 10-3 and 8.7 × 10-7 to 9.6 × 10-5 transconjugant per recipient cell, respectively. The antibiotic resistance genes qnrB, acc(6’)-Ib, blaTEM and blaCTX-M could be transferred from the donors to the recipients during conjugation to impart the recipients with antibiotic resistance phenotypes to kanamycin, ceftiofur, ceftriaxone, ampicillin, streptomycin, and gentamicin after conjugation. Conclusion: The prevalence of IncI1 and IncN plasmid positive Salmonella was relatively low, and the antibiotic resistance phenotypes of the hosts were found to be associated with the types of plasmids they carried to some extent. The antibiotic resistance genes in the plasmids could be transferred from the donor to the recipient by horizontal gene flow among different species to confer antibiotic resistance to the recipient.
Analysis of the Diversity and Function of Lactic Acid Bacteria in Fermented Rice Noodles during Fermentation
XIONG Xiangyuan, ZHANG Lizhao, CHEN Lili, GONG Huike, ZHOU Yue, REN Youhua
2020, 41(18):  85-91.  doi:10.7506/spkx1002-6630-20200113-137
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In order to investigate the diversity of lactic acid bacteria in fermented rice noodles at different fermentation times, the diversity and functional predication of lactic acid bacteria on days 0, 1, 2, 3 and 4 of fermentation were studied by high-throughput sequencing. The results showed that a total of 278 operational taxonomic units (OTU) belonging to 27 genera in 5 phyla were identified in 15 samples of fermented rice noodles. During the entire fermentation process, Lactobacillus was absolutely dominant, and its relative abundance was over 99%. The diversity of lactic acid bacteria decreased with prolonged fermentation time. There were 32 species detected at 0 day, while only 12 species at 4 days at the species level. In addition to Lactobacillus, Lactococcus and Leuconostoc were also present. PICRUSt gene function prediction showed that the predicted functional genes at different fermentation periods were mainly those involved in amino acid transport and metabolism, carbohydrate transport and metabolism, lipid transport and metabolism, coenzyme transport and metabolism, energy production and conversion, and other metabolism-related genes. This study provides a theoretical rationale for further studying the mechanism of the effect of lactic acid bacteria on the eating quality of fermented rice noodles and for the development of fermentation starters for rice noodle.
Microbial Diversity of Yak Yogurt in Agricultural and Pastoral Areas of Tibet Analyzed Based on High-throughput 16S rRNA Gene Sequencing
LIU Yixuan, XU Guoqi, CAO Pengxi, JIN Yanlong, LI Xiaoyan, LIU Xing
2020, 41(18):  92-97.  doi:10.7506/spkx1002-6630-20190714-180
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In this study, the bacterial diversity and its differences in yak yoghurt collected from agricultural and pastoral areas in Tibet were analyzed by high throughput sequencing technology. The influence of environmental factors on the differences was explored. The results of hierarchical clustering and principal coordinate analysis (PCoA) showed that the microbiota structure of yak yogurt was significantly different between agricultural and pastoral areas. Linear discriminant analysis effect size (LEfSe) analysis showed that the dominant bacterial genera in yak yogurt from both agricultural and pastoral areas were Lactobacillus, Lactococcus and Streptococcus under Firmicutes. The three genera and seven other genera with relatively low abundance were significantly different between yak yogurt samples from the different regions (P < 0.05). Regression analysis and distance-based redundancy analysis (db-RDA) of environmental factors showed that elevation explained the difference between samples more than did average annual temperature. This study provides a reference for further studies on the relationship between the natural environment and the microbial diversity in fermented food in the Qinghai-Tibet plateau and for screening for excellent strains.
Metabolic Engineering of Corynebacterium glutamicum for the Production of Methionine
ZHAO Lan, LIU Shimeng, QIN Hanxiong, WANG Yanan, FAN Zhanqing, MIN Weihong
2020, 41(18):  98-104.  doi:10.7506/spkx1002-6630-20190712-173
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Objective: To a new strain for improved microbial production of methionine. Methods: Aspartate kinase (AK), homoserine dehydrogenase (HSD) and homoserine acetyltransferase (HAT), the key enzymes of methionine synthesis pathway, were modified and overexpressed in vitro. In addition, site-directed mutagenesis and characterization of the substrate binding site of the key enzyme, homoserine kinase (HSK) A20, were carried out to reduce the enzymatic activity and to prevent carbon flow from entering the threonine pathway. Results: Compared with the original strain, the expression levels of lysC, hom and metX in strain WTg1/PEC-lysCm-SD-homm-SD-metX increased by 6.896, 2.378 and 1.659 times, respectively. The liquid chromatographic analysis showed that the production of methionine was 4.14 g/L, 2.26 times higher than that of the original strain. The activities of HSK mutants thrB-A20Y, thrB-A20H and thrB-A20L were 39%, 43% and 49% as compared to HSK. Conclusion: In this experiment, the downstream metabolism of aspartic acid in Corynebacterium glutamicum was regulated by overexpression of the key enzymes in the trunk and weakening of the key enzymes in the branch. As a result, the methionine carbon transport system was strengthened, the accumulation of carbon flow was enhanced, and the production of methionine was effectively increased. This study provides theoretical reference for the construction of engineered bacteria for methionine production.
Identification and Flavor Analysis of an Aroma-producing Bacterium Isolated from Fermented Grains of Strong Flavor Baijiu in Songhe Town
LIU Bingbing, ZHOU Kaixin, YE Lijing, YU Yingying, LI Xuesi, YAN Peixun, GUO Shuxian
2020, 41(18):  105-112.  doi:10.7506/spkx1002-6630-20190715-204
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In order to explore aroma-producing bacterial resources from fermented grains of strong flavor Baijiu in Songhe town, the pure culture method combined with sensory evaluation was used to separate one bacterial strain with aroma-producing ability from fermented grains at different fermentation stages. The strain was identified as Cellulosimicrobium cellulans by morphological characteristics and phylogenetic analysis based on 16S rRNA gene, which was first isolated from fermented grains of Baijiu. It was found that the strain had cellulase and amylase activities under liquid-state culture conditions and had protease activity under acidic, neutral or alkaline conditions. It could withstand 9% ethanol concentration. Starch was used as carbon source to conduct fermentation under aerobic or facultative anaerobic conditions, and the flavor substances produced by this strain were detected by gas chromatography-mass spectrometry (GC-MS). The volatile substances in the fermentation broth under anaerobic conditions were mainly ethanol and acetic acid, whereas the compounds produced under aerobic conditions had complex structures, most of which were unknown.
Heterologous Expression of Codon Optimized Thermophilic β-Mannanase Gene (manBK) and Its Application in Degradation of Konjac Mannan
LU Haiqiang, GU Xinxi, YUAN Qiaomin, TAN Suhui, TIAN Hongtao
2020, 41(18):  113-119.  doi:10.7506/spkx1002-6630-20190805-058
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In this study, we optimized the codon usage of the thermophilic β-mannanase gene (manBK) and the fermentation conditions to increase its expression in Pichia pastoris. The enzymatic properties of ManBK were investigated and the effect of metal ions on the enzymatic stability was explored as well as its application potential in enzymatic hydrolysis of konjac mannan. Compared with the wild type manBK gene, 242 base pairs were modified after the optimization, and the GC content decreased to 40%–53%, on average 47%. The codon adaptation index (CAI) was 0.87. The optimized synthetic sequence showed a 76% homology to the wild-type gene, which was successfully expressed in P. pastoris GS115 using the constitutive expression vector pPIC9K. The ManBK activity attained 22 U/mL after a 72-hour incubation at 25 ℃. The ManBK had a molecular mass of approximately 45 kDa. The optimum temperature and pH value for the enzyme activity were 80 ℃ and pH 5.0, respectively. The kinetic parameters Km and Vmax of ManBK were found to be 1.40 mg/mL and 149.25 μmol/(min·mg), respectively. The enzyme was strongly activated by Fe3+ (32%), Ca2+ (52%), Cu2+ (23%) and Zn2+ (459%) but inhibited by K+, Li+, Mn2+and Cu2+. In addition, Zn2+ enhanced the thermal stability of the enzyme, which prolonged the half-life time by 116 min at 70 ℃. The optimal conditions for enzymatic hydrolysis of konjac flour were determined as follows: reaction time 1.5 h, reaction temperature 70 ℃, pH 5.0, and E/S ratio 1:1. The antioxidant activity of the konjac degradation products was 6.6 times as high as that of konjac flour. In conclusion, The ManBK has a great potential application in the preparation of konjac oligosaccharide.
Analysis of Acid-producing Microorganisms from Qu-making Environment and Mature Qu (Baijiu Fermentation Starter)
WANG Chunxiao, YUAN Guoyi, SU Wei, WANG Xiao, QIU Shuyi
2020, 41(18):  120-126.  doi:10.7506/spkx1002-6630-20190819-199
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The aim of this study was to analyze the main acid-producing microorganisms in mature Qu of Xiaoqu Baijiu, saccharified samples made with it and the Qu-making environment, in order to better control the flavor Xiaoqu Baijiu. High performance liquid chromatography (HPLC) was applied to detect the contents of the main acids in the saccharified samples, and high throughput sequencing was applied to analyze the relative abundance and diversity of bacterial community in the mature Qu, and the traditional isolation, screening and sequencing methods were applied to identify the main acetic acid-producing microorganisms in the saccharified samples and Qu-making environment. The acid production ability of the isolated microorganisms was verified in laboratory-prepared saccharified samples. The results indicated that the main acids in the saccharified samples made withmature Qu of Xiaoqu Baijiu were lactic acid and acetic acid. The formation of lactic acid was correlated with the main genera of Lactobacillus, Weissella and Pediococcus, while the formation of acetic acid was caused by Saccharomycopsis fibuligera isolated from the Qu-making environment and saccharified samples. Acetic acid bacteria were scarcely detected and thus hardly correlated with acetic acid formation in this study. This study is the first to report the acetic acid production ability of S. fibuligera, which provides new insights for research on its role and application in acid formation in Baijiu.
Construction and Enzymatic Characterization of Novel Aspartokinase Mutant Y198N/D201M from Corynebacterium pekinense
WEI Zhen, HAN Caijing, GAO Yunna, FAN Zhanqing, WANG Yanan, WANG Zheren, MIN Weihong
2020, 41(18):  127-133.  doi:10.7506/spkx1002-6630-20190724-321
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Aspartate kinase (AK) is the first key allosteric enzyme involved in the metabolic pathway of the aspartate family of amino acids, which is subject to synergistic feedback inhibition by lysine and threonine. This study aimed to increase AK activity and alleviate the feedback inhibition by site-directed mutation. The monomer aspartate kinase from Corynebacterium pekinense was obtained and its structure was analyzed. On this basis, the key residue sites Tyr198 and Asp201 around ATP were selected for site-directed mutation. The double mutant Y198N/D201M was successfully obtained by high-throughput screening, whose enzymatic activity was improved by 18.26 folds compared to that of the wild type (WT) AK. The Km value of the mutant was reduced (2.37 versus 3.58 mmol/L), and the substrate affinity was enhanced compared to the WT enzyme. The n value was reduced (1.58 versus 1.91), and the positive synergy was weakened. The enzymatic properties showed that optimum temperature, pH and half-life of the mutant were 28 ℃, 7.5 and 5.32 h as opposed to 25 ℃, 8.0 and 4.66 h for the WT enzyme. The mutant was less inhibited by lysine and threonine at all tested concentrations than the WT enzyme, and was even activated by Lys + Met, Thr + Met and Lys + Thr + Met at 5 and 10 mmol/L concentrations.
Relationship between the Signal Molecule N-Tetradecanoyl-L-homoserine lactone in Cnterobacter sakazakii and Its Growth
YAN Xiaotong, LUO Xiaojuan, PAN Jieru, YE Haimei, LIN Kan, LI Changcheng, FANG Ting
2020, 41(18):  134-139.  doi:10.7506/spkx1002-6630-20190802-037
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In this paper, the change in the concentration of the signal molecule N-tetradecanoyl-L-homoserine lactone (C14-HSL) during the growth process of Cnterobacter sakazakii CICC21550 at 37 ℃ was fitted to a curve. Based on the macroscopic model of bacterial growth, the growth mechanism of C. sakazakii CICC21550 was further explored from the microscopic perspective. The results showed that C14-HSL was detected in C. akazakii CICC21550 at the early logarithmic phase of growth, which reached the maximum value in the stationary period of bacterial growth, and then showed a gradually declining trend and it was in an obvious density-dependence manner. The maximum concentration Ymax of C14-HSL was 8.535 and the maximum rate of formation μmax was 0.825.
Isolation and Probiotic Properties of Lactobacillus plantarum HUCM115 from Traditional Pickled Chinese Cabbage
GUO Lidong, ZHANG Wenwen, LIU Yan, HUANG Mengling, MEN Yue, ZHANG Yanli, WANG Liqun
2020, 41(18):  140-145.  doi:10.7506/spkx1002-6630-20190807-084
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In this study, the lactic acid bacterial strain HUCM115 was isolated from traditional pickled Chinese cabbage, and identified as Lactobacillus plantarum by morphological characterization and 16S rDNA and pheS gene sequencing. L. plantarum HUCM115 showed excellent tolerance to acid and bile stress. There was no significant change in the viability of the strain after incubation for 3 h at pH 2.5 or in the presence of 0.5 g/100 mL bile. Meanwhile, this strain exhibited good auto-aggregation capacity, and the auto-aggregation percentage reached almost 70% after being kept for 60 min at room temperature. The percentage of cholesterol removal by HUCM115 reached 20.6% after incubation for 24 h in a medium supplemented with cholesterol. Moreover, the concentration of γ-aminobutyric acid was accumulated to 101.3 μg/mL by HUCM115 incubated for 48 h in a medium supplemented with glutamic acid. These results suggested that L. plantarum HUCM115 has good probiotic properties, and can be used as a candidate probiotic bacterium for further research.
Malolactic Fermentation Characteristics of Lactobacillus hilgardii Q19 at Low Temperature and Its Effect on Aroma Components in Wine
BAI Xuefei, JIN Gang, LIU Si, MA Wen, ZHANG Zhong, WANG Huiqing, ZHANG Junxiang
2020, 41(18):  146-152.  doi:10.7506/spkx1002-6630-20190805-053
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In order to study the malolactic fermentation (MLF) characteristics of Lactobacillus hilgardii Q19, isolated from the wine-producing area in the eastern foothill of Helan Mountain, at low temperature and its effect on the aroma components of wine, MLF was carried out in Cabernet?Sauvignon wine at 10, 15 or 20 ℃ after being inoculated with L. hilgardii Q19. Wine without MLF was set as the control. Bacterial growth, the consumption of L-malic acid and the aroma components in wine before and after MLF were tested and compared at different temperatures. The results showed that strain Q19 could start MLF and produce lactic acid at all temperatures. This strain had good fermentation ability at 15 and 20 ℃, and for the two temperatures, the concentration of malic acid in wines decreased to 0.27 and 0.15 g/L at 28 days after inoculation, respectively. However, the fermentation ability was weakened at 10 ℃, and the concentration of malic acid was 0.87 g/L at 28 days after inoculation. For each temperature, the bacterial density decreased during the first 8 days after inoculation and tended to be stable at 106–107 CFU/mL from day 24 onward. The contents of citric acid and total sugars decreased slightly at 28 days, while the content of total volatile acids increased but not significantly, with the smallest increase being observed at 15 ℃. The types and contents of aroma components produced during fermentation at 15 ℃ increased compared to those produced at 10 and 20 ℃. The increase in 1-octanol, ethyl heptanoate, ethyl nonanoate, ethyl lactate, undecanoic acid ethyl ester, decanal and terpenes (terpinene, β-pinene, inalol and citronellol) imparted unique floral and fruity aromas to wine, increasing the complexity of the aroma. L. hilgardii Q19 could complete MLF at 15 ℃ improving the aroma quality of wine, and thus it has good application potential in MLF in Northwest China with low temperature.
Characterization of D-Lactate Dehydrogenase from Leuconostoc mesenteroides ATCC8293
LI Ling, GONG Jinyan, YUAN Haina, FANG Ruosi, CHU Bingquan, XIAO Gongnian, Namsoo HAN
2020, 41(18):  153-158.  doi:10.7506/spkx1002-6630-20190723-301
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In this study, the lactate dehydrogenase gene (ldh) of Leuconostoc mesenteroides ATCC8293 was cloned into pETDuetTM-1 vector to construct a recombinant plasmid, which was then transformed into Escherichia coli BL21(DE3) for expression. The recombinant enzyme was purified using Ni-NTA column chromatography and its enzymatic properties were characterized. As results, the molecular mass of lactate dehydrogenase (LDH) was measured to be 37 kDa, and the optimal pH and temperature were 7.0 and 40 ℃, respectively. Under the optimized conditions, the specific activity of LDH was 67.45 U/mg. It was also confirmed that the enzyme was D-LDH, which converts pyruvic acid to D-lactic acid. Furthermore, the Km values of this enzyme for pyruvate and NADH were 1.27 mmol/L and 0.48 mmol/L, respectively, and the Kcat and Kcat/Km values for pyruvate were 421 s-1 and 3.31×105 L/(mol·s), respectively. Besides pyruvate as a substrate, it also had high activities toward oxaloacetate and phenylpyruvate. The results of this study provide a theoretical basis for the production of lactic acid and phenyllactic acid using L. mesenteroides.
Analysis of Fermentation Tolerance of Native Oenococcus oeni in Hexi Corridor of Gansu Province
ZHU Xia, WANG Lulu, ZHAO Dandan, WANG Shi, HAN Shunyu, YANG Xueshan
2020, 41(18):  159-164.  doi:10.7506/spkx1002-6630-20190721-267
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In order to screen for native Oenococcus oeni with good fermentation tolerance, seven O. oeni strains isolated from the wine-producing area of the Hexi corridor of Gansu province were investigated for their tolerance to fermentation factors such as pH value, alcohol, SO2 concentration and fermentation temperature. Then, the strains with good fermentation tolerance were selected to analyze their adaptability to various combinations of pH value, alcohol content and SO2 concentration. The results showed that there were differences in fermentation tolerance among the seven strains, and that each strain had different resistance to different stress factors. Strains MG-1, MG-7, QL-11 and ZX-1 could grow and proliferate under the conditions of pH 3.2, alcohol content 14% and SO2 concentration 40 mg/L. In decreasing order of importance, the factors affecting the growth of strains MG-1, MG-7, QL-11 and ZX-1 were alcohol content, SO2 concentration and pH value, while those affecting the growth of strains QL-11and ZX-1 were alcohol content, pH value and SO2 concentration. Collectively, we demonstrate that strain ZX-1 has the strongest fermentation tolerance among the tested strains, followed by MG-1, MG-7 and QL-11, all of which have potential for industrial application.
Isolation and Identification of Latent Fungal Pathogens from Chinese Olive Fruit
CHEN Penglian, CHEN Nanquan, LIN Hetong,, LIN Yuzhao, CHEN Yihui
2020, 41(18):  165-171.  doi:10.7506/spkx1002-6630-20191224-276
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In order to clarify the species of latent pathogenic fungi in Chinese olive fruit, the pathogenic fungi from the flower organ and fruit of ‘Changying’ Chinese olive (Canarium album (Lour.) Raeusch cv. Changying) were isolated by using tissue isolation, and their pathogenicity was assayed using Koch’s postulates. The genomic (DNA of the isolated pathogens was extracted via the cetyl trimethyl ammonium bromide (CTAB) method Polymerase chain reaction (PCR) was employed to amplify of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region, and then the amplified product was sequenced and subjected to homology analysis by basic local alignment search tool (BLAST). A phylogenetic tree for each of the six isolates was established using the neighbor-joining (NJ) method with MEGA6.0 software. According to their morphological and phylogenetic characteristics, the six latent pathogenic fungi were identified as Neofusicoccum parvum, Pestalotiopsis microspora, Aureobasidium pullulans, Phomopsis, Phanerochaete, and Guignardia with N. parvum and P. microspora being the dominant ones, which could infect the plant at the blooming stage. The results of this work can provide a scientific basis for controlling postharvest disease of Chinese olive fruit.
Fermentation and Probiotic Properties of Lactic Acid Bacteria Isolated from Dairy Products in Yili, Xinjiang and Optimization of Their Mixtures for Mixed-culture Fermentation
CAI Jingjing, ZHANG Yachuan, LI Xu, ZHANG Yan, NI Yongqing
2020, 41(18):  172-179.  doi:10.7506/spkx1002-6630-20190724-320
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The acidification ability and aroma-producing ability of seven strains of lactic acid bacteria preserved in our laboratory for yogurt fermentation and the texture properties of yogurt made with each of the strains were measured. The probiotic characteristics of these strains were evaluated by their antibacterial properties, gastrointestinal juice tolerance and antibiotic susceptibility. Strains with better performance were selected and combined to obtain the optimal mixed starter culture. The results of single-strain fermentation showed that the coagulation time of yogurts made separately with Leuconostoc lactis XKN1-5 and Lactobacillus delbrueckii subsp. bulgaricus BSTS6-4 was shorter (about 7 h), and the acidity reached around 96 °T; their acidification abilities were better than those of other strains. The yogurts fermented separately by L. delbrueckii subsp. bulgaricus BSTS6-3 and Streptococcus thermophilus XSXN1-2 had excellent texture characteristics. L. delbrueckii subsp. bulgaricus BSTS6-1 and S. thermophilus XSXN1-1 had the best aroma-producing ability and the yogurt fermented by strain XSXN1-1 contained 2.02 mg/L of diacetyl and 16 mg/L of acetaldehyde, indicating significantly improved flavor. The results of probiotic properties showed that L. fermentum BST2-3 and L. delbrueckii subsp. bulgaricus (BSTS6-1, BSTS6-3, and BSTS6-4) could inhibit the growth of pathogenic bacteria, and Lactobacillus fermentum BST2-3, L. delbrueckii subsp. bulgaricus BSTS6-1, BSTS6-4) had greater adhesion ability; the number of viable L. fermentum BST2-3 was 106 CFU/mL after 9 h of simulated gastrointestinal juice treatment. All the seven strains showed sensitivity to most tested antibiotics and were of high safety. Based on the ability to produce acids and aroma compounds, yogurt texture and probiotic properties, the optimal strain combination for mixed-culture fermentation was obtained as L. delbrueckii subsp. bulgaricus (BSTS6-4 and BSTS6-3) + S. thermophilus XSXN1-1 + L. fermentum BST2-3.
Analysis of Prokaryotic Community Structures in Bottom and Wall Muds from Old and Young Pits Used for Production of Strong-Flavor Baijiu
ZHANG Huimin, WANG Yanli, MENG Yajing, LI Anjun, ZHOU Qingwu, HU Xinhang, LIU Guoying, LI Lan, HUANG Yan, XING Xinhui
2020, 41(18):  180-187.  doi:10.7506/spkx1002-6630-20190723-307
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This study analyzed the prokaryotic community structures of bottom (BPM) and wall (WPM) muds from old and young pits used from the production of Chinese strong-flavor Baijiu by Illumina MiSeq sequencing of the variable V4 region of the 16S rRNA gene. The prokaryotic community from young WPM showed the simplest genus composition, mainly including Lactobacillus (85.59%) and Caproiciproducens (5.63%), followed by young BPM, old WPM and old BPM. The prokaryotic composition of old BPM was the most abundant, with the most abundant being Aminobacterium (11.85%), Sedimentibacter (2.08%) and unknown genera (37.6%). Among the four pit muds, old BPM had the highest values of pH (5.69) and NH4+-N (2.54 g/kg) and the lowest values of alcohol, organic acids, and esters, resulted from the complete metabolic chain of the prokaryotic flora in old BPM. The physicochemical properties of the four pit muds could provide a rationale for understanding prokaryotic evolution during the aging process of pit mud. The number of 16S rDNA copies in old pit mud was 1–2 orders of magnitude higher than that in young pit mud, indicating more prokaryotic abundance in old pit mud. Redundancy analysis revealed that the acetic acid (32.9%) and moisture contents (20.6%) were the most significantly correlated with the prokaryotic community of pit mud (P < 0.01), indicating their importance during PM aging.
Effect of Pre-fermentative Addition of Flavanols on the Color Attributes and Phenolic Profiles of ‘Cabernet Sauvignon’ Dry Red Wine
WANG Xiaoyue, ZHANG Shanshan, ZHANG Xinke, CAO Peng, ZHANG Bo, HE Fei
2020, 41(18):  188-195.  doi:10.7506/spkx1002-6630-20190714-187
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In the present study, the effect of pre-fermentative addition of three flavanol cofactors, including myricetin, kaempferol and quercetin, on the color and phenolic profile of ‘Cabernet Sauvignon’ dry red wine was examined during fermentation and aging. The results showed that the addition of flavanols before fermentation could effectively improve wine color density, enhance red tonality, and stabilize wine color during aging. Myricetin addition favored the extraction of polyphenols during fermentation to improve the stability of wine color during aging and showed better co-pigmentation effect than any other flavanol tested. This study provides scientific support for the application of flavanol cofactors in red wine production.
Component Analysis
Optimization of Extraction Process for 5-Methyltetrahydrofolate from Maize and Determination of Its Content by HPLC
WANG Bolun, GU Fengying, ZHANG Fan, LIU Hao, YANG Tingting, SHAO Zhixiao, WANG Feng
2020, 41(18):  196-202.  doi:10.7506/spkx1002-6630-20200319-297
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In this study, the extraction of 5-methyl-tetrahydrofolate (5-MTHF) from maize was optimized, and the accuracy of high performance liquid chromatography (HPLC) for determination of 5-MTHF in maize was evaluated. The extraction parameters were optimized as follows: extraction buffer pH 6.5, α-amylase concentration 12 U/mL, protease concentration 0.028 U/mL , rat serum concentration 14 μL/mL, and amount of eluate collected from solid phase extraction (SPE) column chromatography 2.5 mL. The HPLC method showed a good linear relationship in the concentration range of 0.003 to 1.000 μg/mL with correlation coefficient of 0.999 9. The limit of detection (LOD) was 0.001 μg/mL, and the limit of quantification (LOQ) was 0.003 μg/mL. The precision expressed as relative standard deviation (RSD) was 1.90%, and the recovery of 5-MTHF from spiked sample was 104.57%. The concentrations of 5-MTHF and folate in folate-fortified maize (Jingke Nuo 928) were 181.18 and 253.65 μg/100 g dry mass, respectively. The optimized HPLC method is suitable for the determination of 5-MTHF and folate in maize, and can provide a reference for the determination of folate concentration in other fortified grains.
Analysis of Volatile Flavor Compounds in Stewed Beef with Broth during Storage
LI Su, ZHOU Huimin, ZHAO Bing, PAN Xiaoqian, ZHU Ning, WU Qianrong, LIU Meng, ZHANG Shunliang
2020, 41(18):  203-209.  doi:10.7506/spkx1002-6630-20200119-232
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Changes in the volatile flavor compounds of stewed beef with broth stored for different periods (0, 2, 4, 6, and 8 months) were analyzed using purge and trap-thermal desorption-gas chromatography-mass spectrometry combined with odor activity and principal component analysis (PCA). The results showed that the patterns of change in volatile flavor compounds were different between the beef and the broth. The total content of volatile flavor compounds in the beef was the highest up to 5 380.24 μg/kg when stored for 4 months. The maximum total content (3 877.84 μg/kg) of volatile flavor compounds in the broth sample was reached after 2 months of storage. Alcohols were the most abundant volatile compounds for both samples, followed by aldehydes, and the content of nonanal showed the biggest difference between the two samples. Odor activity value (OAV) analysis showed that aldehydes were the main flavor contributors. PCA analysis indicated that nonanal, octanal and p-anisaldehyde were the main flavor contributors of stewed beef samples.
Identification of Characteristic Aroma Substances of Three Different Mozzarella Cheeses by Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry and Gas Chromatography-Olfactometry
WANG Jiao, XU Lingyun, ZHANG Jinhua, BIAN Ran, WANG Bei, CAO Yanping
2020, 41(18):  210-217.  doi:10.7506/spkx1002-6630-20190911-141
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The volatile flavor components of three different Mozzarella cheeses (fresh buffalo milk cheese, fresh cow milk cheese and semi-hard cow milk cheese) were identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). By gas chromatography-olfactometry (GC-O), a total of 109 compounds were determined in the cheese samples and semi-quantified by GC-MS. The odor activity values (OAV) of 31 compounds were calculated and analyzed by principal component analysis (PCA). Results showed that the major volatile compounds in fresh buffalo milk Mozzarella cheese were hexanal, heptanal, ethyl caproate, (E)-2-nonene aldehyde, furfural and ethyl butyrate. The main volatile flavor substances of fresh bovine milk Mozzarella cheese were 1-octene-3-alcohol, 2-undecanone and δ-dodecalactone. The main volatile flavor substances of semi-hard Mozzarella cheese were n-decanoic acid phenylacetaldehyde and ethyl acetate. According to the PCA results, six Mozzarella samples could be divided into three categories, which indicated that these key aroma compounds could clearly explain the flavor differences among the 3 kinds of Mozzarella cheese. This study provides theoretical guidance for the identification of buffalo milk adulteration in Mozzarella cheese.
Identification of Unknown Aroma-active Compounds of Soy Sauce by Gas Chromatography-Orbitrap-Mass Spectrometry
FENG Yunzi, ZHOU Ting, CAI Yu, CHEN Zijie, ZHAO Mouming
2020, 41(18):  218-225.  doi:10.7506/spkx1002-6630-20190804-048
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Gas chromatography-orbitrap-mass spectrometry (GC-Orbitrap-MS) combined with gas chromatography-quadruple-mass spectrometry-olfactometry (GC-Q-MS-O) was used to identify the ‘unknown’ aroma active compounds of soy sauce in this study. GC-Q-MS and GC-Orbitrap-MS identified 91 and 166 volatile compounds, respectively. The total peak area detected by Orbitrap-MS was 31 times as large as that detected by Q-MS. Four intense aroma-active areas with ‘fruity’, ‘fruity’, ‘grassy’ and ‘flowery and fruity’ notes were not identified in GC-Q-MS-O. In order to identify the 4 unknown compounds, the compound fragments and natural isotopic compositions were estimated by GC-Orbitrap-MS based on the high-precision mass spectral data. In addition, by comparing the mass spectra with the NIST mass spectral library as well as considering the retention index and aroma characteristics, they were identified as ethyl isobutyrate, ethyl butyrate, 3-hexen-2-one and 3,5,5-trimethyl-2-cyclohexenone (isophorone). This study showed that GC-Orbitrap-MS combined with GC-Q-MS-O could effectively reveal the ‘unknown’ aroma-active compounds whose contents were below the detection limit of quadrupole MS, or which could not be determined due to overlapping with other volatiles, which would be of important significance for the identification of aroma-active compounds in the field of flavor chemistry.
Fermented Dark Tea with Pericarpium Citri Reticulatae: Comparison of Chemical Composition with Raw Dark Tea and Bioactivities in Vitro
HOU Can, DU Yuguang, WANG Xi,XIAO Jie, FAN Yihang, DONG Zhizhong, CHANG Guosheng, WANG Wei, LI Song, YING Jian
2020, 41(18):  226-232.  doi:10.7506/spkx1002-6630-20190717-228
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This study was conducted to investigate the chemical composition and potential bioactivity of fermented dark tea with Pericarpium Citri Reticulatae in comparison with those of raw dark tea. The differences in the contents of the main chemical components between raw dark tea from two manufacturers and fermented dark tea with Pericarpium Citri Reticulatae made with each of them. The influence of fermentation on the chemical composition of fermented dark tea with Pericarpium Citri Reticulatae was observed using ultra-high performance liquid chromatography coupled with Q-exactive quadrupole/electrostatic field orbitrap mass spectrometry (UPLC-Q-Exactive-MS) combined with principal component analysis (PCA). Furthermore, the inhibitory effect of the boiling water extract from fermented dark tea with Pericarpium Citri Reticulatae on α-amylase and α-glucosidase was tested. The results showed that the content of tea polysaccharides decreased after fermentation. The content of flavonoids did not change significantly remaining in the range between 2.69% and 3.29%. After fermentation, the content of tea polyphenols decreased from 14.96% to 8.99%–12.44%. The score plot of PCA indicated that fermented dark tea with Pericarpium Citri Reticulatae could be separated from raw dark tea because of the significant variation in the main components between them. A total of 31 compounds that greatly differed between the fresh and aged samples of fermented dark tea with Pericarpium Citri Reticulatae including phenolic acids, flavanols and flavonoids were identified by comparison with the database library. These compounds were detected in all samples of fermented dark tea with Pericarpium Citri Reticulatae and they showed the same trend with aging time, among which, the relative contents of 13 compounds increased, while those of 17 compounds decreased and the remaining one remained basically unchanged. The inhibiting or activating effect of fermented dark tea Pericarpium Citri Reticulatae on α-amylase differed between the two kinds of raw dark tea. Sample 1-1 showed high inhibitory effect on α-amylase at three concentrations tested and its effect increased with increasing concentration. Sample 1-2 had a strong inhibition effect on α-amylase only at low concentration. All samples had inhibitory effect on α-glucosidase; the inhibitory effect of sample 2-1 was weak, while that of the other samples was strong. The half-maximum inhibitory concentration (IC50) of fermented dark tea with Pericarpium Citri Reticulatae for lipase was high, indicating its weak anti-lipase effect.
Effects of Different Pre-Processing Techniques on the Quality and Volatile Flavor Components of Cold Pressed Double Low Rapeseed Oil
ZHANG Huanhuan, ZENG Zhihong, GAO Feihu, HUANG Taocui, ZHANG Ling, ZHANG Xuemei, LI Xue, YANG Shixiong
2020, 41(18):  233-238.  doi:10.7506/spkx1002-6630-20190917-207
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The effects of microwave or roasting preptreatment on the acid value, peroxide value, fatty acid composition, antioxidant activity and volatile flavor components of cold pressed double low rapeseed oils were studied. The results showed that the acid value of cold pressed rapeseed oil in the two pretreatment groups ranged from 0.64 to 0.88 mg/g and the peroxide value ranged from 1.65 to 1.95 mmol/kg. The acid value and peroxide value of cold pressed rapeseed oil were affected by either pretreatment, but both of them remained within the range of the quality standard for cold pressed rapeseed oil. Microwaving and roasting had no significant effects on the fatty acid composition of cold pressed rapeseed oil. The antioxidant activity of rapeseed oils with different pretreatments, as evaluated by 1,1-dipheny1-2-picryl-hydrazyl (DPPH) radical scavenging capacity, could be ranked as follows: roasting at 180 ℃ for 15 min > roasting at 180 ℃ for 10 min > microwaving for 2 min > roasting at 180 ℃ for 5 min > microwaving for 90 s > microwaving for 1 min > control without any pretreatment. A total of 42 volatile flavor components were detected in the seven rapeseed oils, mainly including sulfide, glucosinolate degradation products and heterocyclic compounds. Microwaving and roasting had significant effects on volatile flavor components of rapeseed oils. With the extension of microwaving and roasting time, the characteristic volatile flavor compounds of rapeseed oils changed from glucosinolate degradation products to pyrazines, and the overall flavor changed from sulfureous and green aroma to baked and nutty aroma.
Determination of Organic Acids in Vinegar and Related Products by Ion Chromatography
JI Fengdi, XIA Rong, TAO Huiyuan, WEI Wei, MU Xiaoting, LI Dong, LU Fei
2020, 41(18):  239-244.  doi:10.7506/spkx1002-6630-20191018-192
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An ion chromatography method was developed for the determination of 14 organic acids in vinegar and related products by?conductivity detection. The chromatographic separation was performed on a Dionex Ion Pac AS11-HC column with gradient elution using KOH as the eluent at a flow rate of 1.5 mL/min. Lactic acid, acetic acid, propionic acid, formate, butyrate, pyruvate, iso-valproic acid, pentarate, adipate, succinic acid, tartrate, ketoglutarate, oxalic acid, and citric acid were separated and quantified in 35 min. The optimized method was validated with respect to linearity, precision, accuracy, and limits of detection and quantification. The applicability of the present method has been demonstrated for the determination of organic acids in different vinegar samples.
Analysis of Antioxidant Components from Vine Tea by Online Ultra-high Performance Liquid Chromatography Photo-Diode Array-Mass Spectrometry-2,2’-Amino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) di-ammonium Salt Cation Radical Method
GAN Xiaona, PENG Bo, LI Tingzhao, LI Bo
2020, 41(18):  245-250.  doi:10.7506/spkx1002-6630-20190624-297
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An online ultra-high performance liquid chromatography photo-diode array-mass spectrometry-2,2’-amino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) di-ammonium salt (UPLC-PDA-MS-ABTS) cation radical method was established to identify the antioxidant components in vine tea. Trolox was used as the reference substance and the standard curve method was used to quantitatively analyze the antioxidant activity of dihydromyricetin as the major bioactive constituent in vine tea. Four compounds were identified from vine tea, including dihydromyricetin (DMY), which was the main antioxidant compound of vine tea. The antioxidant activities of 12 batches of commercially available vine tea samples were analyzed. Results showed that the samples produced in Enshi of Hubei province and those produced in Wuyishan of Fujian province had the highest antioxidant activity. The developed quantitative method is fast, stable and reliable for the online assay of the antioxidant activity of vine tea.
Processing Technology
Effect of Addition of Edible Mushrooms on the Quality Characteristics of Emulsified Duck Sausage
CHEN Kunpeng, YU Yimin, XIA Qiang, HE Jun, SUN Yangying, DANG Yali, CAO Jinxuan, PAN Daodong
2020, 41(18):  251-257.  doi:10.7506/spkx1002-6630-20191028-305
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The aim of the work was to evaluate the effect of the addition of Lentinula edodes (LE), truffle (TR) or Tricholoma matsutake (TM) on the quality and sensory characteristics of emulsified duck sausage. Sausage samples were prepared with the addition of 0.8% (m/m) LE, TR and TM powders, respectively, and their color, texture, lipid oxidation, and contents of amino acids and free fatty acids were evaluated. Electronic tongue analysis and sensory evaluation were also performed. Addition of LE, TR and TM significantly increased the contents of free fatty acids and free amino acids (FAA), resulting in 38.0%, 34.0% and 27.0% increase in the total amount of free amino acids, respectively. Lipid oxidation of sausages was effectively inhibited by the addition of these edible mushrooms. They decreased hardness and chewiness. caused changes in L*, a*, and b* values to different degrees, and enhanced umami intensity with the greatest effect being observed by the addition of TM. Sausages added with the edible mushrooms could be discriminated clearly by electronic tongue. Addition of each of the mushrooms imparted better overall sensory acceptance to emulsified duck sausage, and therefore could represent an effective way to improve the quality of emulsified sausage products.
Safety Detection
Determination of Nine Herbicide Residues in Aquatic Products Using QuEChERS Combined with UPLC-MS/MS and Evaluation of Matrix Effects
LI Lichun, LIU Shugui, YIN Yi, WANG Yujie, ZHENG Guangming, MA Lisha, SHAN Qi, DAI Xiaoxin, ZHAO Cheng, XIE Wenping, WEI Linting
2020, 41(18):  258-266.  doi:10.7506/spkx1002-6630-20190903-035
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A quick, easy, cheap, effective, rugged, and safe (QuEChERS) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for determination of 9 herbicide residues (prometryn, acetochlor, butachlor, quinclorac, bensulfuron methyl, metsulfuron methyl, atrazine, linuron and bentazone) in aquatic products. Detection was carried out in the positive ion mode with multiple reaction monitoring and the external standard method was used for quantification. The matrix effects in the determination of the nine herbicides and the factors influencing?them were analyzed. The results showed that good linearity was observed for quinclorac and linuron in the concentration range of 1.00–100 ng/mL and for seven other herbicides in the concentration range of 0.50–100 ng/mL with correlation coefficients above 0.99. The limits of detection (LODs) and limits of quantitation (LOQs) for all herbicides were in the ranges of 0.20–0.50 and 0.50–1.50 μg/kg, respectively. The average recoveries of the herbicides spiked into blank samples of Penaeus orientalis, Cyprinus carpio, Carassius auratus and Aristichthys nobilis were 84.33%–116.37% (n = 6), and the precision expressed as relative standard deviation (RSD) was 0.80%–13.81% (n = 6). This method could be applied to determine the 9 herbicide residues in aquatic products. The type and mass of matrix and herbicide concentration influenced the matrix effect. Quinclorac, bensulfuron methyl and metsulfuron methyl showed strong matrix enhancement effects in P. orientalis and C. carpio, while butachlor, acetochlor, linuron, bentazone and atrazine showed matrix inhibition effects in all four aquatic products. The matrix calibration curve should be used to reduce the matrix effects to ensure the accuracy in the detection of herbicide residues in aquatic products.
Preparation of Plasmid DNA Reference Material and Its Application in Rapid Detection of Goji Berries (Lycium barbarum) Contaminated with Escherichia coli
XIA Dandan, ZHAO Yingying, MA Panpan, WANG Shenlei, MA Changyang, GAO Xiaofeng, KANG Wenyi,
2020, 41(18):  267-274.  doi:10.7506/spkx1002-6630-20200315-231
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The virulence genes escV, stx2 and hlyA of Escherichia coli were selected as target sequences to construct plasmid standards for the rapid detection of E. coli. The recombinant plasmids were verified by colony PCR and sequencing, and after passaging for 15 generations, sequencing was carried out to verify the stability of the plasmid standards. The escV, stx2, and hlyA plasmid standards were prepared, and they were evaluated by limit of detection (LOD), homogeneity, stability and qualitative tests. These plasmid standards were applied to detect goji berries contaminated with E. coli. The results of colony PCR and sequencing showed successful construction of the escV, stx2 and hlyA plasmid standards. The detection sensitivity of the plasmid standards was at the pg level by the PCR system. The PCR results showed that the plasmid standards were specific, and they remained stable for 15 generations. The LODs of the escV, stx2 and hlyA plasmid standards were 3.93 × 106, 2.41 × 105 and 2.14 × 105 copies/mL, respectively, and they had good stability at 25, 4 and ?20 ℃. When these plasmid standards were used to detect E. coli in 34 batches of goji berries, escV and stx2 were undetectable while hlyA was detected in 3 batches with a detection rate of 8.82%. Using the method specified in the national standard GB 4789.6?2016 Food Hygiene Microbiology Test Standard combined with 16S rRNA gene sequencing, E. coli was not detected in the 3 batches of goji berry samples indicating that the detection sensitivity of the plasmid standards was high, and the detection rate was greatly improved as compared with traditional microbial separation and identification.
Simultaneous Determination of 16 Phthalate Esters in Vegetable Oil by QuEChERS-Isotope Dilution-Gas Chromatography Mass Spectrometry
ZENG Shaofu, HU Changying, FENG Zhiqiang
2020, 41(18):  275-282.  doi:10.7506/spkx1002-6630-20190721-269
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An analytical method for simultaneous determination of 16 phthalate esters (PAEs) s in vegetable oils by quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction combined with gas chromatography mass spectrometry (GC-MS) was established. Acetonitrile saturated with n-hexane was used as the extraction solvent and dehydrated with anhydrous magnesium sulfate. The extraction time, extraction solvent volume, and purifying agent type and dosage were optimized. The applicability of this analytical method was verified with corn oil, peanut oil and sunflower oil samples. Meanwhile, the matrix effect of PAEs detection in the three vegetable oils was also evaluated. The mass spectrometer was operated in the selected-ion monitoring (SIM) mode and the matrix-matched internal standard calibration method was used for quantification. Results showed that the best extraction efficiency was obtained after vortex oscillation for 2 min with 4 mL of the extraction solvent, and the best purification efficiency was achieved using 100 mg/mL octadecyl bonded silica gel (C18) and 50 mg/mL ethylenediamine-N-propylsilane (PSA). The calibration curves for all target analytes displayed good linear relationships over the concentration range of 5–500 μg/L, with correlations coefficients of more than 0.999 0. The limits of detection (LODs) and limits of quantitation (LOQs) were 0.002–0.03 mg/kg and 0.008–0.1 mg/kg respectively. The average recoveries of the 16 PAEs at three spiked levels (0.04, 0.2 and 0.4 mg/kg) from corn oil, peanut oil and sunflower oil ranged from 80.2% to 118.6%, with relative standard deviations (RSDs) of 0.2% to 9.8%. The 16 PAEs exhibited an enhanced matrix effect in the three oil matrices, with a percentage enhancement in the range of 11.0% to 158.9%. However, the matrix-matched curve was found to effectively eliminate the matrix effects. The method was efficient, simple rapid, and suitable for determining the residues of 16 PAEs in vegetable oils.
Selection of Characteristic Near Infrared Spectra and Establishment of Prediction Model for Qingzhuan Tea Quality
WANG Shengpeng, GONG Ziming, ZHENG Pengcheng, LIU Panpan, TENG Jing, GAO Shiwei, GUI Anhui
2020, 41(18):  283-287.  doi:10.7506/spkx1002-6630-20190906-089
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Near infrared spectroscopy (NIRS) was used for rapid and nondestructive evaluation of the quality of Qingzhuan tea. Under the premise of ensuring the integrity of samples, spectra were acquired and preprocessed, and the characteristic spectral intervals were selected by synergy interval partial least squares?(siPLS). Furthermore, principal component analysis was performed, and a prediction model was established for the sensory evaluation score of Qingzhuan tea by Jordan-Elman back propagation-artificial neural network. The best pretreatment method was multiple scatter correction + 2nd derivative, the characteristic spectral intervals were 4 377.6–4 751.7, 4 755.6–5 129.7, 6 262.7–6 633.9, and 7 386–7 756.3 cm-1, and the cumulative contribution rate of the first three principal components in the characteristic spectral regions was 99.15%. The transfer function of the model was tanh with root mean square error of cross-validation set (RMSECV) and determinant coefficient for prediction (R2p) of 0.386 and 0.973, respectively. The root means square error and the determinant coefficient were respectively 0.393 and 0.971 for unknown Qingzhuan tea samples. The results showed that NIRS combined with Jordan-Elman back propagation-artificial neural network could allow rapid and accurate evaluation the quality of Qingzhuan tea scoring in the range of 75.00–93.00.
Determination of Cadaverine in Mackerel by Biosensor Based on Unfolded Hemoglobin and Diamine Oxidase
ZHANG Min, WU Junquan, YAO Jiawen, YANG Chunting, BAI Weidong, ZHAO Xiaojuan
2020, 41(18):  288-295.  doi:10.7506/spkx1002-6630-20190603-014
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Unfolded hemoglobin was immobilized onto the surface of a clay-gold nanoparticles (Clay-AuNPs) modified glassy carbon electrode (GCE) by the physical adsorption method, and then diamine oxidase (DAO) was immobilized with 2.5% glutaraldehyde as a crosslinking agent. The resulting electrochemical biosensor had good detection performance for cadaverine. The electrode modification system, DAO concentration, the type of mimic peroxidase, the stability of the modified electrode and the test conditions were optimized. The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Furthermore, the method was used to detect the cadaverine content in mackerel. The results showed that under the optimized conditions, the response current of the sensor had a good linear relationship with cadaverine concentration in the range between 2.0 × 10-12 and 1.0 × 10-11 mol/L (r = 0.991), and the detection limit (RSN = 3) was 6.7 × 10-13 mol/L. The spiked recoveries of cadaverine in mackerel samples by this biosensor method ranged from 82.2% to 109.1%. Thanks to its good reproducibility and stability and high sensitivity, this method is suitable for the determination of cadaverine in fish samples such as mackerel.
Tracing the Geographical Origin of Potato Based on Mineral Elements and Stable Isotopes
ZHANG Xinxin, ZHANG Fujin, LIU Guanghua, ZHANG Yao, WANG Xuejiao, SHA Na, LIAN Haifei
2020, 41(18):  296-302.  doi:10.7506/spkx1002-6630-20190916-187
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In order to investigate the feasibility of identifying the geographical origin of potato based on mineral elements and stable isotopes, we determined the mineral element contents and stable isotope ratios of representative potato samples collected from five main potato-producing areas of China, Inner Mongolia, Heilongjiang, Xinjiang, Sichuan and Guangdong provinces. The results of principal component analysis (PCA), cluster analysis (CA) and discriminant analysis (DA) indicated that 8 fingerprint minerals, including sodium, aluminum, phosphorus, manganese, cobalt, nickel, copper and cadmium, were selected by stepwise method, and in the cross-validation, the correct discrimination rate was 89.3% for these minerals. δ13C and δ15N values could basically distinguish between samples from Inner Mongolia and those from Heilongjiang, Xinjiang and Guangdong. The discriminative accuracy of the model established based on δ13C and δ15N was 82.0%. Based on the fingerprint minerals combined with the stable isotopes, the initial correctness rate for the different geographical origins was 94.7%, and the correctness rate in cross-validation was 93.2%, both of which were higher than those obtained based on either of them alone, indicating good discrimination results. The results obtained in this study show that the combination of mineral elements and stable isotopes allows effective and accurate discrimination of the geographical origin of potato and therefore provides an effective method for geographical origin traceability.
Geographical Origin Identification of Tan Sheep Meat Based on Mineral Element Profiles of Meat and Environmental Samples
WANG Xuerong, LUO Ruiming, LI Yalei, MA Mengbin
2020, 41(18):  303-310.  doi:10.7506/spkx1002-6630-20190722-289
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In this study, the contents of 25 mineral elements in Tan sheep meat and environmental samples from Yanchi county in Ningxia were determined by inductively coupled plasma-mass spectrometry (ICP-MS). The characteristics of these mineral elements were further analyzed by principal component analysis (PCA), cluster analysis (CA) and linear discriminant analysis (LDA) in order to screen for geographical traceability indicators. A discriminant model was established and verified based on the mineral element profiles of both meat and environmental samples. For meat samples, the mineral elements selected were Cr, Ni, Cu, Rb, Sr, Mo, Ba, Mn and Sn, and the overall accuracy rates of back substitution test and cross validation were 97.50% and 95.00%, respectively. For environmental samples, the mineral elements selected were K, P, Ca, Na, Mg, V, Ni, Zn, Ba, Rb, Cr, Sr and Fe. and the overall accuracy rates of back substitution test and cross validation were 93.80% and 97.50%, respectively. The traceability indexes common to Tan sheep meat and the environment were Cr, Ni, Rb, Sr, and Ba. The overall accuracy rates of back substitution test and cross validation were 96.30% and 95.00%, respectively. This study indicated that the selected mineral elements were effective in identifying the geographical origin of random Tan sheep meat samples, and could be used as indicators for fingerprint geographical traceability of Yanchi Tan sheep meat.
Rapid Determination of Tetrodotoxin in Fresh and Processed Aquatic Products by Two-Dimensional Ultra-high Performance Liquid Chromatography-Triple Quadrupole/Linear Ion Trap Mass Spectrometry
ZHANG Xiuyao, CAI Xinxin, ZHANG Xiaoyi, LI Ruifen
2020, 41(18):  311-316.  doi:10.7506/spkx1002-6630-20190617-171
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In this study, a heart-cutting two-dimensional ultra-high performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry method was developed and successfully validated for the determination of tetrodotoxin (TTX) in fresh and processed aquatic products. This method was based on the use of two different stationary phase columns (Hypercarb PGC and Acquity BEH Amide), which were connected through a six-port two-position switching valve. Samples were extracted with 0.2% (V/V) acetic acid aqueous solution, and then the extract was diluted with the solvent, filtered and directly injected for analysis. The separation of TTX was carried out on a Hypercarb PGC (2.1 mm × 100 mm, 3 μm) as the 1st dimension through elution with 0.2% (V/V) formic acid aqueous solution. The fraction containing TTX was switched into a trap column (XBridge BEH Amide guard column (4.6?mm × 10?mm, 2.5?μm)), and acetonitrile was simultaneously merged into the fraction by a dilution pump through a tee. After the trap column retained the TTX completely, the valve switched it into the 2nd dimensional stream, and then the TTX was separated on an Acquity BEH Amide (2.1 mm × 100 mm, 1.7 μm) with gradient elution using acetonitrile-H2O both containing 0.1% (V/V) formic acid, detected by positive electrospray ionization tandem mass spectrometry in the multiple reaction monitoring information-dependent acquisition enhanced product ion scanning (MRM-IDA-EPI) mode, and quantified by standard solvent external standard method. The average spiked recoveries were 82.3%–95.4% with relative standard deviations (RSDs) of 2.1%–14% (n = 6). The limit of quantitation (RSN = 10) was 0.002 mg/kg. This simple, selective and sensitive method has been successfully applied to the determination of TTX in fresh and processed aquatic products.