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Table of Content

25 June 2021, Volume 42 Issue 12
Food Chemistry
Effect of Protein Content on Functional Properties of Yak Whey Protein Concentrate
GAO Ruiping, LIANG Qi, BAI Lili
2021, 42(12):  1-7.  doi:10.7506/spkx1002-6630-20191110-126
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Native whey protein concentrate (NWPC) and sweet whey protein concentrate (SWPC) were prepared by spiral-wound membrane ultrafiltration of native yak whey and sweet yak whey followed by sequential dialysis through regenerated cellulose membranes with different molecular mass cutoffs (MMCOs), respectively, and their major components (lactose, pH and total protein) and functional properties (solubility, water-holding capacity, oil-holding capacity, foaming properties, emulsifying properties and thermal stability) were evaluated. The results showed that the total protein content of the whey protein concentrates (WPCs) obtained after dialysis through 10 000 Da MMCO cellulose membrane, which were found to contain no lactose, was over 80%. The solubility, water-holding capacity (WHC), oil-holding capacity (OHC), foaming properties, emulsifying properties and thermal stability were all significantly higher than those of the WPCs obtained after ultrafiltration through 3 500 Da MMCO rolled membrane and after dialysis through 5 000 Da MMCO cellulose membrane. The higher the protein content in WPC, the better the functional properties. After the same concentration treatment, the foaming capacity, foam stability, emulsifying capacity and emulsion stability of SWPCs were significantly (P < 0.05) higher than those of NWPCs. Yak WPC was the most unstable at 85 ℃, which was higher than 80 ℃ observed for Holstein cow milk WPC. The appropriate thermal treatment could improve the foaming properties, emulsifying properties and thermal stability of yak WPC. The yak WPC with high protein content obtained by membrane processing had good functional characteristics, making it having wide applications. It will play a key role in solving the problem of yak whey resource utilization, protecting the environment, and improving the economic benefits of enterprises.
Fabrication of pH-Responsive Hydrogels Composed of Chitosan-Fulvic Acid Microparticles and Konjac Glucomannan
CHEN Xiaohan, PANG Jie
2021, 42(12):  8-16.  doi:10.7506/spkx1002-6630-20200414-185
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A series of konjac glucomannan (KGM) hydrogels were prepared using chitosan-fulvic acid microparticles (CFMPs) as the reinforcing agents. CFMPs possessed high surface density and oxygen-containing functional groups, which could induce hydrogen-bonding interaction between KGM and CFMPs to form a more dense network structure. The effects of CFMPs on the network structure and releaseing behavior of KGM hydrogels were evaluated. The rheological results revealed that the regular change in the steady shear viscosity and dynamic viscoelasticity of all hydrogel-forming solutions was attributed to the reinforced network structure with strong interaction. Fourier transform infrared spectral analysis indicated that the carboxyl groups (–COOH) in CFMPs successfully formed hydrogen bonds with the hydroxyl groups (–OH) in the KGM polymer chains. Scanning electron microscopic images revealed that the structure of hydrogels was dense with interconnected pores. In addition, the in vitro drug-releasing behavior of KGM/CFMP hydrogels in artificial intestinal and gastric fluids was investigated. KGM/CFMP composite hydrogels possessed excellent pH-responsive property due to the protonated amine groups (–NH3+). Therefore, KGM/CFMP composite hydrogels have a huge potential as a pH-responsive drug delivery system.
Effect of NO on AMPK Activity, Glycolysis and Water-holding Capacity in Beef during Postmortem Aging
LI Qiao, MA Jibing, YU Qunli, HAN Ling
2021, 42(12):  17-23.  doi:10.7506/spkx1002-6630-20200425-327
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The purpose of this study was to explore the cascade regulation of NO on the AMP-activated protein kinase (AMPK) activity, glycolysis and water-holding capacity (WHC) in beef during postmortem aging. Beef quadriceps femoris treated with the NO release agent DETA, the nitric oxide synthase (NOS) inhibitor L-nitro-arginine methyl ester (L-NAME) or the AMPK inhibitor DETA + compound C were tested for NO content, AMPK activity and the key enzyme activities related to glycolysis, protein solublity and WHC after 0, 6, 12, 24, 48 and 72 h of postmortem aging. The results showed that at 0, 6 and 12 h postmortem, AMPK activity was significantly lower in the L-NAME-treated group than in the control group (P < 0.05), while it was significantly higher in the DETA-treated group than in the control group (P < 0.05). The activity of hexokinase (HK) and phosphofructokinase (PFK) in the L-NAME-treated group were significantly lower than those in the control group at 6 and 12 h (P < 0.05), whereas the pH was significantly higher than that in the control group at 6, 12 and 24 h (P < 0.05). Moreover, the total protein solubility was higher than that in the control group at 6, 12, 24, 48 and 72 h (P < 0.05), while the centrifugal loss was lower than that in the control group at these time points (P < 0.05). On the other hand, there was no significant difference in any of the above indexes between the NO-promoted group and the control group (P > 0.05). However, the activity of HK and PFK in the NO-promoted + AMPK inhibited group were significantly lower than in the other three groups at 6 and 12 h (P < 0.05), while the pH was significantly higher than in the other three groups at 6, 12 and 24 h (P < 0.05); the total protein solubility was significantly higher than in the other three groups at 6, 12, 24, 48 and 72 h (P < 0.05), while the opposite was true for centrifugal loss at these times (P < 0.05). From these results, it was shown that at the early post-mortem aging stage, NO enhanced the activity of HK and PFK and glycolysis rate by activating AMPK, which thereby reduced the pH, total protein solubility and WHC of beef. This study provides a theoretical basis for revealing the endogenous factors causing postmortem deterioration of beef quality and for future studies on meat quality control.
Emulsifying Properties of Different Kinds of Aggregates from Whey Protein Isolate and Stability of Pickering Emulsions Stabilized with Them
LIU Pei, CHEN Cheng, QIN Xinguang, ZHANG Haizhi, HU Zhongze, WU Qiong, YAO Renyong, WANG Xuedong, LIU Gang
2021, 42(12):  24-30.  doi:10.7506/spkx1002-6630-20200610-144
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In this study, whey protein isolate (WPI) was heated at 85 ℃ for 12 h under different pH conditions (pH 2.0 and 7.0) to prepare two different forms of protein aggregates, and the morphological characteristics and emulsifying properties at different pH and salt concentrations of the aggregates were evaluated. The microstructure, salt stability and thermal stability of Pickering emulsions stabilized with WPI and the two aggregates were comparatively investigated by transmission electron microscopy (TEM), dynamic light scattering (DLS) and optical microscopy. The results showed that fibrous and spherical aggregates were obtained at pH 2.0 and 7.0, respectively. The isoelectric points (IP) of the two aggregates shifted away from that of WPI, and the emulsifying activity increased at all pHs and salt concentrations tested compared with WPI. Moreover, the Pickering emulsion stabilized by the aggregates had better stability to severe environments. Notably, the spherical aggregates had better thermal stability. This study provides a basis for the application of Pickering emulsions stabilized WPI aggregates in milk beverages.
Effects of Gleditsia sinensis Lam. Gum on the Retrogradation Properties of Corn Starch and Water Distribution in Mixture Systems
ZHANG Xiaoyu, ZHANG Dandan, LI Rongfang, LUO Lei, LUO Denglin, ZHU Wenxue
2021, 42(12):  31-36.  doi:10.7506/spkx1002-6630-20191118-197
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The effect of Gleditsia sinensis Lam. gum (GSLG) added in different proportions (1%, 3%, 5% and 7%, based on the dry mass of corn starch) to corn starch on its retrogradation during cold storage (CS) was investigated in this study by rheological and texture characterization, differential scanning calorimetry (DSC) and low-field nuclear magnetic resonance (LF-NMR) imaging analysis. Dynamic rheological analysis showed that the elastic modulus of corn starch increased with refrigeration time; addition of GSLG decreased the change in elastic modulus and inhibited the short-term retrogradation of corn starch to some extent. The hardness of starch gels increased with cold storage time but decreased with the addition of GSLG. The results of DSC showed that the aging rate of mixed systems decreased with increasing addition of GSLG. Using LF-NMR, a comparative analysis of the proportions of free, bound, and immobile water in starch gels before and after addition of 5% GSLG was conducted, which confirmed that the gum had a large effect on the water distribution in the gels. This study further enriches the theoretical understanding of the effect of plant gums on the aging property of starch.
Effect of Transglutaminase on the Quality of Mandarin Fish (Siniperca chuatsi) Surimi Gel
YANG Mingliu, ZHOU Yingqin, FANG Xubo, YIN Junfeng, CHEN Xiao’e, XIE Ningning
2021, 42(12):  37-44.  doi:10.7506/spkx1002-6630-20201009-055
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In this work, the effect of transglutaminase (TG) on the quality of mandarin fish (Siniperca chuatsi) surimi gel (MFSG) was determined by measuring salinity, water-holding capacity (WHC), whiteness, texture, sensory evaluation, protein secondary structure and microstructure as well as using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that TG could improve the whiteness of MFSG. As compared with the control group, MFSG with TG presented significantly increased gumminess, chewiness and resilience (P < 0.05). Gel hardness increased significantly with increased TG addition (P < 0.05), and WHC reached its maximum value at an addition level of 0.6 U/g. SDS-PAGE showed the main protein bands gradually weakened with increase of TG concentration. By scanning electron microscope (SEM), it was observed that after addition of TG, MFSG generated a denser network structure. Therefore, TG could greatly improve the quality of MFSG, especially at 0.4 and 0.6 U/g. These results may provide a theoretical basis for improving the quality of MFSG.
Quantum Chemistry Calculation of Hydrogen Bond Interactions between Major Compounds in Baijiu during Storage
HUANG Zhangjun, ZHANG Wenhua, ZENG Yunhang, SHI Bi, WANG Songtao, SHEN Caihong
2021, 42(12):  45-51.  doi:10.7506/spkx1002-6630-20200531-375
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In this work, the hydrogen bond interactions between ethanol and water, between ethanol and flavor compounds, between water and flavor compounds, and among ethanol, water and flavor compounds were investigated by quantum chemistry calculation to provide insights into the mechanism of Baijiu aging. The results density functional theory showed that the structural stability of the binary and ternary complexes was in the decreasing order of ethanol/water-acid > ethanol/water-alcohol > ethanol/water-ester, and ethanol-water-acid > ethanol-water-alcohol > ethanol-water-ester, respectively. The carbonyl oxygen atom and hydroxyl hydrogen atom in the organic acids could form hydrogen bonds with the hydrogen atom and oxygen atom in the hydroxyl group of ethanol or water, separately, causing the ethanol/water-acid and ethanol-water-acid complexes to form ring structures and consequently improving the structural stability. This may partially explain why the flavor components of Baijiu showed an increase in acids and a decrease in esters during storage and may also explain why storage could strengthen the hydrogen bond interactions and reduce free molecules in Baijiu.
Structural and Functional Characteristics of Proteins Extracted from Zinc-Enriched and Selenium-Enriched Fruit Bodies of Pleurotus geesteranus
LIU Sishan, GANG Jie, ZHU Shuzhen, CHEN Taotao, DENG Haiping, LIU Yao, QU Pengkun, YUAN Jiacheng, PAN Qin
2021, 42(12):  52-59.  doi:10.7506/spkx1002-6630-20191117-196
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In this study, the structure and functional properties of proteins prepared from zinc-enriched and selenium-enriched Pleurotus geesteranus by ultrasound-assisted alkali extraction followed by acid precipitation were evaluated. The results showed that the total protein content of zinc-enriched P. geesteranus Zn200 was the highest up to 25.41%, 18.35% higher than that of the control group without zinc or selenium enrichment; the crude protein content of selenium-enriched mushroom Se60 was the highest up to 62.92%, 7.63% higher than that of the control group; the extraction rate of proteins from Se20 was the highest up to 29.05%. Sequential extraction revealed that albumin was the most abundant protein (up to 31.02%), followed sequentially by globulin, glutenin, gliadin, gliadin-like protein and glutenin-like protein. The structural characterization was performed by spectrophotometry, which demonstrated that the surface hydrophobicity of proteins from zinc-enriched and selenium-enriched P. geesteranus increased compared with the control group, while the sulfhydryl and disulfide bond contents decreased. Four secondary structures were identified in proteins from zinc-enrich and selenium-enriched P. geesteranus by Fourier transform infrared (FTIR) spectroscopy; the highest content of α-helix and β-sheet were found in Zn500 (18.89%) and the control group (39.24%), respectively. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the relative molecular mass masses of proteins from zinc-enriched and selenium-enriched P. geesteranus were mainly distributed between 32.4 and 40.3 kDa. The protein digestibility, water-holding capacity (WHC) and oil-holding capacity (OHC) of proteins from zinc-enriched and selenium-enriched P. geesteranus were lower than those of the blank group. The proteins from zinc-enriched P. geesteranus had the best foaming capacity, but had the weakest foam stability, while the emulsifying properties of the proteins from zinc-enriched and selenium-enriched P. geesteranus were improved relative to the control group.
Preparation and Structure Characterization of Inclusion Complexes between Flavor Substances with Different Configurations and Octenyl Succinic Acid Debranched Starch
FENG Tao, HU Zhongshan, ZENG Xiaolan, ZHANG Yu, SONG Shiqing, YAO Lingyun, SUN Min, XU Zhimin
2021, 42(12):  60-66.  doi:10.7506/spkx1002-6630-20191112-154
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Octenyl succinic acid debranched starch (OSA-DBS) was prepared from waxy corn starch using pullulanase and octenyl succinic anhydride as an esterifying agent. Flavor substances (n-caproaldehyde, n-heptanal, n-octaldehyde, γ-caprolactone, γ-heptanolactone, γ-caprolactone, 2,5-dimethyl-pyrazine, 2,3,5-trimethyl-pyrazine and 2,3,5,6-tetramethyl-pyrazine) with different configurations were successfully included in OSA-DBS. The inclusion complexes showed improved thermal stability, in the decreasing order: linear < circular < stereoscopic. The inclusion complexes may be spherical, with particle sizes in the range of 90–180 nm, and the particle size distribution of the pyrazine inclusion complex was more uniform than the others. For the same configuration, the inclusion efficiency and drug-loading amount tended to increase with the number of carbon atoms, the highest values (78.50% and 21.41%) being observed for the OSA-DBS-2,3,5,6-tetramethyl-pyrazine complex.
Regulation Mechanism of Epigallocatechin Gallate on the Structure of Soybean Protein
ZHAO Siming, JIANG Lianzhou, WANG Dongmei, SUI Xiaonan, ZHOU Linyi, FAN Zhijun
2021, 42(12):  67-75.  doi:10.7506/spkx1002-6630-20200523-273
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In this experiment, different concentrations of epigallocatechin gallate (EGCG) was induced to interact with soy protein isolate (SPI) to form complexes, aiming to explore the change of SPI structure and the effect of heat treatment on the composite system. The results showed that the mechanism of action of EGCG on SPI was static quenching by non-covalent hydrophobic interaction. Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy showed that binding to EGCG increased the percentage of α-helix and decreased the percentage of β-sheet structure in SPI before and after heat treatment, but resulted in no significant changes in the percentages of β-turn or random coil. The fluorescence and UV-visible spectra demonstrated that the interaction between EGCG and SPI caused a red shift in the maximum emission wavelength of SPI, and heat treatment enhanced this tendency.
Changes in Protein Degradation and Flavor Substance in Sauce Beef during Processing
WU Qianrong, ZHU Ning, CHEN Song, ZHOU Huimin, LI Su, ZHAO Bing, LIU Meng, PAN Xiaoqian, ZHANG Shunliang, QIAO Xiaoling
2021, 42(12):  76-84.  doi:10.7506/spkx1002-6630-20200518-206
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The concentrations, SDS-PAGE profiles, particle sizes and surface hydrophobicities of water soluble and salt soluble proteins, protein degradation index, and volatile flavor compounds in samples collected at different stages of sauced beef processing (raw meat, tumbling, cooking, and sterilization at 90, 105, 110 or 120 ℃) were analyzed using a Kjeldahl nitrogen analyzer, a nanoparticle potential analyzer, and headspace-solid phase micro-extraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results illustrated that as the process proceeded and sterilization temperature rose, protein concentration decreased, and protein degradation index increased significantly (P < 0.05). The change of protein particle size showed a consistent trend with surface hydrophobicity, and the maximum values were found in the tumbled sample, which decreased significantly after heating (P < 0.05). A total of 42, 50, 79, 70, 75, 79 and 74 volatile flavor compounds were respectively detected in the above seven samples, and the highest total amount of volatile compounds was determined in the unsterilized finished product, which declined after sterilization. The total amount of volatile flavor compounds in the sample sterilized at 105 ℃ was significantly higher than that in other sterilized samples (P < 0.05). In conclusion, sauced beef processing and sterilization could promote the degradation of proteins and the formation of volatile flavor compounds, but too high temperature could affect the denaturation and oxidation of proteins, and the flavor of the sample undergoing sterilization at 105 ℃ was the closest to that of the unsterilized sample. The findings of this study will provide a theoretical basis for the industrial production and flavor control of traditional sauced beef products.
Effect of Carrier Oil on Physicochemical Stability and Bioavailability of Quercetin Nanoemulsion
LI Chaoyang, DOU Zhongyou, ZHANG Liping, DIAO Jingjing, JIANG Lianzhou, MA Ping
2021, 42(12):  85-90.  doi:10.7506/spkx1002-6630-20200609-125
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The effects of different types and concentrations of carrier oil on the stability of nanoemulsion and quercetin bioavailability were studied. Quercetin-loaded nanoemulsions were prepared by high pressure homogenization of medium-chain triglycerides (MCT) or long-chain triglycerides (LCT) as the oil phase with pear adzuki bean protein as the emulsifier. The stability was investigated by light scattering particle size analysis, zeta potential, microscopic observation, physical stability, free fatty acid release and quercetin bioavailability. The results showed that compared with the LCT nanoemulsion, the MCT nanoemulsion showed higher stability and a slower degradation rate of quercetin. The average particle size of the nanoemulsion prepared with 5% MCT was (152.80 ± 1.73) nm compared to (180.42 ± 1.98) nm for the one prepared with 5% LCT. The biological retention rate of quercetin measured using a simulated gastrointestinal model was greatly improved in the MCT nanoemulsion system. This finding will be useful for the design of a more effective delivery system for enhanced quercetin bioavailability.
Flavonoid Composition and Antioxidant Activity of Diaphragma juglandis Fructus
SHA Yuhuan, MAO Xiaoying, WU Qingzhi, ZHANG Jian, CHENG Weidong
2021, 42(12):  91-98.  doi:10.7506/spkx1002-6630-20200423-304
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In this study, ultrasonic-assisted ethanol extraction was used to extract flavonoids from Diaphragma juglandis fructus, the dry wooden diaphragm inside walnuts, and AB-8 macroporous resin was used to separate and purify the crude flavonoids, yielding six fractions. Their antioxidant properties including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) cation radical, hydroxyl radical, superoxide anion radical and reactive oxygen species (ROS) scavenging capacity, and iron ion reducing power were determined. The composition and content of flavonoids in the six fractions were analyzed by ultra-high performance liquid chromatography-triple quadruple tandem mass spectrometry (UPLC-QQQ-MS/MS). The results showed that the ABTS cation radical, hydroxyl radical and ROS scavenging capacity of all six samples were significantly higher than those of vitamin C (VC). Quercetin was the most abundant flavonoid with the highest concentration of 2 426.9 ng/mL, and rutin and gallic acid were hardly detectable. In addition to the reported flavonoids naringin, rutin, isoquercetin, hyperoside, dihydroquercetin, catechin, quercetin and gallic acid (a member of the polyphenol family), quercetin, astragaloside and epicatechin gallate were also detected. The correlation analysis between flavonoid content and antioxidant capacity showed that naringin content was significantly negatively correlated with DPPH radical and ABTS cation radical scavenging capacity, while catechin content was positively correlated with ROS scavenging capacity. The results of validation experiment showed that the ABTS cation radical scavenging capacity of quercetin was significantly higher than that of VC and mixed samples. This study lays a foundation for the study of the antioxidant capacity and structure-activity relationship of quercetin, naringin and catechin.
Bioengineering
Directed Evolution of Lactobacillus plantarum and Propionibacterium acnes for Increased Production of Conjugated Linoleic Acid
SHI Xu, LIU Ying, SHI Haisu, WU Junrui, WU Rina, NIU Xueqing, LUO Xue, YUE Xiqing
2021, 42(12):  99-106.  doi:10.7506/spkx1002-6630-20200305-072
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In order to obtain high-yield conjugated linoleic acid (CLA)-producing strains, the myosin-cross-reactive antigen gene (mcra) from Lactobacillus plantarum and the linoleic acid isomerase gene (lai-p) from Propionibacterium acnes were separately cloned and ligated to pCold-SUMO skeleton plasmid to construct recombinant E. coli strains. The expression of the positive recombinant strains was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature. The target proteins were validated by SDS-PAGE, indicating successful expression of the two genes. Error-prone polymerase chain reaction was employed to establish a library of recombinant mutant strains with mcra or lai-p. Recombinant mutants with higher absorbance and CLA production were identified by flow cytometry, including 243 recombinant pCold-ycmcra-gfp mutants, among which the one numbered 155 displayed the highest absorbance value (1.050 6 ± 0.000 4), 6.02 times higher than that of the original strain; and 156 recombinant pCold-yclai-p-gfp mutants, among which the one numbered 39 showed the highest yield of (11.62 ± 0.003 6) μg/mL, 3.99 times higher than the original strain. This study can be helpful for further understanding the mechanism for increased CLA production in the mutant strains.
Separation, Purification and Enzymatic Characterization of Polyphenol Oxidase from Taro
CUI Xiaoying, PENG Xinyan, HE Hongjun, ZHANG Min, ZHANG Xiaotong
2021, 42(12):  107-115.  doi:10.7506/spkx1002-6630-20200928-340
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Polyphenol oxidase (PPO) from taro was obtained and purified through sequential steps of extraction, precipitation with 30%–80% saturated ammonium sulfate, dialysis, ultrafiltration, and chromatographic separation. The PPO was enzymatically characterized and the action pattern of selected inhibitors on it was explored. The results showed that electrophoretically pure PPO was obtained using DEAE-Sepharose and Superdex G-75 column chromatography, and the specific activity was 27 471.26 U/mg, which corresponded to a 6.37-fold purification. SDS-PAGE analysis of the purified PPO showed one band at approximately 24 kDa. The enzyme showed high substrate affinity for catechol. Its binding capacity to various substrates decreased as follows: catechol > resorcinol > phloroglucinol > pyrogallic acid > L-tyrosine. The optimal reaction temperature, pH and time were 30 ℃, 6.8 and 60 s, respectively. The PPO enzyme was inhibited by ascorbic acid and L-cysteine in an uncompetitive and competitive manner, respectively. This study demonstrates that controlling the temperature and pH and adding inhibitors can reduce the occurrence of enzymatic browning during taro processing effectively.
Dynamic Changes of Microbial Community, Amino Acid Composition and Moisture Distribution during the Fermentation of Miao Ethnic Suanzuo Fish
WANG Zehan, TAO Yuhao, SUN Liming, LIU Jia, DU Ming, WANG Zhenyu
2021, 42(12):  116-122.  doi:10.7506/spkx1002-6630-20201023-228
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In this work, the changes of the microbial community structure, taste and water distribution during the fermentation of Suanzuo fish were investigated. The microbial community was detected by Illumina MiSeq sequencing. Lactobacillus, Weissella and Staphylococcus were found to play the dominant role in the fermentation process. The abundance of Staphylococcus attained the maximum value of 64.65% on day 24, followed in decreasing order by Tetragenococcus, Lactobacillus, Macrococcus and Weissella. After completion of the fermentation on day 24, the concentration of total free amino acids increased to 1 406.09 mg/100 g from 380.61 mg/100 g. The taste-active amino acids Asp, Glu, Ala, Val, Met, Phe, Lys and His were detected in Suanzuo fish. In the product, the taste activity value (TAV) of Glu was 30.69, which made Glu the most prominent contributor to the overall taste of Suanzuo fish. It was observed that the percentages of immobile and free water decreased obviously while the percentage of bound water increased gradually during the fermentation process. This change was consistent with the change of fish flesh texture. This study provides theoretical support for the industrial development of traditional ethnic aquatic foods in China.
Identification and Mechanism Analysis of Angiotensin-I Converting Enzyme Inhibitory Peptides from Soybean Meal
LIU Jingbo, WANG Ziqin, YU Yiding, ZHANG Ting, LIU Boqun
2021, 42(12):  123-129.  doi:10.7506/spkx1002-6630-20200416-212
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In this study, ultrasonic-assisted enzymatic hydrolysis, ultrafiltration and ?KTA chromatography were sequentially used to separate and purify angiotensin-converting enzyme (ACE) inhibitory peptides from an enzymatic hydrolysate of soybean meal, and the molecular mass distribution of peptides was investigated. Then, the amino acid sequence of the ACE inhibitory peptides was identified by mass spectrometry (MS) and molecular docking. A consensus peptide sequence was synthesized by a solid-phase method to determine its ACE inhibitory activity and explore its underlying mechanism by molecular docking. The results showed that after ultrafiltration, the molecular mass distribution of peptides in the hydrolysate was mainly under 6 000 Da. The peptides GVRP and IIVTP, selected for their lowest free energy (–8.44 and –9.04 kcal/mol correspongdingly) predicted by molecular docking, could inhibit ACE activity, with half-maximum inhibitory concentration (IC50) values of (84 ± 0.06) and (77 ± 0.08) μmol/L, respectively. The molecular docking results revealed that GVRP and IIVTP could form hydrogen bonds with the S1, S1′ and S2 pockets of ACE. The shared amino acid residues in close contacts (within 3.5 ?) with ACE were His513, Ala354 and Glu384. Based on the?LC-MS/MS?and the molecular docking technology, ?a method to rapidly identify and select active ACE inhibitory peptides from peptides mixture was established. The stable binding of active peptides to ACE and the inhibitory mechanism of its ACE activity were also reflected in this study. The finding of this study may provide further references for studies on ACE inhibitory peptides.
Purification and Enzymatic Characterization of Thermolabile Type I Pullulanase from Bacillus amyloliquefaciens
GAO Zhaojian, HU Xinqiang, SONG Yulin, DING Feihong, ZHAO Yifeng, CHEN Teng
2021, 42(12):  130-137.  doi:10.7506/spkx1002-6630-20200219-194
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A pullulanase (named PulBa) from Bacillus amyloliquefaciens HxP-21 was isolated and purified, and its enzymatic properties were studied to provide a theoretical basis for the application of pullulanase in starch processing. The pullulanase was isolated and purified from the fermentation broth of strain HxP-21 by sequential ammonium sulfate precipitation, anion exchange chromatography and dextran gel filtration chromatography. The yield of PulBa was 53.2%, and the procedure resulted in a 20.8-fold purification and a specific enzyme activity of 176.5 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) measured PulBa to be electrophoretically pure, with a molecular mass of 51.2 kDa. PulBa had high enzyme activity in the range of 45–70 ℃ and pH 3–6, with optimum temperature of 55 ℃ and optimum pH of 4.5. PulBa also exhibited good pH stability and thermostability. More than 80% of its initial activity was retained after being incubated at 40–70 ℃ for 120 min. PulBa was highly stable over an acidic pH range of 3-7, and more than 60 U/mL of its activity was retained after 6 h incubation under these pH conditions. PulBa showed different sensitivities to various metal ions and chemical reagents, and Mg2+ and Ca2+ were able to significantly enhance its enzyme activity. The most suitable substrate for PulBa was pullulan, and it also showed hydrolytic activity on potato amylopectin, corn amylopectin, soluble starch and glycogen, but no effect on α-cyclodextrin and β-cyclodextrin and amylose. When pullulan was used as a substrate, its Km and Vmax were 1.34 mg/mL and 24.6 μmol/(min·mg), respectively. Results indicated that PulBa was a typical type I pullulanase. Thin layer chromatography (TLC) further demonstrated that PulBa specifically cleave the α-1,6-glycosidic bonds of amylopectin to produce maltotriose. Therefore, PulBa had high thermostability and acidic pH tolerance, making it a promising candidate for application in biotechnological industries such as starch processing.
Identification of 4-Ethylguaiacol Producing Yeast and Its Application in Soy Sauce Brewing
ZOU Mouyong, HE Liqin, SUN Qixing, ZHU Xingui
2021, 42(12):  138-144.  doi:10.7506/spkx1002-6630-20200317-259
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A yeast strain named A10-2 producing 4-ethylguaiacol was isolated from soy sauce mash. This strain was identified as Wickerhamiella versatilis by morphological observation, physiological and biochemical test, 18S rDNA ITS sequencing and homology sequence analysis. W. versatilis A10-2 was able to tolerate 18% NaCl and grow normally in soy sauce mash while synthesizing 4-ethylguaiacol. Using ferulic acid as the substrate, A10-2 produced the intermediate product 4-vinylguaiacol through decarboxylation by ferulic acid-decarboxylase, which was subsequently derived into 4-ethylguaiacol by 4-vinylguaiacol reductase. Strain A10-2 was capable of producing 4-ethylguaiacol (0.63 mg/mL) from the soy sauce background precursor, which was increased by 19.1 times with addition of 50 mg/L ferulic acid when compared with the control group. Meanwhile, it was found that the mellow aroma and sauce flavor was enhanced, and the saltiness was mitigated. This study highlights the promising potential of W. versatilis A10-2 for flavor improvement of fermented condiments such as soy sauce and sauce paste.
Analysis of Bacterial Community Succession and Bacterial Sources during Fermentation of Chinese Light/Sauce-Flavored Liquor in Winter
ZUO Qiancheng, HUANG Yongguang, ZHU Jiahe, MA Caifei
2021, 42(12):  145-152.  doi:10.7506/spkx1002-6630-20200420-251
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In this study, high-throughput sequencing was used to analyze the bacterial community structure during the fermentation of Chinese light/sauce-flavored liquor. A total of 14 phyla and 113 genera were detected in the fermented grains. At the beginning of fermentation, Firmicutes, Proteobacteria and Cyanobacteria were dominant with an average relative abundance of higher than 10%. As the fermentation progressed, Firmicutes gradually evolved into the only dominant bacterium. The top 10 most abundant bacterial genera during the fermentation process were Lactobacillus, Bacillus, Weissella, Acetobacter, Gluconobacter, Pediococcus, Kroppenstedtia, Staphylococcus, Enterobacter, and Scopulibacillus. The bacterial community finally evolved to be dominated by Lactobacillus only. In order to explain the sources of bacteria in the fermented grains during thin-layer stacking fermentation, the bacterial community structure in koji was analyzed. Samples without adding koji was set as a control. The bacterial community structure in the fresh and fermented grains as well as the control sample was further analyzed. The results showed that 31.67% of the bacterial genera in the fermented grains came from the environment, 50% from koji, 23.33% from both koji and the environment, and only 5% from the environment. The dominant bacterium Gluconobacter was exclusively from the environment. This study will help in understanding the microecology of bacteria involved in liquor fermentation, which is of great significance to improve the quality, safety and controllable production of liquor.
Component Analysis
Effect of Electron Beam Irradiation with Different Doses on Flavor of Lateolabrax japonicus Meat
LU Jiafang, ZHU Yukang, XU Dalun, ZHANG Jinjie, HUANG Tao, LI Chao, ZHANG Han, YANG Wenge
2021, 42(12):  153-158.  doi:10.7506/spkx1002-6630-20191104-046
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This study evaluated the effect of different electron beam irradiation doses (1, 3, 5 and 7 kGy) on the flavor of Lateolabrax japonicus meat as assessed based on the contents of volatile flavor substances, free amino acids and flavor nucleotides. It turned out that the relative content of hydrocarbons decreased while the relative contents of aldehydes and ketones increased with the increase of irradiation dose. According to the results of electronic nose analysis, the odor changed significantly after irradiation, but it was similar between the 1 kGy and 3 kGy treatment groups, and between the 5 kGy and 7 kGy treatment groups. With the increase of irradiation dose, the content of inosine monophosphate (IMP) increased continuously, whereas the ratio between amino acids responsible for a pleasant flavor and total free amino acids, the content of guanosine monophosphate (GMP) and equivalent umami concentration (EUC) initially increased with irradiation dose up to 3 kGy and then decreased. Thereby, pretreatment with a dose of 3 kGy of electron beam irradiation could kill bacteria in the fish meat while maintaining its flavor to the maximum extent. These results may provide a basis for the preservation of L. japonicus meat by irradiation.
Variation of Naringin Content in Young Guanxi Honey Pomelo (Citrus grandis) Fruit
HU Yang, YAN Xing, WU Ling, LI Lijun, YANG Yuanfan, DU Xiping, WENG Shuyi, NI Hui, LI Qingbiao
2021, 42(12):  159-165.  doi:10.7506/spkx1002-6630-20200423-307
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This study investigated the effects of planting altitude, fruit development stage, fruit location, storage time, and heat drying on the naringin content in young Guanxi honey pomelo fruit as determined by high performance liquid chromatography (HPLC) for the purpose of exploring the optimal harvest time of young Guanxi pomelo fruit to be used for naringin extraction. The results showed that the content of naringin in young Guanxi pomelo fruit planted in the high-altitude area (668 m) was higher than that grown in the low-altitude area (40 m). Naringin contents in the albedo and flavedo decreased with fruit growth. The content of naringin in young fruit picked at the same time increased ceaselessly as the color changed from green to yellow during storage. Heat drying treatment significantly reduced the content of naringin in fresh young fruit. The young Guanxi pomelo fruit at stage I grown in the high altitude area had the highest naringin content after complete yellowing during storage, with values of 28.3% and 26.5% in the epidermis and albedo, respectively. This study provides an experimental basis for the preparation of naringin from young Guanxi pomelo fruit.
Characteristic Aroma Components in Ripe Zijuan Tea during Artificial Fermentation
SU Dan, HUANG Ganghua, LI Yali, REN Hongtao, BAO Yunxiu, ZHOU Hongjie
2021, 42(12):  166-172.  doi:10.7506/spkx1002-6630-20200528-347
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In order to explore the role of artificial fermentation in the formation of the volatile flavor of ripe Zijuan tea, seven samples from the same batch were collected at different processing stages from the raw material piled up (day 0) to the end of fermentation (day 42) to analyze their volatile components by headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). A total of 92 volatile components were identified including alcohols, ketones, aldehydes, hydrocarbons, acids, phenol ethers, and oxygen-containing heterocyclic compounds. The fermentation process enriched the aroma of the tea. According to correlation analysis, the period from day 7 (F1) to day 35 (F5) was key to forming the aroma characteristics. Aroma qualitative analysis and sensory evaluation indicated that dehydrolinalool, β-caryophyllene, and myrrholene were the signature contributors to the spicy aroma of Zijuan tea. During the fermentation process, a nutty aroma namely bitter apricot kernel-like aroma (benzaldehyde), uniquely produced by fermentation, an animal-like aroma namely milky aroma (p-methoxystyrene) and a microbial fermentation-derived odor were produced. Sun-dried Zijuan tea leaves had floral, fruity and spicy aromas, and during fermentation into ripe Zijuan tea, floral (violet flower-like and rosy), sweet, mild woody and faint spicy aroma characteristics were generated. The results of this study lay the foundation for further research on the relationship between the flavor of ripe Zijuan tea and microbial metabolism.
Effect of Fruit Color and Ripeness on Volatile Distribution in Cherry Tomato (Solanum lycopersicum var. cerasiforme) Fruit
CHENG Guoting, CHANG Peipei, WANG Xinyu, LOU Qianqi, Ahmed H. El-SAPPAH, ZHANG Fei, LIANG Yan
2021, 42(12):  173-183.  doi:10.7506/spkx1002-6630-20200419-244
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To analyze the effect of fruit color and ripening on the volatile profile of cherry tomatoes, the volatile compositions of the inbred lines Jinzhu (orange), Heiyingtao 1 (purple), 1 Hao (pink), and Hongzhenzhu (red), were measured at the mature green, turning and red stages by headspace solid-phase micro-extraction (HS-SPME) and gas chromatography-mass spectroscopy (GC-MS). The volatile content of orange tomato fruit was significantly higher than that of red and pink tomato fruit at each stage. The highest content of volatiles appeared in the orange tomato fruit at the mature green stage and in purple tomato fruits at the turning and red stages, and the lowest content of volatiles was always observed in pink tomato fruits during the whole period. The intensity of volatile odor was determined by the concentration and olfactory threshold of volatiles. 3-Methyl-butanal, (E,E)-24-heptadienal, 1-penten-3-one, hexanal, benzaldehyde, and (Z)-3-hexen-1-ol contributed significantly to tomato odor. The strongest odor was observed in red, orange, and purple tomato fruit at the mature green, turning and red stages, respectively. With fruit ripening, the concentration of total volatile components was increased, and the odor became stronger. In conclusion, fruit color and ripeness have a significant effect on the volatile profile of cherry tomatoes.
Analysis of Important Aroma Components in Pixian Bean Paste by Diethyl Ether Extraction
ZHAO Cong, FAN Wenlai, XU Yan
2021, 42(12):  184-188.  doi:10.7506/spkx1002-6630-20200617-237
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In order to clarify the important aroma substances of Pixian bean paste, different extraction methods were applied to extract the aroma substances in Pixian bean paste, and ether was selected as the optimal extraction solvent. The ether extract was fractionated into an acid-water soluble component and a neutral-alkaline component for analysis by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). A total of 46 aroma substances were identified in Pixian bean paste, including 5 alcohols, 13 volatile organic acids, 7 aldehydes and ketones, 11 phenols, 5 aromatics, 3 heterocyclics, and 2 pyrazines. Six aroma compounds including 3,4-dimethyl-2,5-furandione, crotonicacid, 2,6-dimethoxyphenol, 3-methyl-1,2-cyclopentanedione, γ-butyrolactoneand 3,4-dimethylbenzaldehyde were first identified in Pixian bean paste. Acetic acid, 3-methylbutyric acid, 3-methylvaleric acid, 2-phenylethanol, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (HDMF), phenol, 1-octen-3-one, 3-methylthiopropionaldehyde and eugenol were identified as important aroma substances of Pixian bean paste.
Application of Near Infrared Spectroscopy in the Rapid Detection of Total Sugar Content in Lentinula edodes
LU Jie, TIAN Jing, LIANG Zhenhua, WANG Jinmei, KANG Wenyi, MA Changyang, LI Changqin
2021, 42(12):  189-194.  doi:10.7506/spkx1002-6630-20210115-167
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In order to improve the defects of the method specified in the Chinese national standard to determine the total sugar content in edible fungi (Lentinula edodes), such as long detection time and tedious steps, near infrared spectroscopy (NIR) combined with partial least square (PLS) was adopted to establish a rapid method for determining the total sugar content of L. edodes and the predictive model parameters were optimized. In this study, a total of 106 samples were collected, 13 of which were randomly assigned into the validation set for testing model reliability, while the remaining 93 were assigned into the calibration set. In the calibration set, according to the studentized residuals and leverage value, 65 lots of samples with the typical characteristics of L. edodes were selected to optimize spectral range and the number of PLS factors for the establishment of a quantitative model for predicting the total sugar content in L. edodes. It was found that the optimal spectral preprocessing method was multiplicative scatter correction (MSC) combined with second derivatives (SD), and the predictive model with optimal results was constructed using the spectral range of 4 000–10 000 cm-1 and 10 PLS factors, with a root mean square error of calibration (RMSEC) of 1.393 and a root mean square error of prediction (RMSEP) of 1.557 as well as a correlation coefficient for calibration (R2) up to 0.940 04 and a relative percent deviation (RPD) up to 4.08. Finally, the verification results showed the model-predicted value had a good linear relationship with the experimental value with no significant difference between them (P = 0.993). In conclusion, the NIR prediction model could accurately predict the total sugar content of L. edodes.
Quality Analysis and Comprehensive Evaluation of Chinese Congou Black Tea
LIU Panpan, ZHENG Pengcheng, GONG Ziming, YE Fei, FENG Lin, GAO Shiwei, WANG Xueping, WANG Shengpeng, TENG Jing, ZHENG Lin, GUI Anhui
2021, 42(12):  195-205.  doi:10.7506/spkx1002-6630-20200611-150
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This study aimed to explore the characteristics of the contents of the major quality components in Congou black tea and to develop a comprehensive evaluation method for the taste quality of tea infusion. A total of 17 black teas from the main tea-producing regions of China were measured for quality indicators including polyphenols, amino acids, tea pigments and volatile components and comprehensive evaluation of their quality was investigated by principal component analysis (PCA), cluster analysis (CA) and partial least squares discriminant analysis (PLS-DA). The results showed that the composition and contents of quality components varied significantly among the different black teas. Polyphenols and their oxidation products were the major quality components. The ratio of the sum of theaflavins and thearubigins to theabrownins was 1.05–1.12, which constituted the material basis of the fresh and mellow taste and the red bright color of tea infusion. Yinghong, Haihong, Xinyanghong, Yihong, Yuehong, Qianhong and Qihong had soluble sugar contents higher than 4%, which was an important material basis of the sweet and mellow taste of tea infusion. The flavonoid content in large-leafed black tea (Dianhong, Yinghong and Haihong) was higher than 2.7%. Alcohols and aldehydes were the principal aroma compounds, especially terpene alcohols, responsible for the sweet and floral aroma and more abundant than other aroma compounds. PCA was used to establish a taste quality evaluation model. The model prediction was consistent with the sensory evaluation, indicating a significant correlation between them (P < 0.01) with a correlation coefficient of 0.735. The contents of theaflavins and tea polyphenols were important indicators of Congou black tea quality. Cluster?analysis?revealed?that?Minhong belonged to one class, Dianhong, Yinghong and Haihong shared?similar?quality?characteristics,?while?other middle- and small-leafed black teas belonged to a third class. PLS-DA showed a clear discrimination between the large-leafed black tea and the middle- and small-leafed black teas with eight differential quality indicators. Notably, inalol, geraniol and hexanal could be used to discriminate between the two classes of black tea. The results of this study enrich the basic theory of Congou black tea flavor quality and provide new ideas and methods for the scientific evaluation of black tea quality.
Analysis of Flavor Characteristics of Traditional Fish Sauce from Different Regions
JIANG Jinjin, YAN Jing, ZHENG Yuxi, OU Aifen, REN Fang, CHEN Qing, CHEN Fenghua
2021, 42(12):  206-214.  doi:10.7506/spkx1002-6630-20200825-335
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In order to study the flavor characteristics of fish sauce produced in different areas, modern flavor analysis techniques such as headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS), electronic nose, headspace solid phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS), amino acid analyzer and sensory quantitative description analysis (QDA) were used to analyze the volatile and nonvolatile flavor compounds of fish sauce from Shantou and Dongguan, Guangdong province and Fujian province in China as well as Thailand and Vietnam. Their relative odor activity values (ROAVs) were calculated as well. The results showed that GC-IMS and electronic nose could identify the differences in the volatile flavor compounds of samples from different regions. According to QDA, fish sauce from Thailand, Vietnam and Shantou had overall similar volatile flavor components, while they were quite different from those from the other two regions. Totally 37 flavor compounds were identified by GC-IMS, including aldehydes, alcohols, esters, acids and sulfur compounds. In addition, 47 volatile compounds were identified by HS-SPME-GC-MS. Volatile acids accounted for 78.13% and 42.92% of the total volatiles in Shantou and Vietnam fish sauce, respectively. Nitrogenous compounds account for a large proportion of the total volatiles in Vietnamese fish sauce. The content of alcohols was the highest in Fujian fish sauce and the lowest in Vietnam fish sauce. The major flavor components in Thai fish sauce were aromatic substances. Nitrogenous compounds (pyrazines and furans) and esters were the most abundant in Vietnamese fish sauce as the characteristic flavor compounds. Shantou fish sauce had the highest content of sulfur compounds, contributing to the characteristic flavor of Shantou fish sauce, including methyl sulfide and 3-methylthiopropionaldehyde. Amino acids were the precursors of the volatile flavor compounds. The total content of free amino acids was ranked as follows: Vietnam fish sauce > Shantou fish sauce > Thailand fish sauce > Fujian fish sauce > Dongguan fish sauce. However, the proportion of delicious amino acids in Dongguan fish sauce was the highest, with glutamic acid accounting for 93.35% of the total amount. Thailand fish sauce had the highest proportion of sweet amino acids. The content of sulfur-containing amino acids and taurine in Shantou fish sauce was the highest. The difference in the contents of free amino acids may account for the difference in characteristic volatile flavor compounds from another perspective.
Broadly Targeted Metabolomics Analysis of Differential Metabolites between Light Yellow and Purple Walnut Endocarps
FANG Xiansheng, WU Tao, XIAO Liangjun
2021, 42(12):  215-221.  doi:10.7506/spkx1002-6630-20191108-099
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In order to understand the major metabolites of walnut endocarp and compare the difference in the metabolite compositions of walnut endocarps of different colors, broadly targeted metabolomics based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine and compare the metabolite compositions of light yellow and purple walnut endocarps from the same habitat. The obtained data were analyzed by cluster analysis, correlation analysis and orthogonal partial least squares discriminant analysis (OPLS-DA). The results showed that a total of 680 metabolites belonging to 23 classes were detected in the endocarps, of which 658 metabolites were shared by both, while 670 metabolites were present in the yellowish one and 668 metabolites in the purple one. Totally 244 differential metabolites were identified between the two endocarps, accounting for 37.08% of the total metabolites common to them. Furthermore, 93 (38.11%) of the 244 differential metabolites had higher content in the purple endocarp than that in the yellowish one, whereas 151 metabolites (61.89%) showed the opposite result. The significantly differentially expressed 244 metabolites were mainly distributed in 20 metabolic pathways, and the first five dominant ones were the secondary metabolite biosynthesis pathway, flavonoid biosynthesis pathway, flavone and flavonol biosynthesis pathway, environmental microorganism-related metabolic pathway and phenylpropanoid biosynthesis pathway, in which 90 (36.89%), 48 (19.67%), 37 (15.16%), 30 (12.30%) and 23 (9.43%) metabolites were involved, respectively. The types and contents of differential anthocyanin metabolites in the purple endocarp were higher than in the yellowish one, which may be one of the major causes the different colors of walnut endocarp.
Determination of Phenolic Acids and Their Distribution in Freeze-Ground Aerial Parts of Different Sweetpotato Varieties by Ultra-High Performance Liquid Chromatography
ZHAO Shan, ZHONG Lingli, LI Xi, LEI Xinyu, ZHENG Xingguo, GUO Ling’an, LEI Shaorong, ZHOU Hong, HUANG Shiqun, FENG Junyan
2021, 42(12):  222-232.  doi:10.7506/spkx1002-6630-20200519-222
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In this study, an ultra-high performance liquid chromatography (UPLC) method was established to analyze the distribution of phenolic acids in aerial parts of 13 sweetpotato varieties. The results showed that addition of 0.2% sodium hydrogen sulfite to the extraction solvent could improve the recovery and stability of caffeic acid (CA) and caffeoylquinic acids (CQAs). Among the tested pretreatment methods, freeze grinding resulted in the highest content of phenolic acids, followed by freeze drying, oven drying, sun drying and homogenization. The UPLC method was fast, stable and highly sensitive with spiked recoveries of 99.5%?103.1%, and precision (expressed as relative standard deviation, RSD) of 0.3%?1.2%. The limit of detection (LOD) and the limit of quantification (LOQ) were 3.6?21.4 and 12.1?71.3 ng/mL, respectively. The contents of total CA and CQAs including CA, 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA and 3,4,5-triCQA in the leaves, petioles and stems of sweatpotato ranged from 18.0?44.8, 0.7?11.9, and 0.3?9.7 g/kg dry sample, respectively. The contents of phenolic acids in different sweetpotato varieties were obviously different from each other, with dicaffeoylquinic acids (diCQAs) being the most abundant phenolic acid in all varieties. Moreover, diCQAs were the most abundant in the leaves among the three parts.
Detection and Identification of Phenyllactic Acid in Chinese Vinegar and Its Combined Bacteriostatic Effect with Acetic Acid
NING Yawei, HOU Linlin, YU Tongyue, HAN Panpan, FU Yunan, LI Mingrui, WANG Zhixin, JIA Yingmin
2021, 42(12):  233-241.  doi:10.7506/spkx1002-6630-20200420-261
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In this study, phenyllactic acid (PLA) was isolated and purified from Chinese vinegar and identified by nuclear magnetic resonance (1H-NMR and 13C-NMR). Furthermore, a rapid method was established to analyze PLA in 72 vinegar products marketed in China by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS). The results showed that PLA was detectable in all samples. The concentration of PLA in cereal vinegar was significantly higher than that in fruit vinegar, with average and maximum values of 227.89 and 618.69 mg/L, respectively. The concentration of PLA was closely related to the raw material and fermentation mode. Vinegar made from glutinous rice through solid-state fermentation showed the highest PLA concentration. The time-killing curve method and scanning electron microscopy (SEM) were used to evaluate the antibacterial effect of PLA combined with acetic acid, and the results showed that they had a synergistic antibacterial effect on a variety of spoilage and pathogenic bacteria in foods, and the combined application of PLA and acetic acid could damage the morphology of Staphylococcus aureus and Salmonella typhimurium, leading to the leakage of intracellular contents from S. aureus and the shrinkage of S. typhimurium cells. This study provides a new scientific basis for elucidating the health benefits of vinegar, and a theoretical basis for the application of PLA in food bio-preservation.
Analysis of Dynamic Changes of Flavor Components during Large-Scale Brewing of Soy Sauce
LIU Xiaoyan, YE Yuehua, QIAN Min, BAI Weidong, HUANG Hancong, YANG Hong, HE Yilong
2021, 42(12):  242-247.  doi:10.7506/spkx1002-6630-20200514-167
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In order to provide valuable information for improving the flavor of large-scale fermented soy sauce products, the dynamic changes of flavor components in high-salt dilute-state soy sauce during the mechanized brewing process were studied. The types and relative contents of flavor components in the mash at different brewing stages were analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). A total of 210 components were detected, including amino acids and their derivatives, organic acids, sugars and their derivatives, alcohols, esters, amines and other substances. Heat map and principal component analysis (PCA) were used to explore the differences in the flavor of the samples at different brewing stages. The results showed that the fermentation process had a significant effect on amino acids and their derivatives as well as sugars and their derivatives, which contributes to the sweetness of soy sauce, and the sample collected on Day 1 also had a significant effect on organic acids, amines, lipids and other substances, which contribute to the modified flavor. Amino acids and their derivatives were the most abundant flavor compounds in soy sauce.
Safety Detection
Terahertz Spectroscopy for Non-destructive Identification of Quality Change of Edible Oils during Storage
YANG Yufei, LIU Cuiling, WU Jingzhu, SUN Xiaorong
2021, 42(12):  248-254.  doi:10.7506/spkx1002-6630-20191030-330
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In order to evaluate the quality change of edible oils during storage, terahertz attenuated total reflection (THz-ATR) spectroscopy was used for the non-destructive identification of oils with different oxidation degrees. First, the effect of storage conditions on the quality of edible oils was explored, and then according to the national standard definition of edible oil quality, the collected samples were divided into fresh oil, edible oil and inedible oil. Terahertz time-domain spectra of these oils were collected. After data preprocessing, the euclidean distance matching method was used to identify the refractive index spectra of edible oils with an accuracy of 95.65%, and linear discriminant analysis (LDA) was used to identify the absorption coefficient spectra with an accuacy of 91.00%. Comparative analysis demonstrated that both models exhibited desired recognition accuracy. This study may provide a theoretical basis for the analysis of various quality attributes of edible oil by terahertz spectroscopy.
Rapid Detection of Florfenicol Metabolites in Poultry Eggs
PI Jiangyi, HU Jiayong, WANG Wei, LIU Guojiao, LIN Jin, ZHOU Taohong
2021, 42(12):  255-260.  doi:10.7506/spkx1002-6630-20200427-347
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A rapid method for the detection of a marker residue for florfenicol and its major metabolite florfenicol-amine in poultry eggs was established using immunochromatography. Florfenicol and florfenicol-amine were extracted from poultry eggs with ethyl acetate, and florfenicol-amine was derived into florfenicol by dichloroacetic anhydride. Finally, florfenicol was detected by a colloidal gold test strip. The results showed that florfenicol-amine was successfully converted into florfenicol in ethyl acetate, and the detection limits of florfenicol and florfenicol-amine were 1 and 3 μg/kg, respectively. In addition, the method was successfully applied to actual positive egg samples and spiked samples with results consistent with those obtained by the existing national standard method. This method has the characteristics of simple operation, fast detection and low cost, which is suitable for the on-site screening of massive samples, improving food safety supervision efficiency.
Quantitation and Source Analysis of Mineral Oil Hydrocarbons in Traymates
ZHANG Yicai, LIN Qinbao, CHEN Sheng, ZHONG Huaining, CHEN Yanfen, ZENG Ying, WU Zechun, LI Zhong
2021, 42(12):  261-267.  doi:10.7506/spkx1002-6630-20191119-221
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In this study, the quantitative analysis of mineral oil hydrocarbons (MOH) in 15 kinds of traymates was conducted, and source analysis was performed on one sample with known printing ink. The sample pretreatment was accomplished by off-line solid phase extraction using a mixed solution (n-hexane:ethanol = 1:1, V/V) as the extraction solvent. Mineral oil saturated hydrocarbon (MOSH) and mineral oil aromatic hydrocarbon (MOAH) were separated and purified on 0.3% silver nitrate silica gel column, quantified by gas chromatography-flame ionization detection (GC-FID), and qualified by gas chromatography-mass spectrometry (GC-MS). The results showed that MOH contents of four kinds of traymates purchased online were higher than those of traymates collected from a fast food restaurant. The maximum content of MOSH was 3 397.6 mg/kg, and the maximum content of MOAH was 374.6 mg/kg, which both exceed the limit value. Through source analysis, it was confirmed that MOH in printed traymates came from the ink. The limit of detection (LOD) and limit of quantitative (LOQ) of the proposed method were 2.73 and 8.19 mg/kg, respectively. The spiked recoveries were 92.1%–115.1%, with relative standard deviation (RSD) of 3.12%–11.85%. This method proved to be accurate, easy to operate and useful for quantitation and source analysis of MOH in food contact paper.
Matrix Applicability of Chinese National Standard Methods for Determination of Some B Vitamins in Infant Formula Foods for Special Medical Purposes
GUO Meijuan, SHI Guohua, ZHANG Bin, LI Yangweiyu, ZHANG Yan, ZHANG Lantian
2021, 42(12):  268-274.  doi:10.7506/spkx1002-6630-20200910-134
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In this study, the applicability of the Chinese national standard methods to detect vitamin B1, vitamin B2, vitamin B6, and niacin in infant formula for special medical purposes was explored. High performance liquid chromatography (HPLC) was used to determine vitamin B1, vitamin B2, vitamin B6 and niacin in eight samples of three types of infant formula foods for special medical-purpose and infant formula milk powder quality control samples in seven laboratories. The recovery rate was also measured. The results showed that the national standard methods were suitable for the determination of vitamin B1, vitamin B2, vitamin B6, and niacin in infant formula milk powder, and for the determination of vitamin B1, vitamin B2 and vitamin B6 in infant formula for special medical purposes. The recovery rate of vitamin B1 was low. The recovery rate of vitamin B2 in deep hydrolysis formula and amino acid formula products was low, and the test data for niacin was generally unsatisfactory. This study provides technical support for the establishment of methods for testing infant formula for special medical purposes.
Simultaneous Determination of Abietic Acid and Dehydrogenated Abietic Acid in Livestock and Poultry Skin by Liquid Chromatography-Tandem Mass Spectrometry
SHEN Jincan, HUANG Changxiong, XIAO Chengui, WU Zhangjie, ZHAO Fengjuan, KANG Haining, YUE Zhenfeng
2021, 42(12):  275-280.  doi:10.7506/spkx1002-6630-20200428-378
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A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to determine abietic acid and dehydrogenated abietic acid in livestock and poultry skin. Samples were extracted with acetonitrile, and the extract was purified with an HLB solid-phase extraction (SPE) cartridge before analysis by LC-MS/MS. The chromatographic separation was accomplished on a C18 column (100 mm × 2.1 mm, 2 μm) with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid-methanol solution (10:90, V/V) at a flow rate of 0.3 mL/min. The analysis was achieved by using an electrospray ionization (ESI) source in the positive ion mode with multiple reaction monitoring. The linear range, detection limit and quantification limit were 2.0–100 μg/L, 1.1 μg/kg, and 3.6 μg/kg for abietic acid; and 5.0–250 μg/L, 3.9 μg/kg, and 13 μg/kg for dehydrogenated abietic acid, respectively. The recovery rates of spiked samples at three different concentration levels of 5.0, 10 and 50 μg/kg for abietic acid and of 20, 40 and 200 μg/kg for dehydrogenated abietic acid were in the range of 81.7%–93.7%, with relative standard deviations (RSDs) within 12%. The developed method was successfully applied for the analysis of several real samples. Both abietic acid and dehydrogenated abietic acid were detected in some samples including duck skin dehaired with rosin glyceride. This analytical method proved to be simple, fast, sensitive and reliable, and could be used for confirmatory and quantitative analysis of abietic acid and dehydrogenated abietic acid in livestock and poultry skin.
Preparation and Application of Dummy Template Molecularly Imprinted Polymer in the Detection of Triazines in Fruits and Vegetables
GONG Bichuan, HU Qiuhui, SU Anxiang, XU Hui, LIU Jianhui, PEI Fei, YANG Wenjian
2021, 42(12):  281-287.  doi:10.7506/spkx1002-6630-20201203-039
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Molecularly imprinted polymers (MIPs) were prepared by bulk polymerization with cyromazine as the virtual template molecule, methacrylic acid (MAA) as the functional monomer, trimethylolpropane trimethacrylate (TRIM) as the ternary crosslinking agent, and acetonitrile as the porogen. The optimum molar ratio of cyromazine to MAA to TRIM was determined to be 1:4:4. The as-prepared polymer was evaluated and characterized by equilibrium adsorption test, infrared spectroscopy, and scanning electron microscopy (SEM). The adsorption performance test and the Scatchard equation showed that the MIP had the best adsorption capacity toward prometryn. Two types of adsorption sites existed, and the maximum apparent binding capacity was 3.13 mg/g. A molecularly imprinted solid phase extraction (MISPE) column was prepared with the MIP, and the elution conditions were optimized. On this basis, a method for the determination of four triazine pesticides in cucumber, apple and corn was established by combining high performance liquid chromatography (HPLC). The results showed that 10% acetic acid-methanol was the best eluent for the target pesticides. The detection limits of this method were 0.01–0.03 ng/mL, and the average recoveries were 81.5%–104.3%, with relative standard deviations (RSDs) of 1.3%–5.8% (n = 3). The method could meets the requirements for the detection of pesticides with greatly improved efficiency and accuracy, which will provide theoretical support for the detection of triazine pesticide residues.
Determination of Mineral Oil Saturated Hydrocarbons and Aromatic Hydrocarbons in Chocolate by On-line Liquid Chromatography-Gas Chromatography
XIE Yaoqing, LIU Lingling, LI Bingning, OUYANG Jie, WU Yanwen
2021, 42(12):  288-294.  doi:10.7506/spkx1002-6630-20200125-275
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A method for the determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in chocolate was established using on-line liquid chromatography-gas chromatography (LC-GC). Samples were extracted with n-hexane while sonicating at 60 ℃ for 20 min, and the collected supernatant was treated with silica gel for lipid removal. Then epoxidation was employed with m-chloroperoxybenzoic acid in order to oxidize the endogenic olefins in samples. Finally, the separation and determination were conducted by LC-GC, in which LC was used to retain both oil and olefin epoxide, and the isolated MOSH and MOAH were separately introduced into the two parallel GC channels through the LC-GC interface consisting of transferring valves, retention gaps and solvent release valves and detected with a flame ionization detector. The calibration curves for MOSH and MOAH were linear in the range of 0.5–85.5 mg/L (R2 = 0.998), and the limit of quantification (LOQ) of the method was 0.5 mg/kg. The spiked recoveries were in the range of 80.3%–93.8% with relative standard deviation (RSD) of 1.58%–8.22%. This method was used to determine the mineral oil in 28 commercial chocolate products, of which 20 samples were found to contain MOSH in the range of 1.83–22.23 mg/kg and 1 sample was found to contain MOAH at 1.57 mg/kg. The analysis of on-line LC-GC chromatograms demonstrated that some chocolate products contained polyolefin oligomeric saturated hydrocarbons (POSH) migrating out from plastic packages. POSH and MOSH could not be separated from each other by on-line LC-GC, so the measured results were actually the total amount of MOSH and POSH.
Construction of Down-conversion Fluorescence-Aptamer Immunochromatographic Strip for Rapid Detection of Aflatoxin B1
WANG Zouluqi, LI Lihuang, LI Danyang, AI Chaochao, REN Lei, SUN Benqiang
2021, 42(12):  295-301.  doi:10.7506/spkx1002-6630-20191030-337
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In this study, a down-conversion fluorescence-aptamer immunochromatographic strip was constructed for the rapid and efficient detection of aflatoxin B1 (AFB1) in foods. The presence of AFB1 in the system will weaken the binding ability of down-conversion-aptamer fluorescent nanoparticles to the hapten AFB1 when down-conversion-aptamer fluorescent nanoparticles reach the T-line, thus leading to the attenuation of down-conversion fluorescence signal and consequently highly efficient detection of AFB1. In the range of 1–40 ng/mL, the concentration of AFB1 had a good linear relationship with the fluorescence signal, showing a correlation coefficient of 0.994, and the detection limit for AFB1 was 0.287 ng/mL. By taking advantage of the long-lived luminescence and the near infrared fluorescence characteristics of rare earth doped fluorescent nanoparticles, this method effectively reduced the interference of biological background fluorescence and improved the specificity of the detection system, making it a promising candidate for application in the rapid and sensitive detection of AFB1.
Rapid Screening of 27 Plant Growth Regulator and Antibiotic Residues in Bean Sprouts by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
YAN Weihua, ZHOU Ying, GUO Haowei, WANG Xiaoxiao
2021, 42(12):  302-308.  doi:10.7506/spkx1002-6630-20191224-277
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Objective: An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for rapid screening of 27 plant growth regulator and antibiotic residues in bean sprouts. Methods: The analytes were separated on an Agilent Eclipse Plus-C18 chromatographic column (2.1 mm × 100 mm, 1.8 μm) using gradient elution with 0.01% formic acid as mobile phase A and methanol as mobile phase B at a flow rate of 0.4 mL/min. Qualitative and quantitative analysis were conducted by tandem mass spectrometry in a multiple reaction monitoring (MRM) mode. Results: The method had a wide linear range and good linearity, with correlation coefficient (R2) ≥ 0.998 5, and the precision expressed as relative standard deviation (RSD) ranged from 1.1% to 3.5% (n = 6). The recoveries at three spiked concentrations ranged from 80.5% to 114.9%. The limits of detection (LOQ) and limits of quantitation (LOQ) were 0.5–50 and 1.5–150 μg/kg, respectively. Conclusion: This method is rapid, accurate and highly sensitive, and can be used for simultaneous detection of the 27 plant growth regulator and antibiotic residues in bean sprouts.
Spectroscopic Determination of Trace Nitrite in Water and Meat Following Solid Phase Extraction
LI Chaoyi, LI Long, ZHU Ying, WU Ting, DU Yiping
2021, 42(12):  309-314.  doi:10.7506/spkx1002-6630-20191104-043
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A spectroscopic method was used for the quantitative analysis of sodium nitrite after solid phase extraction. The analytical wavelength was set at 472.5 nm, and the derivatization conditions were optimized as follows: pH 4.74, derivatization time 15 min, and derivatization reagent composition sodium nitrite:p-aminobenzenesulfonic acid:1-naphthol molar ratio of 1:3:3. Enrichment was accomplished using a nylon membrane with a loading volume of 10 mL. In the concentration range of 0.002–0.030 mg/L, the absorbance (y) had a quadratic relationship with nitrite concentration (x), expressed as y = ?268.76x2 + 14.55x + 0.03, with a correlation coefficient (R2) of 0.999, and the limit of detection (LOD) of the presented method was 1.29 μg/L. The method was applied to the detection of NaNO2 in tap water with spiked recoveries of 91.3%–114.0%, and relative standard deviations (RSDs) of 4.21%–7.90%. The results of this method for sodium nitrite in bacon agreed with those of ion chromatography, with standard deviations of 8.51% and 9.98% in parallel experiments on American-style and Shuanghui-branded bacons, respectively. This study has provided a rapid, sensitive, accurate and precise method for the determination of NaNO2 with high selectivity.
Determination of Chlorate and Perchlorate in Infant Formula Powder by Ion Chromatography Coupled with Tandem Mass Spectrometry Based on Captiva EMR-Lipid Clean-up
ZHANG Hao, JIANG Xiaohong, HUO Zongli
2021, 42(12):  315-321.  doi:10.7506/spkx1002-6630-20191112-149
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The aim of this study was to develop a method for the determination of chlorate and perchlorate in infant formula powder by using non-suppressed ion chromatography-tandem mass spectrometry. After being dissolved in pure water, proteins in the samples were precipitated with acetonitrile. After centrifugation, the supernatant was cleaned up by using Captiva EMR-Lipid, which could remove lipids from the matrix. The resulting filtrate was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). An aqueous solution containing 0.7 mol/L methylamine and acetonitrile were applied as the mobile phase. The separation was performed on an IonPac AS16 column. Data were collected by multi-reaction monitoring (MRM) in electrospray negative ionization mode. The quantitative analysis was carried out by the internal standard method. The calibration curves for chlorate and perchlorate showed a good linearity in the range of 5–200 and 1.25–50 μg/L with a correlation coefficient greater than 0.999, respectively. The recoveries of chlorate and perchlorate in positive samples at three different spiked levels were in a range of 98.8%–101% and 97.2%–112% with relative standard deviations (RSD) of 1.1%–2.6% and 1.3%–6.8%, respectively. The limits of detection (LOD) and limits of quantification (LOQ) of this method were 4.8 and 16 μg/kg for chlorate, and 1.6 and 5.0 μg/kg for perchlorate respectively. This method is suitable for the routine detection of chlorate and perchlorate in infant formula powder with the advantages of simple pretreatment and high accuracy.
Rapid Detection of Murine-Derived Ingredients in Mutton Products Using Loop-Mediated Isothermal Amplification
CHEN Zhenjin, ZHANG Huang, SHI Lei, YE Lei, CAI Kaixian, YAN Donglin, WEI Tao, XIE Naizhen, CHEN Xiaocong
2021, 42(12):  322-327.  doi:10.7506/spkx1002-6630-20201016-146
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Loop-mediated isothermal amplification (LAMP) was employed for the qualitative detection of murine-derived ingredients in mutton products. Specific primers targeting the murine mitochondrial genes (rat COX gene KP244683.1 and mouse 16S rRNA gene KY018919.1) were designed, and the reaction conditions were optimized to determine the optimal reaction system. The specificity, sensitivity, and stability were evaluated using simulated mixed samples. LAMP was used to detect commercially available mutton products, and the obtained results were compared with those obtained with real-time PCR. The results showed that the method established in this study had good specificity and stability. The limits of detection (LODs) for rat- and mouse-derived ingredients were 0.1% and 0.5%, respectively. The results of LAMP were highly consistent with those of real-time fluorescent PCR. Therefore, the LAMP method proved simple, efficient, and accurate, which could provide a new choice for rapid diagnosis of murine meat adulteration and primary diagnosis, and could be used as a new rapid detection method for murine-derived ingredients in mutton products.
Simultaneous Determination of Eight Sweeteners in Dry Food Wrappings by Using Matrix Matched Solvent Calibration-High Performance Liquid Chromatography-Tandem Mass Spectrometry
LIAO Huiyun, WU Yang, MA Mengjie, MAO Shurui, ZHU Huaiyuan, CHEN Jingbo, ZHANG Hua, CAO Yi, ZHU Longjie, YUAN Yilai
2021, 42(12):  328-333.  doi:10.7506/spkx1002-6630-20191128-278
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A method for simultaneous determination of eight sweeteners in dry food wrappings, acesulfame, saccharin sodium, sodium cyclamate, sucralose, aspartame, neohesperidin dihydrochalcone, neotame, and stevioside, was developed by using matrix matched solvent calibration-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The matrix matching solvent was prepared by using the base paper. The analytes in samples were ultrasonically extracted with triethylamine buffer solution, separated on an XDB-C18 column by gradient elution using methanol-5 mmol/L ammonium acetate solution as the mobile phase. The mass spectrometry was performed in the positive and negative ion switching mode by multiple reaction monitoring mode with an electrospray ion source, and an external standard method was used for quantification. The results showed that under the optimal conditions, the developed method exhibited excellent linearity for the eight sweeteners (R2 ≥ 0.995). The limits of detection (LODs) and quantification (LOQs) were in the range of 0.13–2.50 mg/kg and 0.43–8.33 mg/kg, respectively. The spiked recoveries ranged from 85.49% to 108.81%, with relative standard deviations (RSDs) of 2.24%–6.03%. When the method was used to test ten different commercial dry food wrappings for different brands of candy and chocolate, one sample was detected to contain acesulfame and neotame at 0.56 and 0.015 g/kg, respectively. This method had the advantages of rapidity, high efficiency and good reproducibility, making it a promising candidate for simultaneous quantitative determination of the eight sweeteners in dry food wrappings.