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Table of Content

25 December 2020, Volume 41 Issue 24
Food Chemistry
Preparation and Stability of Calcium-Chelating Peptide and Its Absorption Characteristics in Caco-2 Cells
QIAN Yuewei, XU Hanlin, LÜ Qiyan, CHEN Zhichao, TENG Hui, CHEN Lei
2020, 41(24):  1-8.  doi:10.7506/spkx1002-6630-20200706-078
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In this study, pepsin, trypsin, and chymotrypsin were used in combination to prepare eel bone collagen peptide (EBCP) with high calcium-binding ability. The optimal preparation conditions of EBCP-calcium chelate that provided maximum calcium chelation rate of over 80% were determined as follows: 40 ℃, pH 7.5, 40 min, and mass ratio of peptide to calcium 5:1. Fluorescence, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopic (SEM) observation indicated that the carboxyl and amino nitrogen atoms of EBCP could chelate calcium to form EBCP-Ca. EBCP-Ca was stable toward temperature, pH and simulated gastrointestinal digestion. In addition, this chelate exhibited low toxicity to mouse Caco-2 cells. In addition, the Caco-2 cell monolayer model was used to evaluate the calcium uptake-promoting activity of EBCP-Ca in vitro. The effects of EBCP-Ca concentration and culture time on the calcium content and alkaline phosphatase (AKP) activity in Caco-2 cells were investigated. It was demonstrated that EBCP-Ca enhanced the AKP activity, thereby promoting the absorption of calcium in the human gastrointestinal tract. This research provides a scientific basis for the development of new calcium supplements and the high-value utilization of eel bones.
Effects of Bamboo Leaf Extract on the Quality and Nitrite Content of Low-Nitrite Western-Style Smoked Ham
ZHOU Yajun, LI Wenlong, CHEN Yan, WANG Shujie
2020, 41(24):  9-15.  doi:10.7506/spkx1002-6630-20200621-282
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In order to study the effect of bamboo leaf extract on improving the quality of low-nitrite western-style smoked ham and reduce nitrite content, the color, texture, sensory score, pH, thiobarbituric acid reactive substances (TBARS) value, total volatile basic nitrogen (TVB-N) content and residual nitrite content of ham samples added with 0.01%, 0.02%, 0.03%, 0.04% and 0.05% bamboo leaf extract were evaluated. Samples added with only 0.009% and 0.015% sodium nitrite were used as controls for comparison. The results showed that with increasing addition of bamboo leaf extract, the redness value a* of ham increased (P < 0.05), and the level of residual nitrite gradually and significantly decreased (P < 0.05). The maximum removal rate of nitrite of 62.47% was obtained upon addition of 0.05% bamboo leaf extract. In addition, the texture of ham added with bamboo leaf extract during storage was improved compared with that of the controls. Adding more than 0.04% bamboo leaf extract caused no significant differences in hardness, elasticity or chewiness during the entire storage period compared with the control groups (P > 0.05). The TBARS value and TVB-N content of the bamboo leaf extract added samples during storage were significantly lower than those of the control groups (P < 0.05). At the end of the 20-day storage period, the TBARS and TVB-N values of the samples added with more than 0.03% bamboo leaf extract were similar to or even lower than those of the second control group. From these results, it was seen that the bamboo leaf extract can effectively inhibit the spoilage of western-style smoked ham, significantly reduce nitrite residue and improve the color and texture of the product.
Characterization of Rabbit Skin Collagen and Modeling of Critical Gelation Conditions
ZHANG Xiaoliang, ZHANG Yi
2020, 41(24):  16-21.  doi:10.7506/spkx1002-6630-20190619-212
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Collagen was extracted from rabbit skin by acetic acid, and its structure and properties were characterized. The results showed that the extraction yield of rabbit skin collagen (RSC) was 69.18%. Amino acid analysis showed that RSC was composed of 18 amino acids. UV and Fourier transform infrared spectroscopy (FTIR) analyses showed that RSC was characterized as type I collagen; thermal analysis showed that the denaturation temperature of RSC was 35.8 ℃. The RSC gel was prepared by dialysis method, the relationship of gel formation with pH, temperature, collagen concentration and was analyzed rheologically. It was found that the formation of gel was positively correlated with pH and collagen concentration, but was negatively correlated with temperature. After that, a number of experiments were carried out to establish a multivariate nonlinear model for storage and loss moduli versus pH, temperature and collagen concentration. The fitting degree of the model was good. This study can provide a theoretical basis for the formation of collagen gel.
Bioengineering
Analysis of Differential Metabolites in Cabernet Sauvignon Skins from Different Rootstock-Scion Combinations by Non-Targeted Metabolomics
ZHANG Zhijun, LIU Huaifeng, SUN Junli, ZHAO Baolong, PAN Lizhong, HE Wang, LIU Jingjing
2020, 41(24):  22-30.  doi:10.7506/spkx1002-6630-20190617-172
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This study aimed to analyze the differences in the metabolite profiles and related metabolic pathways among different rootstock-scion combinations of Cabernet Sauvignon (CS). Liquid chromatography-mass spectrometry (LC-MS)-based non-targeted metabolomics was used to analyze the metabolites in grape skins from CS grafted to 1103P, 5C, SO4, 3309C, 140R and CS rootstocks. Multivariate statistical analyses including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal-partial least squares discriminant analysis (OPLS-DA) showed that a total of 32 metabolites significantly differed (P < 0.05, VIP ≥ 1) among the six rootstock-scion combinations, and 22 related metabolic pathways were found. The results showed that the rootstocks could affect the contents of metabolites such as organic acids and polyphenols in grape skins by controlling metabolic pathways involved in the biosynthesis of isoquinoline alkaloids, flavonoids (flavonols), pantothenic acid and coenzyme A, and the citric acid cycle, thereby altering the quality of grapes. This study can provide a theoretical basis for the selection of grape rootstock-scion combinations, and also provide important inspiration for improving the quality of grape-based products.
Isolation of Salt-tolerant and Aroma-producing Yeasts from Soybean Paste Starter and Their Fermentation Characteristics
MENG Fanbing, WANG Zhongwei, LI Yuncheng, MA Changzhong, LIU Dayu
2020, 41(24):  31-38.  doi:10.7506/spkx1002-6630-20190929-355
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In the present study, two salt-tolerant and aroma-producing yeast strains, named Waro03 and Saro47, were isolated from soybean paste starter. Ribosomal DNA sequencing indicated that they were highly homologous with Wickerhamomyces anomalus and Saccharomyces cerevisiae, respectively. Both strains showed good pH and NaCl resistance. Waro03 showed good growth in the medium containing 14% NaCl (m/m) whereas Saro47 and commercial Zygosaccharomyces rouxii AS2.180 could grow well only in the medium containing 12% NaCl (m/m). Saro47 produced large amounts of alcohols, esters, aldehydes and acids, while Waro03 produced large amounts of esters, alcohols, phenols and acids. NaCl could significantly inhibit the production of alcohols such as 3-methyl-1-butanol and phenylethyl alcohol, esters and furanones by Waro03, but significantly increased the production of aldehydes, acids and phenols. NaCl did not affect ethanol production. Saro47 showed similar aroma-producing characteristics and NaCl tolerance to AS2.180. The results of this study suggested that W. anomalus Waro03 looks promising as a starter culture for soy sauce and paste fermentation due to its capability of producing esters and tolerance to salts
Comparative Study on the in Vitro Functional Characteristics of Three Strains of Lactic Acid Bacteria Isolated from Fermented Sausages
FENG Meiqin, LUAN Xiaoxu, SUN Jian
2020, 41(24):  39-45.  doi:10.7506/spkx1002-6630-20200721-272
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To obtain functional meat starters with high antioxidant activity, three strains of lactic acid bacteria (Lactobacillus plantarum NJ107 and KM119, and L. fermentum GZ114) isolated from Chinese traditional fermented sausages were selected to compare their in vitro functional characteristics. The in vitro antioxidant activities of the fermentation supernatant, intact cells and cell-free extracts of each strain were evaluated. The in vitro probiotic properties were evaluated by surface hydrophobicity, and the ability to tolerate simulated gastroenteric fluid and bile salt. The results showed that all three strains had good antioxidant activity in vitro. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity and reducing capacity of the fermentation supernatant were significantly higher than those of the intact cells and the cell-free extracts (P < 0.05), and among the three strains, GZ114 displayed the highest DPPH radical scavenging activity. The hydroxyl radical scavenging capacity, superoxide anion radical scavenging capacity and ferrous ion chelating capacity of the intact cells were significantly higher than those of the fermentation supernatant and the cell-free extracts (P < 0.05), and the highest hydroxyl radical scavenging capacity, superoxide anion radical scavenging capacity and ferrous ion chelating capacity were observed in KM119, GZ114 and NJ107, respectively. The superoxide dismutase (SOD) activity of the fermentation supernatant were significantly higher than those of the intact cells and the cell-free extracts for all three strains (P < 0.05). The activities of glutathione peroxidase (GSH-PX) and catalase (CAT) in NJ107 and GZ114 were significantly stronger than those in KM119 (P < 0.05). In terms of decreasing surface hydrophobicity, these strains were ranked as follows: NJ107 > GZ114 > KM119. All the three strains showed high tolerance to simulated gastroenteric fluid and 0.5% bile salt.
Optimization of Culture Conditions for Production of Raw Starch-Degrading Amylase by Lactobacillus paracasei L1 and Analysis of Its Metabolites
GUO Haonan, ZHANG Lili, FENG Linlin, WANG Tianqi, WANG Cheng, ZHAO Xinqi, XU Yunhe
2020, 41(24):  46-53.  doi:10.7506/spkx1002-6630-20190916-191
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In order to study the starch-metabolizing ability of lactic acid bacteria with specific starch-binding activity, Lactobacillus paracasei L1 was evaluated for its ability to produce raw starch-degrading amylase by utilizing raw starch. The conditions for enzyme production were optimized and the molecular mass of the amylase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The production and utilization of carbohydrates and organic acids were determined by high performance liquid chromatography (HPLC). The results showed that raw starch-degrading amylase existed on the cell surface and in the culture supernatant of L1. The enzyme could hydrolyze raw starch from different sources to a certain extent, and raw starch could induce the production of the enzyme. The highest enzyme activity of (191.64 ± 0.63) U/mL was achieved under the following conditions: initial medium pH 6.5, 68% starch in total carbon source, and 2.03% carbon source. Glucose and maltose were the final products of carbohydrate hydrolysis, and malt oligosaccharide was the intermediate product. Lactic acid was the main product of organic acid hydrolysis while citric acid was the secondary product. This study proved that L1 can combine and utilize raw starch, which is of great significance to the application of lactic acid bacteria in starch matrices.
Proteomics Analysis of Breast Milk Whey Based on Immunoaffinity Chromatography and ProteoMiner
JIN Dengpeng, ZHOU Xiong, LIU Huan, YANG Jiao, HUANG Zhuoquan, BU Lingling, KE Qianhua, LIU Chunhong
2020, 41(24):  54-60.  doi:10.7506/spkx1002-6630-20190810-112
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After removing high-abundance proteins from breast milk, we applied proteomics to analyze low-abundance proteins with special important biofunctions in breast milk. Mature milk about 1 month old was used for this study, and high-abundance proteins were removed by immunoaffinity chromatography or ProteoMiner low-abundance protein enrichment kit, followed by proteomic analysis. The results showed that both methods were effective in removing high-abundance proteins. A total of 186 low-abundance proteins were identified by the two methods, 49 proteins of which were identified by immunoaffinity chromatography, and 172 by ProteoMiner low-abundance protein enrichment kit. According to bioinformatics analysis and gene ontology (GO) functional annotation analysis, the functions of the identified proteins mainly included serving as cellular components, participating in biological processes in cells and exerting binding activities. Using the Pathway database as a reference, these proteins were found to be involved in 201 metabolic pathway branches, including the PI3K-Akt signaling pathway and the phospholipase D signaling pathway. Compared with the Cluster of Orthologous Groups of proteins (COG) database, these proteins mainly participated in such biological activities as carbohydrate transport and metabolism, and signal transduction mechanisms. These results not only help to understand the functions of low-abundance proteins in breast milk, but also provide a theoretical basis for healthy growth and disease prevention in infants and young children as well as provide technical support for research on the protective mechanism of breastfeeding and on breast diseases.
Analysis of the Relationship between Bacterial Community Composition and Changes in Organic Acids during Natural Fermentation of Mixed Fruits
LI Xiyu, GAO Jie, LI Yunjiao, WANG Zhaoling, ZHANG Zhaoxi, SANG Yaxin
2020, 41(24):  61-68.  doi:10.7506/spkx1002-6630-20191015-141
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The purpose of this study was to analyze the changes in the bacterial community composition and organic acids in naturally fermented mixed fruits added with different amounts of brown sugar (fruit to sugar ratio of 1:1, 2:1 and 3:1, denoted as A, B and C, respectively) . High performance liquid chromatography (HPLC) was used to determine the change of organic acid contents. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the changes in bacterial community, and the results were analyzed by redundancy analysis (RDA). It was found that the Shannon-Wiener index first decreased and then increased in the fermentation process. Lactic acid, malic acid, citric acid and oxalic acid were the main organic acids during fermentation. A total of 19 specific bands were detected by PCR-DGGE, including 7 strains of lactic acid bacteria, 2 strains of Enterobacter, 1 strain of Enterococcus, 2 strains of Pseudomonas, and 3 strains of unidentified microorganisms. RDA showed that in terms of their decreasing contribution to organic acid production, the bacteria were ranked as follows: Lactobacillus plantarum > uncultured Brevundimonas sp. > Enterobacter xiangfangensis > uncultured Lactococcus sp. for citric acid production; and L. plantarum > L. lactis subsp. > E. xiangfangensis > L. lactis subsp. > uncultured Lactococcus sp. for lactic acid production.
Bioactive Ingredients and Microbial Diversity of Fuzhuan Tea Produced from Different Raw Materials
ZENG Qiao, LÜ Shenghua, LI Xiang, HU Xin, LIANG Yan, FAN Cheng, DUAN Jie, DENG Tongling
2020, 41(24):  69-77.  doi:10.7506/spkx1002-6630-20190927-333
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In order to investigate the bioactive ingredients and microbial diversity of Fuzhuan tea produced from different raw materials, the bioactive ingredients of Fuzhuan tea produced from Anhua black tea, Dianhong black tea, Eucommia ulmoides leaves or dandelion under identical conditions were detected, respectively. Additionally, the microbial community structure was determined by high-throughput sequencing, and correlation analysis between the microbial community structure and the bioactive ingredients was further performed. The results showed that there was a significant difference in the contents of bioactive ingredients among the four tested samples, but no significant difference was found in the fungal community structure at the genus level. Aspergillus was the absolutely dominant group with a relative abundance of over 99.99% in all tested samples. The bacterial communities were abundant in the four samples, consisting of 30 genera in 9 phyla, and a significant difference in bacterial community structure was found. Interestingly, at the level of phylum, Proteobacteria was the dominant bacterium in all tea samples. However, at the genus level, the bacterial community in the sample produced from Anhua black tea was mainly composed of unclassified_f__Enterobacteriaceae and Pseudomonas. In the sample produced from Eucommia ulmoides leaves, Pseudomonas showed the highest abundance. In the sample produced with Dianhong black tea, Burkholderia-Caballeronia-Paraburkholderia was the most abundant. In the product produced from dandelion, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium was the most abundant. In addition, Pseudomonas and Burkholderia-Caballeronia-Paraburkholderia appeared in higher abundance in the four samples. The correlation analysis indicated that unclassified_f__Enterobacteriaceae was positively correlated with polyphenol, catechin, epicatechin, epigallocatechin and gallic acid. Pseudomonas was positively correlated with flavonoid, whereas Burkholderia-Caballeronia-Paraburkholderia was negatively correlated with epigallocatechin gallate, and all these correlations were statistically significant (P < 0.05). This study provides a deeper understanding of the microbial diversity in Fuzhuan tea produced from different raw materials and provides the basis for the development of new products of Fuzhuan tea.
High-throughput Sequencing Study on Changes in Microbial Community Structure in Different Production Stages of Chishui Sun-Dried Vinegar
LI Rongyuan, LU Hongmei, QIN Xing, CHEN Li, WANG Sha, WU Zhen
2020, 41(24):  78-86.  doi:10.7506/spkx1002-6630-20191022-228
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The structure and abundance of the microbial community in samples collected from different production stages of Chishui sun-dried vinegar including herbal Qu, alcoholic fermentation, acetic acid fermentation and sun-dried fermented grains were detected by high-throughput sequencing. The results showed that the sequencing depth effectively covered all microbial species in the samples. The dominant bacterial genera of herbal Qu were Bacillus, Streptomyces and Thermoactinomyces, and the dominant fungal genera were Aspergillus and Thermomyces. During alcoholic fermentation, the dominant bacterial genus was Lactobacillus and the dominant fungal genera were Aspergillus and Thermomyces. The dominant bacterial genera were Lactobacillus and Acetobacter, and the dominant fungal genera were Aspergillus and Trichosporon during acetic acid fermentation. Sun-dried fermented grains had the greatest number of microbial species with uniform relative abundance distribution, and there were no absolutely dominant bacteria and fungi in this stage. The relative abundances of Bacillus, Thermoactinomyces, Proteus, Gluconacetobacter and Rhodococcus were higher than those of other bacterial genera identified, and the relative abundances of Aspergillus, Cladosporium, Sterigmatomyces, Acremonium and Alternaria were higher than those of other fungal genera identified. Clustering heat map analysis and β diversity showed that the microbial community structure in the first three stages basically did not change, while that in the last one significantly varied among samples from the same batch, which may related to the presence of salt and lack of oxygen in the jar. The microbial community structure in the stages of alcoholic fermentation and acetic fermentation was similar to each other, but was significantly different from that in the other two stages, indicating that the microbial community structure in the former these two stages is continuous due to environmental similarity.
Comparative Effects of Fermentation with Three Species of Lactic Acid Bacteria on Polyphenol and Proanthocyanidin Contents and Antioxidant Activity of Blueberry Fruit
WANG Chuyan, ZHANG Jigang, YANG Liuqing, LI Kexin, CAI Jingmin, HU Yong, OU Xiaohua
2020, 41(24):  87-94.  doi:10.7506/spkx1002-6630-20200326-387
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In this paper, blueberry fruit slurry was inoculated with Lactobacillus acidophilus, L. casei or L. paracasei for fermentation. Changes in the contents of free and bound polyphenols and proanthocyanidin composition as well as the antioxidant activity of polyphenols were monitored before and after fermentation. The results showed that fermentation with L. paracasei and L. casei significantly increased the contents of blueberry polyphenols, and all three species of lactic acid bacteria (LAB) significantly decreased the content of free proanthocyanidins. LAB fermentation also had a great impact on the contents of 9 characteristic phenolic acids and 3 proanthocyanidin monomers; the three species all increased the contents of protocatechuic acid, vanillic acid, syringic acid, catechins and quercetin, especially the last one, which was extremely significantly increased, while the content of proanthocyanidins B1 was significantly increased exclusively by fermentation with L. paracasei. Antioxidant tests showed that fermentation with L. paracasei significantly enhanced the ability of blueberry polyphenols to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation, and L. casei also improved the scavenging ability of ABTS radical cation. Collectivley, compared with the other two strains, L. paracasei could better improve the content and antioxidant activity of polyphenols in blueberries. This study provides a reference for the development of fermented blueberry beverage.
Effect of Mixed Starter Cultures on the Quality of Fermented Sausage
HUANG Junyi, WANG Fengna, WU Xiang, LIU Shan, LI Cong, LI Xinfu, XU Baocai
2020, 41(24):  95-101.  doi:10.7506/spkx1002-6630-20191023-241
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The effect of mixed starter cultures of Lactobacillus acidophilus and Staphylococcus carnosus (isolated from Jinhua ham) inoculated at different concentrations on the physicochemical and sensory qualities of fermented sausage was evaluated. The results showed that pH and water activity in fermented sausage declined significantly more slowly during inoculated fermentation as compared to natural fermentation (P < 0.05). Inoculated fermentation enhanced the flavor of fermented sausage and increased the number of flavor compounds. The a*/b* ratio of the sausage fermented by a 1:3 mixture of L. acidophilus and S. carnosus at an inoculum size of 107 CFU/g was not different from that of commercial sausage, and the hardness, elasticity, cohesiveness and chewiness were significantly higher than those of naturally fermented sausage (P < 0.05) and were not different from those of commercial sausage. In addition, the sausage fermented by the mixed starter had the highest overall acceptance. Therefore, this mixed starter culture can effectively improve the quality of fermented sausages, and thus is promising for commercial application.
Analysis of Microbial Community Structure Changes of Wheat Bran during Fermentation by High-throughput Sequencing
HE Chenghu, ZHAO Haizhen, LU Zhaoxin, LÜ Fengxia, BIE Xiaomei, ZHANG Chong
2020, 41(24):  102-109.  doi:10.7506/spkx1002-6630-20191014-111
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The objective of the study was to examine the structure of the microbial community in spontaneous and back-slopping fermented wheat bran in order to provide a foundation for understanding the relationship between microbial community diversity and the quality of fermented wheat bran. Four samples were obtained through spontaneous fermentation for one day (1#) followed by seven cycles of back-slopping fermentation for one day each (3#, 5#, and 7# from the third, fifth and seventh cycles, respectively). Results showed that for the four samples, the Shannon index of fungi was higher than that of bacteria, indicating that the diversity of fungi is richer than that of bacteria; while the Chao 1 index of fungi was lower than that of bacteria, indicating that the relative abundance of fungi is lower than that of bacteria. At the phylum level, the dominant bacteria in fermented wheat bran were Protebacteria and Firmicutes while the dominant fungus was Ascomycota. At the genus level, the dominant bacteria were Lactobacillus and Pediococcus, while the dominant fungi were Aspergillus and Alternaaria. Principal component analysis revealed that the microbial community structures of samples 3#, 5# and 7# were similar to each other but different from that of sample 1#. The heatmap analysis showed that the dominant bacteria in the early fermentation stage were Cronobacter, Pantoea, Enterobacter and Clostridium, while the dominant bacteria in the later fermentation stage were Lactobacillus and Pediococcus; Aspergillus and Alternaria were the dominant fungi throughout the fermentation process. The results of this study indicated that fermentation methods and back-slopping fermentation cycles had significant effects on the microbial community structure and diversity of fermented wheat bran. Back-slopping fermentation promoted the growth of lactic acid bacteria while inhibiting the growth of pathogenic bacteria.
α-Glucosidase and Dipeptidyl Peptidase IV Inhibitory Activity and Peptide Composition of Porphyra yezoensis Protein Hydrolysate
HUANG Qinqin, TIAN Yaping
2020, 41(24):  110-116.  doi:10.7506/spkx1002-6630-20191014-114
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Porphyra yezoensis protein hydrolysate, prepared enzymatically under specific and controlled conditions, was found to be dominated by small peptides with molecular mass less than 1 000 Da. It had inhibitory activities against α-glucosidase and dipeptidyl peptidase IV (DPP-IV) and was able to tolerate pH, temperature and gastrointestinal digestion. The enzymatic hydrolysate was separated and purified consecutively by ultrafiltration, nanofiltration and gel filtration chromatography. Two bioactive peptides, Ala-Pro and Pro-Gly-Gly-Val, were identified by ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS). Peptides corresponding to the sequences with a purity of 99.69% were separately synthesized and their inhibitory activities against the two enzymes were characterized. The results showed that Pro-Gly-Gly-Val was a double-enzyme inhibitory peptide with an IC50 of 18.60 mmol/L for α-glucosidase and of 17.80 mmol/L for DPP-IV. Ala-Pro had an inhibitory effect only against DPP-IV with an IC50 of 4.65 mmol/L.
Antimicrobial Activity of Soy Whey Fermented by Lactobacillus plantarum D1501 and Purification and Identification of Bacteriocin from It
LU Zhou, DAI Yiqiang, Hafiz Abdul RASHEED, WU Han, XIA Xiudong, DONG Mingsheng
2020, 41(24):  117-124.  doi:10.7506/spkx1002-6630-20190929-362
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Lactobacillus plantarum D1501 was inoculated into soy whey and cultivated under static conditions at 37 ℃ for 24 h to prepare fermented soy whey (FSW). Antimicrobial tests showed that FSW had obvious antimicrobial activity against both Gram-positive and Gram-negative bacteria tested, and had weak antimicrobial activity against Candida, but exhibited no inhibitory effect on Saccharomyces cerevisiae or two mold strains tested. The bacteriostatic substance in FSW was speculated to be bacteriocin by enzymatic hydrolysis and neutralization experiments. The cell-free neutral supernatant of L. plantarum D1501 was separated by 30, 10 and 3 kDa ultra filtration tubes sequentially and concentrated by rotary evaporation. Then it was subjected to RESOURCE-Q anion exchange chromatography and reverse-phase chromatography with 300SB-C18 for further purification. The purity of the purified bacteriocin was determined to be high by high performance liquid chromatography (HPLC), and it was named as Lp100. The molecular mass of Lp100 was determined by LC-MS/MS to be approximately 1 434.90 Da, and its amino acid sequence was MCCKVLLLLSRR. The specific activity of the purified product was 7 247.00 IU/mg with 79.94-fold purification and 1% recovery.
Component Analysis
Geographical Origin Traceability of Chinese Mitten Crabs Based on Mineral Elements and Stable Isotopes
ZHANG Zhengquan, HUANG Dongmei, CAI Youqiong, MENG Xianjing, SHI Yongfu, KONG Cong, HUANG Xuanyun, TANG Yunyu, ZHANG Xuan, YANG Guangxin
2020, 41(24):  125-130.  doi:10.7506/spkx1002-6630-20191222-254
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This study was undertaken in order to explore the potential of mineral elements and stable isotopes for the geographical origin traceability of Eriocheir sinensis. Microwave digestion-atomic absorption spectrometry and stable isotope ratio mass spectrometry were used to determine the contents of eight mineral elements (K, Ca, Na, Mg, Mn, Zn, Cu and Fe) and the abundances of C and N stable isotopes in the muscle tissue of E. sinensis from Chongming of Shanghai, and Yangcheng Lake and Xinghua of Jiangsu. By using multivariate statistical analysis, we established a discriminant model for distinguishing the three geographical origins from each other. The correct discrimination rates for samples from Chongming, Yangcheng Lake and Xinghua were 100%, 95% and 100%, respectively. The initial overall discriminant accuracy was 98.3%, and the cross-validation accuracy was 98.3%. Four principal components were selected by principal component analysis (PCA), cumulatively contributing to 74.8% of the total variance. In the three-dimensional PCA scatter plot of PC1 versus PC2 versus PC4, the three samples were clearly separated. It is proved that mineral elements combined with stable isotopes can distinguish Chinese mitten crabs from different regions of the Yangtze River.
Effects of Different Heat Treatment Conditions on the Flavor of Pasteurized Milk
YANG Shanshan, DING Ruixue, SHI Haisu, LUO Xue, YANG Mei, YUE Xiqing, WU Junrui
2020, 41(24):  131-136.  doi:10.7506/spkx1002-6630-20191206-069
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The flavor characteristics of milk undergoing low-temperature long-time (LTLT) pasteurization (62, 65 and 68 ℃; 20, 25, 30 and 35 min) or high-temperature short-time (HTST) pasteurization (72, 75, 80, 83, and 85 ℃; 15, 20, and 30 s) were evaluated by electronic tongue, gas chromatography-mass spectrometry (GC-MS) and automatic amino acid analyzer, in order to study the effect of different heat treatment conditions on the flavor of pasteurized milk. The results showed that compared with LTLT pasteurization, HTST pasteurization could better retain the flavor of raw milk. By GC-MS, a total of 19 volatile flavor compounds were identified in raw milk, and 11 of these disappeared after heating, but 42 compounds were newly produced. In the LTLT pasteurized milk, the volatile flavor substances were mainly alcohol, ester, acid and alkane; in the HTST pasteurized milk, phenols, aldehydes and ketones were more abundant. It was found that LTLT pasteurization was beneficial to the production of flavor amino acids, while HTST pasteurization increased the content of bitter amino acids in milk, and the content of sweet amino acids reached the maximum value after treatment at 75 ℃ for 20 s, which basically did not change with varying sterilization conditions. This study provides a theoretical basis for improving the quality of pasteurized dairy products and the pasteurization process.
Changes in Some Volatile Bioactive Ingredients of Fermented Grains of Chinese Baijiu during Distillation
CHEN Lu, SHI Dongmei, HE Hongkui, LU Wei, HAN Xinglin, SUN Jinyuan
2020, 41(24):  137-143.  doi:10.7506/spkx1002-6630-20191223-263
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In this paper, by using two different pretreatment methods, solid-phase microextraction (SPME) and vortex-assisted liquid-liquid microextraction (VA-LLME), we studied changes in volatile bioactive ingredients of fermented grains of Gujinggong Baijiu during distillation. It was found that VA-LLME was more suitable for this study. Among the volatile bioactive constituents detected using VA-LLME, the contents of pyrazines and most phenols in distillation fractions increased with the extension of distillation time, the content of ethyl linoleate decreased, while the contents of dimethyltrisulfide and 3,5-di-tert-butyl-4-hydroxybenzaldehyde did not change significantly during the distillation process. The experimental results showed that some volatile bioactive ingredients can be generated by chemical reactions that occur during distillation, and the distillation pattern of compounds is related to their structures.
Analysis of Changes in Volatile Components during Processing of Handmade Fuzhuan Tea
LI Junjie, XU Yuanhao, CHEN Mengjuan, DENG Gaowen, WU Kai, JIANG Liwen
2020, 41(24):  144-154.  doi:10.7506/spkx1002-6630-20191226-316
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Gas chromatography-ion mobility spectroscopy (GC-IMS) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) were used to analyze the changes in volatile components in Fuzhuan tea during fermentation and drying. The results of GC-IMS showed that there were 44 small-molecular volatile components (C3–C10) that differed between the two processing stages. The contents of volatile components increased continuously during the fermentation process. The contents of volatile components did not change significantly during the first four days of fermentation, but changed significantly by the eighth day, and then tended to change slowly later. During the drying process, the contents of volatile components increased gradually. Further, by HS-SPME-GC-MS, a total of 57 of C8–C15 organic compounds were identified, belonging to seven chemical classes, mainly terpenes, alcohols, aldehydes, esters, ketones and ethers. The contents and kinds of volatile components increased with fermentation and drying time. The contents and kinds of terpenes with characteristic wood-like and grassy odor decreased gradually, and the contents and kinds of alcohols, esters ketones and aldehydes with fruity, herbal and mushroom-like aroma as the characteristic odor increased. Finally, the odor of tea samples was characterized as mushroom-like and floral aromas integrating herbal, wood-like, flowery and fruity aroma notes. The results of GC-IMS and HS-SPME-GC-MS showed some differences from each other. Most of the components detected by GC-IMS were small molecules at low levels, while most of the components detected by HS-SPME-GC-MS were large molecules at high levels. The general trend of changes in volatile components determined by HS-SPME-GC-MS was consistent with that determined by GC-IMS, and the change in odor-active volatile components was basically consistent with the sensory evaluation results. To sum up, combined use GC-IMS and HS-SPME-GC-MS can make up for each other’s limitations and more comprehensively reflect changes in volatile components of samples.
Analysis of Flavonoids in Fruit of Jujube (Ziziphus jujuba) by UPLC-MS-based Metabonomics
WANG Lefei, GU Shaobin, WU Ying
2020, 41(24):  155-161.  doi:10.7506/spkx1002-6630-20191018-195
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Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) based metabolomics was used to analyze the relationship between the varieties and contents of flavonoids and color change during the fruit development of jujube (cv. Sanbianhong). Results showed that as fruit development proceeded from S1 to S2 to S3, the color changed from purple red to green white to deep red, and the total flavonoid content of jujube peel showed an increase-decrease-increase trend, which was 4.94, 0.95 and 4.45 mg/g dry at mass at the three time points, respectively, indicating that the content of total flavonoids in jujube peel was positively correlated with the color change. Principal component analysis (PCA) and orthogonal partial least square-discriminate analysis (OPLS-DA) showed clear differences in the composition and contents of flavonoids at the three developmental times. The contents of quercetin 3-O-rutinoside, methyl quercetin O-hexoside, luteolin O-sinapoylhexoside, luteolin O-sinyl hexoglucoside, naringenin O-malonylhexoside, 3,4,5-trihydroxyflavone O-rutinoside, 6-C-hexosyl-luteolin O-hexoside and isovitexin at S1 were much higher than those at S2 and S3, and they were identified as characteristic flavonoids at the S1 stage, while cyanidin O-hexose, apigenin O-hexosyl-pentoside, quercetin 5-O-malonylhexosyl-hexoside, sirolimus O-glucuronoic acid (salicyl alcohol) ether O-dialuronic acid, and chrysoeriol were considered as characteristic flavonoids at the S3 stage. This study is of great significance to reveal the mechanism of fruit coloration in “Sanbianhong” jujube.
Changes in Odor-Active Compounds and Analysis of Off-Flavor Compounds in Chinese Sausage Added with Black and White Pepper during Storage
ZHOU Huimin, ZHAO Bing, WU Qianrong, LI Su, PAN Xiaoqian, ZHU Ning, QIAO Xiaoling, WANG Shouwei, LIU Bowen, ZHANG Shunliang
2020, 41(24):  162-171.  doi:10.7506/spkx1002-6630-20200315-236
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The pattern of changes in the odor-active compounds of Chinese sausage added with black and white pepper during storage (at time points of 0, 7, 21, 35, 49, and 77 d) was investigated using purge & trap-thermal desorption-gas chromatography-olfactometry-mass spectrometry (P&T-TDS-GC-O-MS) combined with odor activity value (OAV), sensory evaluation, and multivariate statistical analysis. The characteristic off-flavor compounds were identified, and an effective method for flavor deterioration and shelf life prediction was proposed. The results showed that a total of 56 volatile flavor compounds with OAV ≥ 0.1 were detected in all samples. Among them, aldehydes showed the highest total OAV (1 724.58–4 682.24), contributing to 85.85%–90.35% of the overall flavor, and totally 15 aldehydes were identified, accounting for the highest proportion of the total number of volatile flavor compounds. As the storage time increased, the OAV values of all flavour substances except esters and olefins continued to increase, especially between days 35 and 49. Furthermore, the color became dark, the cured meat-like and pepper-like flavor became light, the off-flavor became strong, and the acceptability decreased. The correlation analysis showed that 17 odor active substances were significantly correlated with storage time and sensory flavor score, indicating that these compounds can be seen as the optimal indicators to predict the storage period and flavour deterioration. According to OAV analysis and GC-O identification, hexanal, E,E-2,4-decanodienal, heptanal, octanal, E-2-octenal, nonanal, 1-octene-3-ol and E-2-decenal might be the main sources of sausage off-flavor. Based on odor-active compounds with OAV > 1, an objective method was established to distinguish sausage samples added with black and white pepper at different storage times using principal component analysis (PCA) and cluster analysis.
Effects of Sterilization and Preservative Treatments on the Flavor Components and Comparison on the Flavor Fidelity of Zaojiang, a Traditional Naturally Fermented Food in China Made Mainly from Ginger Roots
JIA Qian, CHENG Chao, TIAN Cheng, LI Wei, ZHOU Zhi, MO Kaiju, WANG Xingping
2020, 41(24):  172-178.  doi:10.7506/spkx1002-6630-20200625-337
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The flavor compounds and characteristics of Zaojiang undergoing six different treatments including sterilization, preservatives and control were determined by head-space solid phase micro-extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and electronic nose. Subsequently, principal component analysis (PCA) and cluster analysis were carried out on the data obtained to comprehensively analyze the effect of different treatments on the flavor maintenance of Zaojiang. A total of 117 volatile flavor components were identified, including 74 terpenes, 20 terpene alcohols, 7 carbonyl compounds, 7 esters, 3 acids, 2 phenols and 4 aromatic compounds. Heat treatment caused changes in the kinds of terpenes alcohols, carbonyl compounds and aromatic compounds, leading to significant changes in the flavor of Zaojiang. Of the preservatives tested, 0.1% potassium sorbate was the most effective in preserving the flavor of Zaojiang.
Comprehensive Quality Evaluation of Different Rice Varieties Based on Principal Component Analysis
JING Ruiyong, WEI Jiaqi, WANG Liyan, SONG Weimin, ZHENG Guiping, GUO Yongxia
2020, 41(24):  179-184.  doi:10.7506/spkx1002-6630-20191218-198
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In order to explore the difference in the comprehensive quality of different rice varieties, 11 rice varieties from Heilongjiang province, China and 7 rice varieties from Japan were selected for quality evaluation in terms of brown rice rate using subordinate function combined with principle component analysis (PCA). These varieties were classified by cluster analysis. The results showed that among the 18 varieties, Longjing 39, Kongyu 131 and Mudanjiang 32 had the best processing quality, the Japanese rice cultivar Aichiasahi had the best appearance quality, and Suijing 18, Songjing 9 and the Japanese early-maturing cultivar Aomori had the highest amylose content and taste values. According to subordinate function analysis, the quality of the 18 rice varieties could be?ranked?in?decreasing order as follows: Longjing 39, Mudanjiang 32, Kongyu 131, Kenjing 8, early-maturing Aomori, Longjing 36, Aichiasahi, Suijing 18, Songjing 9, Hoshinoyume, Kenjing 6, Longqingdao 1, Xinyueguang, Longqingdao 3, Shangyu 418, Longqingdao 2, Longjing 43, and Fujusu. Cluster analysis showed that these varieties were classified into three groups: Group I, with the best quality, including Longjing 39; Group II including Mudanjiang 32, Kongyu 131, Kenjing 8, early-maturing Aomori, Longjing 36, Aichiasahi, Suijing 18, Songjing 9, Hoshinoyume, Kenjing 6, Longqingdao 1, Xinyueguang, Longqingdao 3, Shangyu 418, Longqingdao 2, and Longjing 43; and Group III, with the worst quality, including Fujusu.
Determination of Inositol in Infant Formula and Milk by Ion Chromatography-Tandem Triple Quadrupole Mass Spectrometry
WANG Yan, WANG Juan, WU Chunmin, ZHANG Chunlin, WANG Dong, GAO Zhuo, ZHANG Leilei, ZHANG Yan
2020, 41(24):  185-189.  doi:10.7506/spkx1002-6630-20191225-298
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In this study, inositol contents in infant formula and milk were determined using ion chromatography-tandem triple quadrupole mass spectrometry. The sample pretreatment procedure included extracted by dissolution, extraction, pH adjustment, centrifugation and filtration. The analysis was performed on a Dionex CarboPac MA1 column (2.0 mm × 250 mm, 7.5 μm) using gradient elution with KOH generated online with an automatic eluent generator as eluent. Electrospray ionization was operated in the negative ion mode to detect the analyte. The results showed that good linearity with correlation coefficient (r) ≥ 0.999 was observed for inositol in infant formula and milk within the concentration range of 0.01–3.00 μg/mL. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.023 and 0.076 mg/100 g, respectively, both lower than those specified in the national standard of China. The recoveries of spiked samples were between 90.3% and 109.5% with relative standard deviations (RSDs) of 0.7%–9.1% (n = 6). The method was simple to operate, stable and sensitive and could be suitable for the determination of inositol in infant formula and milk.
Influence of Partial Replacement of NaCl with KCl on the Volatile Compounds of Xuanwei Ham Investigated by Gas Chromatography-Ion Mobility Spectrometry Combined with Multivariate Statistical Analysis
DING Xilin, WANG Guiying, ZOU Yingling, ZHAO Yaying, GE Changrong, LIAO Guozhou
2020, 41(24):  190-198.  doi:10.7506/spkx1002-6630-20191226-311
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In order to reduce the content of sodium salt in Xuanwei ham, the effect of partial replacement of NaCl with KCl on the volatile compounds of Xuanwei ham was studied. In this study, 100% NaCl was used as the control, and 30%, 40%, 50% and 60% KCl were used to replace NaCl and considered as treatment groups. By gas chromatography-ion mobility spectrometry (GC-IMS), the volatile compounds of Xuanwei ham were analyzed. The results showed that GC-IMS could quickly identify differences in volatile flavor compounds among ham samples. A total of 49 volatile compounds were detected from all samples, mainly including aldehydes, ketones, alcohols and esters. Multivariate statistical analysis including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) could clearly distinguish Xuanwei ham samples with different levels of KCl substitution. In addition, based on PLS-DA analysis, a total of 24 compounds were selected and identified as characteristic markers (VIP > 1), including ethyl propanoate, 2-pentanone (monomer), ethyl acetate (dimer) and acetal, which were the important components contributing to the difference in flavor among Xuanwei hams with different levels of KCl substitution. According to the results of heat map clustering analysis, the control group and the 30% KCl substitution group were similar in flavor. Therefore, in order to maintain the flavor of traditional Xuanwei ham, the KCl substitution level should not exceed 30%.
Analysis of Characteristic Flavor Compounds of Chinese Light-Soy Sauce Aroma Type Liquors
SUN Youlan, HUANG Yongguang, ZHU Xiaochun, MA Yu, JIANG Xiang, YIN Sumei
2020, 41(24):  199-208.  doi:10.7506/spkx1002-6630-20191203-035
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Sensory quantitative description analysis was performed to investigate the flavor characteristics of four kinds (Jingdian, Hongtu, Xingfu and Fengnian) of Chinese light-soy sauce aroma type liquors. Our research results showed that Jiangdian had a stronger light aroma but weaker soy sauce aroma compared to Hongtu, while the other two had similar sensory characteristics. By headspace-solid phase microextraction/liquid-liquid microextraction-gas chromatography-mass spectrometry, a total of 80 volatile flavor compounds were detected at high concentrations in the four types of liquors, the numbers of esters, alcohols and acids being greater than that of the other compounds. According to odor activity values (OAV), there were 31 characteristic compounds contributing to the aroma of light-soy sauce aroma type liquors. Partial least squares regression (PLSR) was employed to investigate the correlation between sensory attributes and characteristic aroma compounds. It turned out that the soy sauce-like and burnt aroma was well correlated with furfural, 2-undecone, γ-valerolactone, and n-nonanol, while the light aroma displayed a good correlation with isobutyric acid, ethyl butyrate, and acetic acid-2-phenylethyl ester. Furthermore, the floral and ester aroma have good correlation with isoamyl acetate, isobutanol, isoamyl alcohol, 3-hydroxy-2-butanone and ethyl heptanoate. This study provides a basis for the quality evaluation and improvement of light-soy sauce aroma type liquors.
Analysis of Phenolic Composition and Antioxidant Activities in Fruits, Stems and Leaves of Rubus chingii Hu
CHEN Qingqing, LI Ke, TANG Xiaoqing, GENG Li, WANG Lei, PENG Yaping
2020, 41(24):  209-215.  doi:10.7506/spkx1002-6630-20190929-351
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The phenolic compounds in the fruits, stems and leaves (tender leaves and old leaves) of Rubus chingii Hu were separated and detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Their antioxidant activities were determined by spectrophotometry. The results showed that the contents of total phenol, total flavonoid and proanthocyanidins in the above-ground parts were significantly different, the highest contents of total phenol (69.49 mg/g), total flavonoid (87.21 mg/g) and proanthocyanidins (36.70 mg/g) being in young leaves, old leaves and fruits, respectively. The composition of phenolic compounds in young leaves was similar to that in old leaves, mainly including kaempferol derivatives as well as quercetin and its derivatives, but significantly differed from that in fruits and stems. The antioxidant activities of the three parts of R. chingii Hu followed the decreasing order of young leaves > old leaves and stems > fruits. According to correlation analysis, the total phenolics contributed to the antioxidant activity. From this study, it could be seen that the young leaves of R. chingii Hu are a potential source of natural phenolic antioxidants that is promising for the development of health foods. This study provides theoretical reference for the development and utilization of this plant’s stems and leaves to promote its comprehensive utilization.
Processing Technology
Optimization of the Production Process for Direct Vat Set of Lactobacillus fermentum FYa1 for Pickle Fermentation
KANG Jiao, ZHANG Yarong, YANG Congxu, XUE Qiaoli, HU Yongjin
2020, 41(24):  216-222.  doi:10.7506/spkx1002-6630-20200430-397
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Lactobacillus fermentum FYa1, a strain isolated from Fuyuan pickles in Yunnan was used to make direct vat set (DVS) for pickle making. The optimal centrifugation conditions were determined as follows: speed of 3 000 r/min, time of 15 min, and temperature of 15 ℃. Under these conditions, the survival rate of FYa1 was 34.57%. By using Plackett-Burman and Box-Behnken designs, the optimal formulation for freeze-drying protectants was found to consist of trehalose 8.5 g/100 mL, sucrose 8.5 g/100 mL and sodium glutamate 2.5 g/mL. The survival rate of lyophilized bacterial powder prepared by the optimized formulation was (68.53 ± 0.09)%, and the bacterial content was (2.0 ± 0.05) × 108 CFU/g. The DVS had excellent fermentation performance and showed good resurrection ability in the fermentation process. These results provide a scientific basis for the processing and quality improvement of Yunnan traditional pickles.
Processing Optimization and Preservation of High-Quality Preserved Grass Carp Products
ZHANG Yujie, GUO Xingyue, DOU Gaole, AI Youwei, WANG Limei, WANG Hongxun, HOU Wenfu
2020, 41(24):  223-232.  doi:10.7506/spkx1002-6630-20191116-188
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This study aimed to develop a process for producing high-quality prepared grass carp products. The effects of glutamine transaminase (TG), soy protein isolate (SPI) and NaCl concentration, blending time, and fish meat to surimi ratio on the gel strength, water-holding capacity, whiteness and sensory score of grass carp surimi were investigated using one-factor-at-a-time method, and further all these parameters except fish meat to surimi ratio were optimized using response surface methodology based on gel strength. Furthermore, we evaluated the efficacy of the ethanolic extract of Euryale ferox salisb seed shells in inhibiting the quality deterioration of the prepared fish product during refrigerated storage. The results showed that TG, SPI and NaCl concentration and blending time had significant effects on the gel strength of grass carp surimi (P < 0.05). There was a significant interaction among NaCl and TG concentration, and blending time (P < 0.05). The error between the experimental and the model-predicted values was within 5%, and the optimized process parameters were as follows: SPI 3.20%, TG 3.83%, NaCl 3.62%, blending time 27 min, and ratio of surimi to fish 4:1. The extract of Euryale ferox salisb seed shell when used at a concentration of 5 mg/mL could inhibit the quality deterioration of the prepared fish product during cold storage and prolonged the shelf-life. The prepared fish product in this study had good texture and gel strength and long shelf life under refrigerated conditions. This study provides theoretical support for the development of prepared grass carp products.
Ultrasonic-Assisted Aqueous Enzymatic Extraction and Chemical Composition of Rice Germ Oil
WU Fei, LI Zhao, ZHOU Qi, LIU Chunhua, PAN Mingzhe, YU Dianyu, YAO Kai
2020, 41(24):  233-241.  doi:10.7506/spkx1002-6630-20200426-343
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Rice germ oil rich in tocopherol was extracted by ultrasonic-assisted aqueous enzymatic method. Under the following conditions: mass ratio of cellulase to neutral protease 1:1, enzyme concentration 1.2%, material-to-liquid ratio 1:5, and hydrolysis time 3 h, the extraction efficiency was (87.4 ± 0.04)%. Through transmission electron microscopy, it was found that obvious separation was observed between fat and protein after ultrasonic treatment of rice germ. Response surface analysis showed that ultrasonic power had the greatest effect on the extraction efficiency, followed sequentially by temperature and duration. Under the conditions of temperature of 55 ℃, ultrasonic power of 400 W, and time of 20 min, the extraction efficiency of rice germ oil was (92.1 ± 0.03)%. Saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids accounted for (20.62 ± 0.14)%, (39.62 ± 0.96)% and (37.46 ± 0.43)% of the total fatty acids in rice germ oil extracted by the ultrasonic-assisted aqueous enzymatic method, respectively. The contents of γ-oryzanol, phytosterols and total tocopherols in the oil were (490.00 ± 2.12), (384.19 ± 5.14) and (93.01 ± 2.75) mg/100 g, respectively.
Preparation of Meaty Flavoring Base from Enzymatic Hydrolysate of Morel Mushroom by Maillard Reaction
GAO Juan, DU Jiaxin, WU Xian, YANG Qian, FANG Donglu, ZHENG Huihua, ZHAO Liyan, HU Qiuhui
2020, 41(24):  242-250.  doi:10.7506/spkx1002-6630-20191212-129
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In this study, an enzymatic hydrolysate of morel mushroom (Morchella sextelata) was used as a substrate to prepare seasoning base with strong meat flavor through Maillard reaction (MR). The effects of different reaction conditions on the flavor and color of Maillard reaction products (MPRs) were analyzed by one-factor-at-a-time and orthogonal array design methods. The browning intensity of MPRs was measured, and sensory evaluation was carried out using Friedman test combined with fuzzy mathematics. The overall flavor and color characteristics of MRPs were assessed by color meter, electronic tongue and electronic nose. According to the sensory evaluation results, the optimal MR conditions were determined as follows: reducing sugar 10%, ratio of D-xylose to D-glucose 2:1, cysteine 2%, temperature 115 ℃, initial pH 7.0, and time 30 min. MRPs prepared under these conditions showed a well-balanced flavor with rich meaty aroma and pleasant taste. According to the results of instrumental analysis, the intensity of sensor responses to the umami and saltiness of the enzymatic hydrolysate increased while those to bitterness and astringency as well as unpalatable volatile flavors, including ammonia, amine compounds and hydrogen sulfide decreased after being subjected to MR. Besides, MR enriched the aroma profile and color characteristics of the hydrolysate.
Safety Detection
Preparation of Single-chain Variable Fragment Antibody against Furaltadone Metabolite and Development of Indirect Competitive Enzyme-linked Immunosorbent Assay for Its Detection in Shrimp Samples
YANG Wuying, WANG Hong, HONG Yanping, WANG Dan, XU Zhenlin, SHEN Yudong, KE Fajun, CHEN Xinzhu, SUN Yuanming
2020, 41(24):  251-258.  doi:10.7506/spkx1002-6630-20190817-191
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In this study, the genes of heavy chain and light chain variable fragment were cloned from hybridoma cells secreting a monoclonal antibody against a derivative of the furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ). The gene of single-chain variable fragment (scFv) antibody against this derivative was constructed by splicing overlap extension-polymerase chain reaction (SOE-PCR) through the ligating peptide (G1y4Ser)3, inserted into the expression vector pET-22b(+) and expressed in E. coli BL21(DE3). The expressed product was purified by Ni affinity column chromatography. The concentration of coating antigen and the dilution factor of scFv antibody were optimized by chessboard titration to establish and evaluate an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this scFv antibody for AMOZ residues. The results showed that the optimal concentration of coating antigen and the dilution factor of scFv antibody were 0.062 5 μg/mL and 20, respectively. The IC50 value, linear range and limit of detection (LOD) of the established icELISA method were 8.65 μg/L, 2.90–53.28 μg/L and 1.48 μg/L, respectively. In addition to the cross-reactivity with the original drug furantadone, the cross-reactivity rates of the scFv antibody with other nitrofuran antibiotics and their metabolites were lower than 0.1%. The average recoveries of AMOZ from the spiked shrimp samples ranged from 74.3% to 86.6%. Good correlation (R2 = 0.999 2) was obtained between the results of icELISA and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The icELISA method established in this study is suitable for the rapid screening of AMOZ residues in shrimp samples and other aquatic products.
A Multiplex Real-time Polymerase Chain Reaction Combined with Melting Curve Analysis Method for Simultaneous Identification of the Meat of Bluefin Tuna, Sablefish and Oilfish
XU Suigen, LI Jiapeng, LI Jinchun, CHEN Xi, YANG Junna, XIONG Suyue, HUANG Xin, QIAO Xiaoling, QU Chao, WANG Shouwei
2020, 41(24):  259-266.  doi:10.7506/spkx1002-6630-20200528-353
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In order to establish a rapid method for the identification of adulterated fish and fish products, we designed three pairs of species-specific primers targeting the mitochondrial displacement-loop (D-loop) region of bluefin tuna (Thunnus thynnus), the mitochondrial 16S rRNA gene of sablefish (Anoplopoma fimbria) and the mitochondrial ND4L gene of oilfish (Lepidocybium flavobrunneum), respectively, yielding amplicons having significantly different melting temperatures (Tm). A multiplex real-time polymerase chain reaction (real-time PCR) combined with melting curve analysis method was established for the rapid identification of bluefin tuna-derived, sablefish-derived and oilfish-derived ingredients in fish and fish products, and it was evaluated in terms of specificity and sensitivity and then applied in the detection of commercial meat products. The results suggest that the method had good specificity and sensitivity, and was able to detect 0.001 ng of DNA from single species and 0.1 ng of DNA from mixed species. Meanwhile, it was indicated that the relative detection limits of bluefin tuna, sablefish and oilfish were 0.5%, 1% and 0.5%, respectively. The method was successfully applied to commercially available samples.
Fully 13C Isotope Labeled Internal Standard for the Accurate Quantification of Zearalenone in Cereals and Oils by Ultra-high Performance Liquid Chromatography-Tandem Triple Quadrupole Mass Spectrometry after Automated Purification with Immunoaffinity Column
WU Yu, YE Jin, LI Li, LI Sen, XUAN Zhihong, CUI Hua, LIU Hongmei, WANG Songxue, MA Haihua, ZHANG Feng, HE Jianhong
2020, 41(24):  267-272.  doi:10.7506/spkx1002-6630-20190927-339
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An accurate method for the quantification of zearalenone in cereals and oils was developed by automated purification with immunoaffinity column combined with ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) using a fully 13C isotope labeled standard. The results showed that the quantification limit (LOQ) and detection limit (LOD) of the internal standard method were 0.33 and 1 μg/kg, respectively, with a correlation coefficient R2 of 0.999 5. The recoveries were 92.7%–101.3% with relative standard deviation (RSD) of 0.02%–3.74%. The results obtained by this method for three quality control samples were within the range of certified values. Its accuracy and precision were better than those of the external standard method. The internal standard method allowed the automated and efficient detection of massive samples without the need for nitrogen gas blowing and could avoid the errors caused by manual operations.
Detection of Lepidocybium flavobrunneum- and Ruvettus pretiosus-Derived Components in Aquatic Products by Real-time PCR
XIN Hongmei, YAO Lin, LU Jianping, QU Meng, JIANG Yanhua, LI Fengling, GUO Yingying, WANG Lianzhu
2020, 41(24):  273-280.  doi:10.7506/spkx1002-6630-20190729-394
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Objective: To develop a real-time polymerase chain reaction (real-time PCR) method for the detection of Lepidocybium flavobrunneum- and Ruvettus pretiosus-derived components. Methods: The mitochondrial cytochrome C oxidase subunit I (COI) gene sequences of 18 cod species including L. flavobrunneum, R. pretiosus and Gadus morhua and other species commonly used as adulterants were aligned with one another, and L. flavobrunneum- and R. pretiosus-specific primers and probes were designed and used to establish a real-time PCR method for the detection of the two fishes. The specificity, absolute sensitivity and relative sensitivity of the PCR method were assessed. In order to evaluate the practicability, 50 commercial products labeled as cod were tested by this method. Results: This method showed high specificity, with absolute sensitivity of 0.002 and 0.000 2 ng/μL for L. flavobrunneum and R. pretiosus, respectively, and relative sensitivity of 0.01% for both so that it could be applied to detect actual samples. The two fishes were separately detected in one of the 50 samples, consistent with sequence alignment. Conclusion: The proposed method in this study displayed good specificity and sensitivity towards the detection of L. flavobrunneum- and R. pretiosus-derived components, and could provide technical support for the detection of adulterated aquatic products to protect consumer rights.
Rapid Detection of Tetracycline and Penicillin in Milk by Lateral Immunochromatography
WU Yuhan, CHEN Wei
2020, 41(24):  281-286.  doi:10.7506/spkx1002-6630-20190705-074
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A rapid double colloidal gold immunochromatographic test strip was developed for the simultaneous detection of residual tetracycline and penicillin in milk samples. The results showed that the detection limit for both tetracycline and penicillin was 2 ng/mL, and the detection time was 10 min. This method had good specificity, and exhibited no cross-reaction with other analytes commonly found in milk. The method is simple and reliable, and provides a simple and effective tool for the rapid and on-site detection of antibiotic residues in milk.
Determination of Residues and Migration of 17 Acrylate Monomers in Food Composite Packaging Materials by Gas Chromatography-Mass Spectrometry
ZHANG Qinjun, BEI Ronghua, ZHANG Hong, HU Changying
2020, 41(24):  287-294.  doi:10.7506/spkx1002-6630-20191113-164
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A gas chromatography-mass spectrometry (GC-MS) method was developed for simultaneous determination of 17 acrylate monomers in food composite packaging films and their migration to isooctane as an oily food simulant. The adhesive samples were diluted with methanol, and the composite film samples were extracted ultrasonically with methanol at 25 ℃ for 1 h. After the positive composite film samples were migrated to isooctane at 60 ℃ for 1 h or at 40 ℃ for 0.5 h, chromatographic separation was performed on an HP-INNOWax column prior to analysis in the selective ion monitoring (SIM) mode. An external standard method was used for quantitation. Under the optimized conditions, the limits of detection (LODs) and limits of quantitation (LOQs) for the 17 monomers were 0.02–0.1 and 0.05–0.50 mg/L, respectively. At four spiked concentration levels of 0.1, 0.5, 1.0 and 2.0 mg/L, the recoveries for acrylate monomer residues and migration were in the range of 70.1%–109.2%, with relative standard deviations (RSDs, n = 6) ranging from 0.8% to 9.2%. This method required simple sample pretreatment, exhibited good separation performance, and could simultaneously detect the 17 acrylate monomers within 17 minutes. When it was applied to determine the residual amounts of acrylate monomers in 6 kinds of adhesives and 35 kinds of food composite packaging films and the migration amounts of positive composite films to isooctane, acrylate monomers were detected in all the adhesives and in only eight of the food composite packaging films, from which no migration of acrylate monomers was found.
Screening of Aptamers for Aflatoxin B1 and Establishment of a Method for Aflatoxin B1 Detection
ZHANG Min, ZHANG Xianzhou, LI Cong, GAO Hao, LIU Jianhui, TIAN Yiling, MA Wen, LI Yingjun, TAN Jianxin
2020, 41(24):  295-303.  doi:10.7506/spkx1002-6630-20190628-369
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In this paper, we aimed to develop a sensitive and rapid method to detect aflatoxin B1 (AFB1). Based on the fact that graphene oxide (GO) can efficiently adsorb single-strand oligonucleotides (ssDNA) through π-π conjugation, but hardly adsorb ssDNAs bound to the targets, a scheme for screening aptamers for AFB1 was designed and performed. After 10 rounds of screening, a total of 45 ssDNA sequences were obtained. Secondly, six representative sequences were selected for further affinity analysis, whose dissociation constants (Kd) were 16.29, 27.88, 18.54, 23.11, 47.94, and 22.31 nmol/L, respectively. Subsequently, the specificity of the ssDNAs AFB-5 and AFB-8, with higher affinity, were determined. The results revealed that the selected aptamers could bind to AFB1 with high specificity. Finally, AFB-8 was used as recognition element to construct a gold nanoparticles (AuNPs)-based colorimetric assay for AFB1。Under the optimized detection conditions, AFB1 concentration (x) showed a good linear relationship with A520 nm (y), which was expressed by the following equation: y = ?0.007 09x + 0.461 42 (R2 = 0.997 9). The linear range and the detection limit were 0.025–10 ng/mL and 0.025 ng/mL, respectively. The specificity of this method was evaluated and the recoveries for spiked rice samples were in the range of 83.36%–105.74%. The results obtained proved the feasibility of the method.
Establishment of a Real-time PCR Method for Detecting Pseudomonas fluorescens in Food Samples
MIN Ke, ZHANG Zheng, ZHOU Yulei, HOU Wenfu, WANG Hongxun, ZHOU Min
2020, 41(24):  304-309.  doi:10.7506/spkx1002-6630-20191018-185
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In this study, we designed primers and probes targeting the gyrB gene of Pseudomonas fluorescens, prepared standard plasmids, drew standard curves, and established a real-time polymerase chain reaction (PCR) system for the detection of P. fluorescens. Our results confirmed that the PCR system was specific to P. fluorescens without detection of other tested bacteria. The sensitivity was 14.3 fg/μL and 3.0 × 102 CFU/mL for pure DNA and pure culture, respectively, and was not affected by the background interference at low concentrations. In artificially contaminated samples, P. fluorescens could be detected by appropriate enrichment. The TaqMan-based real-time fluorescence quantitative PCR method proved to be highly specific, sensitive and resistant to interferences.
A Fluorescence Method Based on AccuBlue and Nucleic Acid Aptamer for Detection of Enrofloxacin in Animal-Derived Foods
LIU Ruobing, HAO Yihuan, YANG Xi, JIAO Wenya, WANG Xianghong
2020, 41(24):  310-315.  doi:10.7506/spkx1002-6630-20190716-214
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AccuBlue fluorescent dye displays only weak fluorescence when being free or coexisting with single-stranded DNA, but significantly enhanced fluorescent signals when coexisting with double-stranded DNA. In this study, a novel fluorescence method for enrofloxacin detection was established based on the reaction between AccuBlue fluorescent dye and a nucleic acid aptamer. The optimal detection conditions were as follows: in a buffer solution at pH 8.5, the double strand was incubated for 10 min, and the fluorescent dye was allowed 6 min to recognize the double strand. Results indicated that the calibration curve was linear in the concentration range of 20–1 000 μg/L. Validation of the method yielded a limit of detection (LOD) (RSN ≥ 3) of 9.96 μg/L and a limit of quantification (LOQ) (RSN ≥ 10) of 29.97 μg/kg. The intra- and inter-plate variation coefficients were in the ranges of 4.97%–9.31% and 5.83%–10.96%, respectively. Recoveries measured for the extraction of enrofloxacin spiked into milk powder, shrimp and beef were between 76.54% and 98.79%. The method developed proved to be specific, stable, and repeatable, and could be easily implemented for rapid detection of enrofloxacin in real samples.
Determination of Major Soybean Allergens by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
LI Lifang, HUANG Wensheng, ZHANG Jiukai, WANG Yanbo, FU Linglin, HAN Xiaoxiang, CHEN Ying
2020, 41(24):  316-324.  doi:10.7506/spkx1002-6630-20190923-278
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In order to develop an accurate method to detect the major soybean allergens using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the signature peptides of the five main soybean allergen proteins were selected and verified. Total protein was extracted from samples under optimized conditions, and it was hydrolyzed by sequencing-grade trypsin. The resulting hydrolysate was analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) combined with computer search of the Uniprot database. Targeted identification of peptide fragments of the five major allergens (Glycinin subunits G1, G2, G3, G4 and G5, β-conglycinin α’, α and β subunits, Gly m Bd 30K, Gly m Bd 28K, and Kunitz trypsin inhibitors) in 24 soybean cultivars was performed. Finally a total of 29 signature peptides with high response value and good reproducibility were obtained, and they were verified by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS) in the multiple reaction monitoring (MRM) mode. The results showed that the specificity, sensitivity and stability of these peptides were in line with the requirements of mass spectrometry detection. In conclusion, this study provide a theoretical basis and technical support for the comprehensive and accurate detection of soybean allergens in foods.
Preparation and Application of Magnetic Molecularly Imprinted Polymer for the Detection of Bitertanol in Foods
TIAN Jingsheng, ZHANG Zhanzhan, QIN Sinan, GAO Wenhui
2020, 41(24):  325-332.  doi:10.7506/spkx1002-6630-20190717-234
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A magnetic molecularly imprinted polymer (MMIP) was prepared by surface imprinting technology using Fe3O4@SiO2 grafted with the silane coupling agent methacryloxy propyl trimethoxyl silane (KH570) as the magnetic carrier and bitertanol as the template molecule. MMIP prepared under optimized conditions was characterized by infrared spectoscopy, transmission electron microscopy and magnetic measurement. The adsorption properties of the MMIP for bitertanol were evaluated by static adsorption experiments. The MMIP was used as the solid phase extraction (SPE) adsorbent to extract bitertanol from various food samples such as millet and wheat, which was then purified before being detected by high performance liquid chromatography (HPLC). The results indicated that the MMIP, with a core-shell structure, could recognize bitertanol and its structural analogues. The proposed method showed good linearity with correlation coefficient ≥ 0.999 6. The limit of detection was 0.01–0.02 μg/mL, and the average recoveries were between 87.7% and 104.3%, with relative standard deviations from 1.39% to 3.46% (n = 5). The MMIP was suitable for the rapid separation and purification of triazole pesticides from foods, and the developed analytical method could selectively and fast detect triazole pesticide residues in foods.
Simultaneous Determination of Pb, As, and Cd in White Hypsizygus marmoreus by Microwave Digestion-Inductively Coupled Plasma-Mass Spectrometry and Health Risk Assessment
XU Mingfang, YUE Tian, FU Lijun, LIU Li, ZHANG Xiumin, MA Yongzheng, SHEN Linyan, SUN Yong
2020, 41(24):  333-339.  doi:10.7506/spkx1002-6630-20200613-184
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The contents of arsenic (As), lead (Pb) and cadmium (Cd) in white Hypsizygus marmoreus sold in the market of Guangzhou were determined by microwave digestion-inductively coupled plasma-mass spectrometry (MD-ICP-MS) and compared with those in other edible fungi (Lentinus edodes, Agaricus blazei and Pleurotus ostreatus). The potential health risks of heavy metals in these edible fungi to adults and children were evaluated by the target hazard quotient method target hazard quotient (THQ) and total target hazard quotient (TTHQ). The results showed that the MD-ICP-MS method exhibited a good linearity with a correlation coefficient greater than 0.999 5 for As, Pb and Cd within the concentration range of 0–100 μg/L. The recoveries from spiked samples were from 98.84% to 100.92% indicating good repeatability. The average content of heavy metal in white H. marmoreus was lower than that in L. edodes, A. blazei and P. ostreatus, and it was the highest in A. blazei. The contents of As and Cd in these edible fungi were higher than the national standard limits. Our data indicated that the various mushrooms had significantly different abilities to accumulate heavy metals and there were risks of Cd and As contamination for these mushrooms. The THQ values of As and Cd were higher than 1 and the TTHQ values of the three heavy metals were also higher than 1. The health risk of heavy metals in white H. marmoreus and the other three kinds of edible fungi to adults and children was mainly caused by As, followed by Cd. These results suggested that controlling the heavy metal contents in the cultivation materials, soil and wate is of important and practical significance for ensuring the safety of edible fungi. This study will provide a scientific theoretical basis for the development of processed white H. marmoreus products and for quality and safety evaluation.
Rapid Determination of Aspartame and Alitame in Food by Reversed-Phase High Performance Liquid Chromatography with Mixed Standard and Sample Increment Method
GAO Xiangyang, ZHANG Fang
2020, 41(24):  340-345.  doi:10.7506/spkx1002-6630-20200729-367
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A new rapid method for the simultaneous qualitative and quantitative analysis of aspartame and alitame in beverage and yogurt was established using reversed-phase high performance liquid chromatography with mixed standard and sample increment method. The results showed that the minimum detection limits of aspartame and alitame in beverages and liquid dairy products were 0.21 and 0.020 mg/kg, respectively. The recoveries ranged from 90.5% to 98.3%, with relative standard deviation (RSD) less than 5%. The results obtained from the proposed method showed no significant difference compared with the national standard method. This method does not need to measure blanks and draw a standard curve. It is simple, fast and low-cost, and allows the simultaneous qualitative and quantitative analysis of standard and sample solutions under the same conditions. Satisfactory results can be obtained when it is used for the rapid determination of aspartame and alitame in food.