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18 January 2021, Volume 42 Issue 2
Food Chemistry
Preparation and Antigenicity of Site-specific PEGylated Beta-lactoglobulin
LIU Chengmei, JIANG Xinlin, LI Dongmei, ZHOU Lei, ZHONG Junzhen, JI Li, LUO Shunjing
2021, 42(2):  1-7.  doi:10.7506/spkx1002-6630-20200116-198
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In order to investigate the effect of different covalent modification sites on the antigenicity of β-lactoglobulin (β-LG), β-LG was modified by using cysteine-specific PEGylation at the thiol group of Cys121. Using one-factor-at-a-time method, the optimal modification conditions were determined as follows: β-LG and mPEG-MAL (molar ratio, 1:30) were dissolved together in 0.1 mol/L phosphate buffer containing 0.3 mol/L tris-(2-carboxyethyl) phosphine (TCEP), 25% (V/V) ethanol and 2 mmol/L ethylenediaminetetraacetic acid (EDTA) at pH 7.0 and incubated at 4 ℃ for 24 h, yielding a maximum modification efficiency of 64.1%. The resulting product was purified to a purity of 94%. The free sulfhydryl content of β-LG decreased from 52.3 to 0.3 μmol/g after the site-specific PEGylation. It was demonstrated that mPEG-MAL was specifically conjugated with β-LG at the thiol group of Cys121. The antigenicity of β-LG increased by 25.9% after the cysteine-specific PEGylation, contrary to results previously obtained by modification at other amino acid positions. Accordingly, site-specific PEGylation can allow effective regulation of β-LG antigenicity.
Ultrasonic-assisted Preparation and Stability Evaluation of W/O Emulsion Containing Chitosan
MAO Zhijuan, ZHANG Kunming, HUANG Yongchun, GUO Yan, YANG Feng, REN Xian’e, AI Shuo
2021, 42(2):  8-16.  doi:10.7506/spkx1002-6630-20191026-296
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W/O emulsions were prepared ultrasonically using Newtonian pseudoplastic chitosan solution as the aqueous phase, palm oil as the oil phase and Span-80 as the emulsifier. The preparation process was optimized using a combination of one-factor-at-a-time method and response surface methodology. The stability of the emulsion prepared under optimized conditions was examined. The results showed that the proportion of inner aqueous phase was a critical factor affecting the mean droplet size, and excessively high ultrasonic power led to an increase in particle size and wider particle size distribution. The optimal preparation conditions were obtained as follows: ultrasonic power 300 W, ultrasonic time 15 min, inner aqueous phase 13%, and Span-80 6%, giving a mean droplet size of (156.1 ± 20.0) nm and a polydispersity index (PDI) of (0.43 ± 0.03). The stability test and the micro-morphology observation indicated that the prepared emulsion was stable at 25 ℃ and showed creaming?index of 0.6%, while the emulsion stability decreased sharply at 50 ℃ and the creaming?index increased up to 7.0%. The increase in particle size and more uneven particle size distribution were detrimental to the emulsion stability, and aggregation was the main cause of the decrease in the emulsion stability.
Preparation and Properties of Rice Oil-based Oleogel with Sugarcane Wax
WANG Weining, WANG Ying, YU Yang, JIANG Yuting, WU Fei, HAN Cuiping, YU Dianyu, SHI Yongge
2021, 42(2):  17-22.  doi:10.7506/spkx1002-6630-20200330-438
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A plastic oleogel was made from rice oil with sugarcane wax added. The effect of the amount of sugarcane wax added on the hardness, thermodynamic properties, solid fat content (SFC), X-ray diffraction (XRD) profile and microstructure of oleogels was studied. The results showed that when the amount of sugarcane wax added was not lower than 7%, gel-like behavior appeared at 20 ℃. The hardness, SFC, melting enthalpy and crystallization enthalpy of the oleogel increased with the increase in sugarcane wax concentration. The XRD results showed that α-, β- and β’-type crystals were found in the oleogel, with type β being dominant. The amounts of α and β’ crystal type increased with increasing sugarcane wax concentration. The crystals were spherical and evenly distributed. As the concentration of sugarcane wax increased, the number of crystals increased while the size decreased, resulting in increased distribution density. The higher concentration of sugarcane wax resulted in greater hardness, stronger structured vegetable oil-forming ability and better structural stability of the oleogel. These results showed that trans fatty acids (TFAS)-free, natural nutrient-rich oleogels with the advantages of appropriate hardness and good structural stability could be formed by incorporating sugarcane wax into rice oil.
Effect of Limited Alcalase Hydrolysis on Functional Properties of Blue Round Scad (Decapterus maruadsi) Protein Isolate
SUN Lechang, LIU Weifeng, LIN Yichen, ZHAO Ayun, ZHANG Lingjing, WENG Ling, CAO Minjie
2021, 42(2):  23-29.  doi:10.7506/spkx1002-6630-20191029-326
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In the present study, limited hydrolysis of protein isolate from blue round scads was performed with alcalase. The effects of the degree of hydrolysis (DH) on the solubility, oil-holding capacity and functional properties including emulsifying and foaming properties of hydrolysates were investigated. The results showed that the molecular masses of the proteins were remarkably decreased by alcalase hydrolysis. The solubility of the proteins was significantly improved and increased as DH increased. The emulsifying and foaming properties of the proteins significantly increased after hydrolysis, showing a trend of increased first and then decreased with increasing DH. At pH 4.0, the worst emulsifying and foaming properties were recorded for the hydrolysates at different DHs. The hydrolysate with 5% DH had the highest emulsifying capacity ((100.9 ± 0.7) m2/g) and highest foaming capacity ((227.3 ± 3.8)%) at pH 10.0 and 7.0, respectively. All hydrolysates except 20% DH showed a notable increase in oil-holding capacity, with the highest value of 3.50 g/g (oil/protein) being found at 5% DH. In conclusion, a certain degree of hydrolysis could remarkably improve the functional properties of protein isolate from blue round scads. The present work provides a theoretical reference for application of fish protein isolate as a protein ingredient in foods.
Effect of Lysine-Galactose Mixture on the Degradation of Trimethylamine N-Oxide in TMAO-Fe (II) System
LI Yingchang, LI Shuangyan, CAO Nana, LIU Xuefei, CAI Youqiong
2021, 42(2):  30-35.  doi:10.7506/spkx1002-6630-20191031-357
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In order to investigate the effect of lysine-galactose (Lys-Gal) mixture on the thermal decomposition of trimethylamine N-oxide (TMAO) and consequently provide a theoretical basis for controlling the degradation of TMAO, we established an in vitro TMAO-Fe (II) model system and studied the mechanism of action of Lys-Gal on the thermal decomposition of TMAO by differential scanning calorimetry (DSC). The results showed that Lys, Gal and Lys-Gal had a promoting effect on TMAO degradation. Under the conditions of higher reaction temperature, longer reaction time and higher concentration ratio, Lys-Gal was more effective in this regard, generating significantly more formaldehyde, dimethylamine and trimethyl. The DSC curve of TMAO thermal decomposition in the Lys-Gal-TMAO-Fe (II) system showed the most significant change along with three endothermic peaks compared to two endothermic peaks with the addition of either Lys or Gal, and the thermal decomposition temperature was lower, promoting the thermal decomposition of TMAO. This effect of Lys-Gal might be related to its ability to reduce the thermal decomposition temperature of TMAO.
Preparation and Microencapsulation of Perilla Seed Oil Emulsion Supplemented with Corn Oligopeptide
LIU Yifeng, HOU Zhanqun, TIAN Qiaoji, XIA Kai, YU Youqiang, PAN Cong, DUAN Shenglin, LUO Anlai, HAN Xiaofeng
2021, 42(2):  36-45.  doi:10.7506/spkx1002-6630-20191106-082
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In this study, five different emulsifiers, gum arabic, soluble soybean polysaccharide, starch sodium octenylsuccinate (HI-CAP 100), sodium caseinate and soybean protein isolate (SPI) were separately used to prepare perilla seed oil emulsions with the addition of different concentrations of corn oligopeptide. We selected the optimal emulsifier and corn oligopeptide concentration for further preparation of microcapsules with high oil loading by spray drying. We chose and evaluated the optimal wall material for maximum oil loading. The results showed that the droplet size of perilla seed oil emulsion prepared using HI-CAP 100 as the emulsifier was mainly distributed between 0.1 and 2 μm; upon the addition of 5% corn oligopeptide, the particle size was small, only (0.76 ± 0.02) μm, and the instability index was 0.275. The surface oil percentage of the microcapsule prepared using HI-CAP 100 (with oil loading ≥ 50%) as the wall material was 3%, indicating good microencapsulation efficiency, and the microcapsule exhibited uniform particle size distribution and smooth surfaces. Accelerated storage tests proved that combination of corn oligopeptide and tea polyphenol palmitate could effectively improve the antioxidant capacity of perilla seed oil microcapsules.
Effect of Laver (Porphyra umbilicalis) Powder on Gel Properties and Protein Structure of Giant Squid (Dosidicus gigas) Surimi
XU Anqi, YANG Rong, ZHU Yukang, SHENG Zhenzhen, SONG Linlu, JIA Ru, HUANG Tao, YANG Wenge
2021, 42(2):  46-52.  doi:10.7506/spkx1002-6630-20191110-121
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In order to improve the gel quality of giant squid (Dosidicus gigas) surimi and illustrate the underlying mechanism, this study determined the effects of laver powder addition on the sensory quality, whiteness, gel strength, water-holding capacity (WHC), microstructure and the conformation and secondary structure of myofibrillar protein (MP) in giant squid surimi. The results showed that laver powder had a significant effect on the whiteness of surimi gel, which made the surimi gel turn slightly yellow-green. The gel strength and WHC of surimi gel significantly increased with increasing addition of laver powder up to 0.4%–0.6%, and then declined and did not change significantly, respectively. Furthermore, compared with the control group, the addition of 0.4%–0.6% laver powder caused an increase in the α-helix content and a decrease in the β-sheet content of MP in the surimi gel. Laver powder could reduce the transformation from α-helix to β-sheet in MP, as well as strengthen the three-dimensional network structure of surimi gel and improve the gel strength and WHC. In conclusion, appropriate laver powder addition (0.4%–0.6%) could significantly affect the gel characteristics and secondary structure of MP in giant squid surimi, thereby improving product quality.
Effect of Nostoc commune on Gelation and Rheological Properties of Chicken Breast Meat Batters
LI Ningning, LI Zhao, ZHAO Shengming, MA Hanjun, ZHANG Wenzhen, KANG Zhuangli, ZHU Mingming, WANG Zhengrong, HE Hongju
2021, 42(2):  53-59.  doi:10.7506/spkx1002-6630-20191031-348
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In this study, the effects of Nostoc commune addition on the cooking yield, water-holding capacity, color, texture properties, gel strength, dynamic rheology and microstructure of chicken breast meat batters were investigated. The results indicated that cooking yield and water-holding capacity increased firstly, reaching a maximum of 95.71% and 95.33% at an addition level of 1.2%, respectively, and then decreased with increasing level of N. commune addition. L*, a* and b* values significantly decreased (P < 0.05), while hardness, springiness, cohesiveness and chewiness rose constantly except that the difference in hardness and springiness was not statistically significant between addition levels of 1.2% and 1.5% (P > 0.05). Upon heating, the storage modulus G’ at 20 and 80 ℃ increased with increasing addition of N. commune; dynamic frequency scanning showed that G’ increased first until reaching a maximum at 1.2% addition level and then decreased with N. commune addition at the same oscillation frequency. The results of scanning electron microscopy showed that compared with the control group, the microstructure of chicken breast meat batters added with N. commune was more compact and uniform. In summary, addition of an appropriate amount of N. commune can significantly improve the gel properties of chicken breast meat batters. This study can provide a theoretical basis and technical support for the application of N. commune in minced meat products.
Effects of pH on the Types and Taste Characteristics of Flavor Peptides in Soy Sauce Produced under Low Temperature Stress
WANG Zhuo, CHEN Churui, XU Liqiang, ZHUANG Peirui, ZHOU Wensi, CUI Chun
2021, 42(2):  60-65.  doi:10.7506/spkx1002-6630-20190716-210
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Solid phase extraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to separate and identify the acidic components of soy sauces fermented at different pH values and their taste characteristics were evaluated. The results showed that a total of 14 flavor peptides were identified from the soy sauce from moromi with pH adjustment, and 12 flavor peptides without pH adjustment. Eight flavor peptides were common to both. The first soy sauce sample contained many small peptides greatly contributing to the taste, among which ED, EE, EF, ESAY and AELY had rich umami and sour tastes with the lowest threshold value at a concentration of 500 mg/L. FLET and SV had kokumi taste at 500 mg/L. In the second soy sauce sample, SV, LLVVQ and ALVLL exhibited a strong kokumi taste, and SV had the lowest threshold value, 500 mg/L. QLLN, LLVVQ, ESAY, AELY, FLET, FLTW, ALVLL, QVELF and SV were synthesized and added to soy sauce at 0.03 g/100 mL each. ESAY was identified to significantly reduce the salty taste and to enhance the kokumi taste of soy sauce. QLLN significantly enhanced the umami taste, while LLVVQ, AELY and FLET did so only slightly, and SV had distinct kokumi-enhancing effect. This study showed that the flavor peptides in the soy sauce fermented by adjusting pH had good flavor characteristics, and imparted rich and well-balanced umami and kokumi tastes to the soy sauce.
Bioengineering
Sequence Analysis and Binding Activity of Salmonella Phage LPST144 Tail Fiber gp38
YANG Qile, DING Yifeng, ZHANG Yu, NIE Ruonan, LI Yameng, WANG Jia, WANG Xiaohong
2021, 42(2):  66-73.  doi:10.7506/spkx1002-6630-20191031-359
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In this study, the tail fiber gene orf38 of Salmonella Podoviridae LPST144 was sequenced and expressed and the product (gp38) was purified, whose specific receptor binding activity was successfully verified, yielding a potential probe for Salmonella detection. Bioinformatics was used to analyze the amino acid sequence, physicochemical properties, genetic evolution relationship and C-terminal secondary structures of LPST144 tail fiber gp38. Results showed that the tail fiber gp38 contained 646 amino acid residues, and two conserved regions with high similarity to T7 existed at its N-terminal. However, the C-terminal sequence was extremely different, and there were large numbers of β-sheet structures similar to the C-terminal secondary structures of T7 tail fiber. gp38 had many features of phage receptor binding proteins such as modular properties, low sequence similarity and β-sheet-rich C-terminal. The ability of the recombinant protein to specifically bind to the host Salmonella typhimurium (ATCC13311) was detected by whole bacterial cell coating ELISA. Results showed that the absorbance value of the experimental group was 0.94 ± 0.02, as compared to 0.17 ± 0.01 for the negative control, indicating strong binding ability to the host cells. The recombinant gp38 could also be combined with six other serotypes of Salmonella tested. When Escherichia coli (T10), Salmonella outer membrane protein, Staphylococcus aureus (6538) and PBS buffer were used instead of the host cells, the absorbance values were 0.58 ± 0.03, 0.56 ± 0.01, 0.59 ± 0.03 and 0.53 ± 0.005, respectively. The observed significant difference between the experimental and control groups indicated that the tail fiber gp38 has the ability to specifically bind to the host S. typhimurium (ATCC13311) and the other serotypes of Salmonella. This study can lay an experimental foundation for the development of Salmonella detection methods using phage receptor binding proteins as a molecular probe.
Comparative Assessment of Modified Sodium Dodecyl Sulfate-Phenol Method and Other Methods for Total RNA Extraction from Fermented Grains of Chinese Strong-Flavor Baijiu
HU Xiaolong, WANG Kangli, SONG Lili, HOU Jianguang, CAO Zhenhua, WU Lili, NIU Guangjie, MA Geli, ZHAO Shumin, ZHAO Dong
2021, 42(2):  74-82.  doi:10.7506/spkx1002-6630-20191110-106
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A modified sodium dodecyl sulfate (SDS)-phenol method was proposed for total RNA extraction from fermented grains of Chinese strong-flavor Baijiu, a complex microecological environment unique to China. This method was compared with other common methods including commercial kit, Trizol method, hexadecyl trimethyl ammonium bromide (CTAB) method and sodium laurate method in regard to cost, extraction time, RNA concentration and purity, electrophoresis results, reverse transcription efficiency and high-throughput sequencing analysis. The results showed that the SDS-phenol and CTAB methods were inexpensive, while the kit method was the most time saving but most expensive. The total RNA concentration extracted by the kit method was the highest, which was 1.84, 2.33, 19.14 and 3.09 times as high as that by the modified SDS-phenol, Trizol, CTAB and sodium laurate methods, respectively. The OD260 nm/OD280 nm and OD260 nm/OD230 nm ratio of total RNA extracted by the SDS-phenol and kit methods were greater than 1.8 and 2.0, respectively. The electrophoresis showed more intact bands for total RNA extracted by the SDS-phenol method as compared to that extracted by the other methods, and but no bands for total RNA extracted the by the CTAB method. RNA extracted by the modified SDS-phenol, kit and Trizol methods could be efficiently reversely transcribed into cDNA and used for high-throughput sequencing analysis. The sequencing results showed that the dominant microbial genera (Lactobacillus and Clostridium) obtained by the three RNA extraction methods and the change of microbial population abundance in fermented grains at different fermentation times were basically the same. Among the three methods, the SDS-phenol method gave the highest diversity of microbial communities and could be useful to determine the differences in microbial groups among fermented grains at different fermentation times. Both the Trizol and kit methods could preferentially detect one more genus, but at very low levels (0.01%). In general, the modified SDS-phenol method was advantageous over the other RNA extraction methods. This study provides a theoretical basis for molecular biological research on fermented grains of Chinese Baijiu based on RNA in the future, and it is of reference significance for RNA extraction from environmental samples rich in sugar, humus and phenols.
Enantioselective L-Phenylglycine Production from D,L-Mandelic Acid Using Engineered Escherichia coli Whole Cells
JIA Yuanyuan, LI Xiang, ZHANG Zhenhua, ZHANG Shan, YANG Lulu, TANG Cunduo
2021, 42(2):  83-89.  doi:10.7506/spkx1002-6630-20191104-030
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In this study, a recombinant Escherichia coli strain carrying D-mandelate dehydrogenase, L-leucine dehydrogenase and mandelate racemase encoding genes was established by using a dual plasmid co-expression system of pACYCDuet-1 and pET28a, which was named as E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-LhDMDH:pET28a-ArMR. Under low temperature and in the presence of low concentrations of the inducer, the recombinant strain successfully expressed three recombinant enzymes with catalytic activities, and the activities of D-mandelate dehydrogenase, L-leucine dehydrogenase and mandelate racemase in its fermentation broth were 195.8, 56.2 and 174.5 U/mL respectively. Using the induced whole cells as the catalyst and D,L-mandelic acid as the substrate, the yield of L-phenylglycine was 77.48% after reaction at 30 ℃ for 48 h in 500 mmol/L NH4Cl-NH3·H2O buffer at pH 9.5 with an agitation rate of 180 r/min with an enantiomeric excess (e.e.) value of greater than 99%. This study has potential for industrial application, laying a solid foundation for large-scale biosynthesis of L-phenylglycine.
Overexpression and Enzymatic Characterization of Leucine Aminopeptidase (LapA) in Aspergillus niger
LIN Xiaotong, DONG Liangbo, ZHENG Junwei, WANG Bin, PAN Li
2021, 42(2):  90-96.  doi:10.7506/spkx1002-6630-20191101-005
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Considering that the industrial production of food-grade leucine aminopeptidase is low in China, in this study, we investigated the recombinant expression of the five leucine aminopeptidase encoding genes (lapA, lap1O, lap2, lap1S and lap1N) from Aspergillus oryzae, A. sojae and A. niger in aconidial A. niger strain HL-1 with a low-background of protein secretion. The encoding region was improved via signal peptide replacement, and the expression vector was constructed with the hybrid promoter PnaII and the auxotroph marker pyrG. The homologous recombination method combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was used to improve gene integration efficiency and protein expression. The LapA activity of the recombinant strain was 11 701.2 U/mL, about 4.7 times higher than that (only 2 476.0 U/mL) obtained without using CRISPR. The recombinant enzyme was purified using the 6 × His tag for enzymatic characterization. The molecular mass of the purified LapA was shown to be 35.0 kDa. The optimum reaction temperature and pH for the recombinant LapA were 65 ℃ and 8.5, respectively. Overall, the recombinant LapA was successfully overexpressed in A. niger.
Effect of Mixed Culture Fermentation with Rhodotorula mucilaginosa and Saccharomyces cerevisiae on the Aroma and Color of Red Wine
MA Na, WANG Xingchen, KONG Cailin, TAO Yongsheng
2021, 42(2):  97-104.  doi:10.7506/spkx1002-6630-20191112-158
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This study aimed to investigate the effect of different mixed culture fermentation strategies with Rhodotorula mucilaginosa and Saccharomyces cerevisiae on the aroma profile, polyphenols and color of dry red wine produced in the monsoonal region of China, and to optimize wine aroma enhancement. Merlot grapes produced in Heyang country, Shaanxi province were used to make dry red wines by simultaneous or sequential inoculation of the two strains. After completion of fermentation, the aroma attributes of wines were determined by solid-phase microextraction coupled to gas chromatography-mass spectrometry (SPME-GC-MS) and a well-trained panel. A total of 13 color parameters and polyphenols were quantitatively determined by spectroscopy. Results showed that simultaneous inoculation with higher proportion of R. mucilaginosa significantly increased the concentration of the varietal aroma compounds, especially C6 compounds and thiol, whereas sequential inoculation of R. mucilaginosa and S. cerevisiae (1:1) was more conducive to the release of terpenols and β-damascenone. In the case of simultaneous inoculation, the contents of higher alcohols, C6–C12 fatty acids and their ethyl esters elevated with increasing proportion of R. mucilaginosa, while sequential inoculation was markedly advantageous in increasing the contents of acetates, other esters and phenyl ethyls. Both simultaneous inoculation with R. mucilaginosa/S. cerevisiae ratio = 4:1 and sequential inoculation remarkably increased the contents of total anthocyanins, flavonols, and tartaric esters, as well as the proportion of co-pigmented anthocyanins. Sensory analysis revealed that simultaneous inoculation with higher proportion of R. mucilaginosa remarkably improved the intensities of fruity and floral aroma as well as green taste and animal-like flavor. Sequential inoculation moderately enhanced the floral and fruity aroma while reducing off-odor intensity. In summary, significant variations in color and flavor characteristics existed among wines produced using the two inoculation strategies. Sequential inoculation moderately increased wine aroma without producing off-odor, and also improved color quality.
Process Optimization for the Development of Fermented Yak Milk Containing Lactobacillus fermentum HY01 and Dynamic Analysis of Main Flavor Components
YU Susu, YANG Jiajie, MA Xiangyang, JIA Xindong, LIU Libo, DU Peng, LI Aili
2021, 42(2):  105-113.  doi:10.7506/spkx1002-6630-20191104-025
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The probiotic strain Lactobacillus fermentum HY01 combined with the commercial starter culture MY105 of Streptococus thermophiles and Lactobacillus delbrueckii subsp. bulgaricus was used to produce fermented yak milk. The fermentation conditions were optimized through the combined use of one-factor-at-a-time method and response surface methodology, and the quality changes of the yogurt prepared under optimized conditions were observed during storage. The changes in aroma components at different post-ripening times were analyzed by electronic nose (EN). The results showed that the optimal process conditions were obtained as follows: HY01 inoculation amount 4%, fermentation temperature 40 ℃, and time 6 h, and the viable count of probiotics in the prepared yogurt was 8.90 × 108 CFU/mL and it scored 89.72 points in sensory evaluation. During the storage period of 28 days, the number of probiotics was maintained higher than 107 CFU/mL and the acidity lower than 118 °T. Texture properties (viscosity, hardness, consistency and cohesiveness), the contents of diacetyl and acetaldehyde and sensory score remained basically unchanged from day 0 to 14 d during storage, but decreased then. In addition, principal component analysis performed on the EN data showed that the characteristic aroma of yak yogurt was composed of nitrogen oxides, methyl compounds, sulfides and alcohols, whose contents were significantly different from those of the yogurt. Therefore, yak yogurt containing L. fermentum HY01 not only has good performance and flavor, but also is expected to be beneficial for intestinal health.
Metabolic Engineering of Escherichia coli for Efficient Synthesis of L-Phenylalanine
MEN Jiaxuan, XIONG Bo, HAO Yanan, LI Xuan, LIU Yining, XIE Xixian
2021, 42(2):  114-120.  doi:10.7506/spkx1002-6630-20191020-200
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In order to obtain an engineered strain with good genetic stability and high ability to produce L-phenylalanine without plasmid, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to introduce the deinhibited bifunctional enzyme (PheA) of chorisate mutase and prephenate dehydratase into E. coli W3110, and then used the promoters with different strengths to regulate the enzymes involved in the shikimate pathway. The optimal expression intensity of the enzymes in the shikimic acid pathway was determined by shaking flask fermentation. Finally, strain PHE12 with the best performance of L-phenylalanine production was obtained by increasing the supply of the precursor phosphoenolpyruvate. The L-phenylalanine titer of PHE12 was 20.5 g/L after 24 h shaking flask fermentation. And after fed-batch fermentation for 48 h, L-phenylalanine production reached 81.8 g/L, which increased by 12.2% as compared with the highest yield reported in the literature. The production intensity and the conversion rate of sugar and acid were 1.7 g/(L·h) and 0.24 g/g glucose, respectively, indicating that this strain is promising for industrial application.
Screening of Lactic Acid Bacteria for Antagonistic Activity against Drug-Resistant Escherichia coli and Underlying Mechanism
SUN Yue, LIU Jiayi, CHEN Lu, DU Hong, BAI Fengling, LÜ Xinran, ZHANG Defu, GUO Xiaohua, LI Jianrong
2021, 42(2):  121-127.  doi:10.7506/spkx1002-6630-20191112-162
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The aims of this study were to screen lactic acid bacteria (LAB) strains isolated from pickled Chinese cabbage for antagonistic activity against drug-resistant Escherichia coli, and to explore the underlying mechanism of action. Six target strains were selected by the oxford cup agar well diffusion method, among which strain XCT1-1 exhibited the largest diameter of inhibition zone against drug-resistant E. coli and was identified by physiological and biochemical tests and 16S rDNA sequence analysis. To shed light on the antibacterial mechanism of the ethyl acetate extract of the cell-free culture supernatant of XCT1-1 against drug-resistant E. coli, the effect of treatment with azithromycin and/or the culture supernatant extract on the electrical conductivity and extracellular proteins, and ultraviolet (UV) absorbing materials of drug-resistant E. coli. The results showed that the diameter of inhibition zone of strain XCT1-1 against drug-resistant E. coli was 20.31 mm and it was identified as Lactobacillus plantarum. After 10 h treatment with the culture supernatant extract of XCT1-1 alone and combined with azithromycin, the electrical conductivity increased by 20.54% and 21.93%, the content of extracellular protein by 25.24% and 27.93%, and the content of UV absorbing materials by 63.56% and 77.12%, respectively. The scanning electron microscope (SEM) images showed that the cell wall and membrane of drug-resistant E. coli were damaged by the culture supernatant extract alone and in combination with azithromycin. The bacterial cell surface became wrinkled after the former treatment, while the latter treatment resulted in the collapse of the cell structure. These results indicated that the culture supernatant extract of XCT1-1, azithromycin and their combination exerted antagonistic effects by damaging the cell membrane. In addition, the culture supernatant extract of strain XCT1-1 increased the susceptibility of drug-resistant E. coli to azithromycin.
Microbial Diversity and Quality Characteristics of Huizhou Stinky Mandarin Fish and Antioxidant Activity of Its Enzymatic Hydrolysates
WU Yongxiang, WANG Tingting, ZHANG Mengting, ZHANG Yao, GUO Xiaocheng, SUN Hanju, CHEN Xiangyang
2021, 42(2):  128-134.  doi:10.7506/spkx1002-6630-20191016-154
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The microbial community composition and diversity in Huizhou stinky mandarin fish from Huangshan, Anhui province were investigated by high-throughput sequencing, and the nutritional components, physicochemical properties and texture properties were evaluated in comparison with those of fresh mandarin fish. Meanwhile, the antioxidant activity of stinky mandarin fish hydrolysates prepared with different proteases was tested. The results showed that the microbial community composition in stinky mandarin fish was greatly different from that in fresh mandarin fish, and so was the dominant bacteria. Pseudomonas and Carnobacterium were the predominant genera in fresh mandarin fish, which accounted for 69.30% and 17.33% of the total genera, respectively. Psychrobacter and Vagococcus were the predominant genera in stinky mandarin fish, accounting for 59.96% and 26.97% of the total genera, respectively. The contents of ash, soluble polypeptide and total soluble sugar, and hardness of stinky mandarin fish were higher than those of fresh fish, while the contents of moisture and crude protein, and water activity significantly decreased. Moreover, significant variation in the color parameters a*, b* and L* was found between fresh and stinky mandarin fish. It was found that the composition and abundance of the microflora had a great influence on physicochemical indexes of stinky fermented mandarin fish, which were more largely influenced by Vagococcus than by other bacteria. Scanning electron micrographs (SEM) revealed more significant cell shrinkage and cell shape curling in stinky mandarin fish compared with the fresh fish. After protein hydrolysis of stinky mandarin fish with acid protease, neutral protease, alkaline protease or papain, soluble polypeptide contents significantly increased (P < 0.05). All resulting hydrolysates possessed stronger 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and reducing power than those of the fermented fish (P < 0.05). In conclusion, this study provides a theoretical reference for the deep processing and utilization of Huizhou stinky mandarin fish.
Effect of Suantangzi Sourdough on the Quality of Bread
BAI Xue, ZHAO Shanshan, XIAO Zhigang, ZHANG Yifan, ZHAO Xiuhong
2021, 42(2):  135-142.  doi:10.7506/spkx1002-6630-20191115-182
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This study aimed to study the microbial diversity of eleven samples of Suantangzi sourdough collected from different areas by high-throughput sequencing technology and to analyze the correlation between the population of the predominant microorganisms and bread quality. The results showed that six genera of lactic acid bacteria were identified from these samples, including Lactobacillus, Leuconostoc, Lactococcus, uncultured Streptococcaceae, Pediococcus and Streptococcus. Lactobacillus was detected in most of the samples. Thus, it was speculated to be the dominant bacterial genus of Suantangzi sourdough. Moreover, seven genera of yeast were also identified from these samples, including Candida, Pichia, Dipodasous, Zynosaccharomyces, Naumovozyma, Metschnikowia and Sugiyamaella. Both Candida and Pichia were detected in most of the samples, and they were speculated to be the dominant fungal genera of Suantangzi sourdough. The populations of lactic acid bacteria and yeast in Suantangzi sourdough were high, and the higher the yeast population, the lower the hardness, stickiness, and chewiness of bread, and the smaller the change in hardness and moisture during storage, indicating better bread quality.
Screening and Mutagenesis of Broad-Spectrum Antagonistic Bacillus licheniformis and Purification and Identification of Antimicrobial Substances Produced by Its Mutant
GAO Zhaojian, WANG Qiufen, DING Feihong, XU Xiang, ZHAO Yifeng, JIAO Wei, CHEN Teng
2021, 42(2):  143-150.  doi:10.7506/spkx1002-6630-20191112-161
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The aims of this study were (1) to isolate and characterize bacterial strains capable of producing broad-spectrum antimicrobial substances and (2) to separate and identify these antimicrobial compounds. Strain identification was performed based on morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis. To obtain genetically stable strains able to produce a high yield of antimicrobial compounds, we carried out mutagenesis by ultraviolet irradiation combined with diethyl sulfate treatment. Then, the antimicrobial substances were isolated and purified sequentially using methyl alcohol extraction, gel filtration chromatography (Sephadex G-10) and semi preparative high performance liquid chromatography (HPLC), and were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). In this study, a total of 67 Bacillus strains were isolated from soils, eight of which showed strong antimicrobial effects, including strains XF32, which showed significant broad-spectrum antimicrobial and was identified as B. licheniformis. It displayed an antibacterial activity against seven pathogenic bacterial species, including Staphylococcus aureus, B. subtilis, B. cereus, Escherichia coli, Salmonella sp., Shigella sp., and Pseudomonas aeruginosa. The strain also exhibited antifungal activity against Saccharomyces cerevisiae, Aspergillus niger, Trichoderma atroviride and fusarium oxysporum. Finally, mutant XF32-22 was obtained, whose antibacterial activity against S. aureus was increased significantly as compared to that of the original strain (XF32). Then, one antimicrobial substance was isolated and purified from the fermentation broth of XF32-22, and was identified as fengycin, a lipopeptide. The mutant produced significantly more fengycin than did its parental strain. Fengycin could strongly inhibit multiple bacterial pathogens as well as plant pathogenic fungi. Mutant XF32-22 holds great promise for application in food preservation and agricultural biological control.
Screening and Identification of Probiotics with Hyaluronidase Inhibitory Activity in Vitro
LEI Wenping, ZHOU Hui, CHEN Qi, ZHOU Xingrong, WU Kun, WANG Jiaqi, LIU Chengguo
2021, 42(2):  151-157.  doi:10.7506/spkx1002-6630-20191104-038
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Probiotic strains were isolated by the traditional lactic acid bacterial isolation procedure and the in vitro probiotic potential evaluation test and rapidly screened for potential anti-allergic activity by the in vitro hyaluronidase inhibition test. The results showed that five probiotic strains tolerant to acid and bile salt and were isolated from milk samples, which were initially identified as safe strains, and all the strains displayed high hyaluronidase inhibitory activity, which could be considered as a selection criterion for probiotic strains with anti-allergic activity. Strains L02 and L15 showed significant (P < 0.05) in vitro tolerance, cell surface characteristics and hyaluronidase inhibitory activity, indicating that they have anti-allergic probiotic potential. The strains were identified by 16S rDNA sequencing as Lactobacillus plantarum.
Composition of Lactic Acid Bacteria in Traditional Chinese Pickles and Analysis of Acid Production and Acid Tolerance Characteristics of Excellent Strains
LUO Qiang, LI Xingyang, CHEN Lianhong, ZHANG Ming, LIU Qiao, ZHANG Dawei, LUO Fan
2021, 42(2):  158-163.  doi:10.7506/spkx1002-6630-20191020-207
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Using the traditional separation and purification method, we isolated a total of 435 strains suspected of being lactic acid bacteria from 20 samples of naturally fermented pickles collected from 4 different regions of northern China. We used 16S rDNA sequence homology analysis for species identification. The results showed that all the isolates belonged to lactic acid bacteria, including 394 strains of Lactobacillus modestisalitolerans, L. sakei, L. coryniformis, L. plantarum, L. brevis and L. delbrueckii, 38 strains of Enterococcus, 2 strains of Leuconostoc mesenteroides, and 1 strain of Pediococcus siamensis. The composition of microbial flora in pickle samples from different regions was different. Among them, the most abundant microbial flora was found in samples from Liaoning and Henan provinces, consisting of six species. The number of acid-producing bacteria found in pickle samples from Jilin was the highest. Nine of these strains were found to have a strong ability to produce acid over 24 h, and their survival rates were above 70% after 3 h of exposure to simulated gastric juice (pH 2.0). This study provides a theoretical guidance and technological support for the quality improvement of traditional pickles and the development of high-quality strain resources.
Effect of Maturity on the Quality of Noni (Morinda citrifolia L.) Fruit Jiaosu (Fermented Fruit Juice)
ZHAO Wenke, XIAO Chunqin, ZHANG Xian, CUI Chun
2021, 42(2):  164-169.  doi:10.7506/spkx1002-6630-20191018-191
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The quality changes of immature (green fruit) and mature noni fruit (yellow fruit) during natural fermentation (the fermented fruit product is called Jiaosu in Chinese) were studied in term of superoxide dismutase (SOD) activity, tyrosinase inhibitory activity, pancreatic lipase inhibitory activity, and the contents of total sugar and total polyphenol. The results showed that pH value, total number of colonies, yeast and mold counts in fermented immature noni fruit were higher than those in fermented mature yellow fruit, whereas the contents of soluble solids, total sugar, reducing sugar and total polyphenol were lower. During the late stage of fermentation, the inhibition rate of tyrosinase in immature fruit was higher than that in mature fruit. During the entire fermentation process, the inhibition rate of pancreatic lipase in immature noni fruit remained at a high level greater than 85%. The inhibition rate of pancreatic lipase in mature fruit increased with the prolongation of fermentation time, indicating that noni fruit Jiaosu has the potential in skin whitening and preventing obesity.
Engineering of Corynebacterium glutamicum for Biosynthesis of Glyoxylic Acid Using Clustered Regularly Interspaced Short Palindromic Repeats Interference
LIANG Yongsi, SHEN Kaijia, FAN Xuyun, HAN Wuyang, LI Tianming
2021, 42(2):  170-176.  doi:10.7506/spkx1002-6630-20191018-179
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The aim of this study was to improve glyoxylic acid production in Corynebacterium glutamicum ATCC 13032 by repressing the genes on the branch metabolic pathways of glyoxylic acid biosynthesis using clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology. First, the gene lldh encoding lactate dehydrogenase, the key enzyme involved in tributary metabolism, was knocked out by homologous recombination. Then the expression levels of the key enzyme isocitrate dehydrogenase gene (icd) and malate synthase gene (ms) were down-regulated by CRISPRi. At the same time, the isocitrate lyase gene (icl) was overexpressed using a free plasmid, icl-pXMJ19GZ, which increased the synthesis of glyoxylic acid. The results of fermentation experiments showed that there was almost no significant difference in the growth status of the engineered and wild-type strains. The level of glyoxylic acid in the fermentation broth of the engineered strain was as high as 5 mg/mL, which was much higher than that of the wild-type strain (almost zero). This study provides reference for the industrial production of glyoxylic acid using C. glutamicum.
Component Analysis
Changes in Aroma Components during Fermentation of ‘Pingguoli’ Pear Jiaosu (Fermented Fruit Juice)
FAN Hao’an, SHA Ruyi, DU Ning, DAI Jing, FANG Sheng, CUI Yanli, MAO Jianwei
2021, 42(2):  177-184.  doi:10.7506/spkx1002-6630-20191106-077
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In this study, changes in the contents of methanol and ethanol during the fermentation of Jiaosu (fermented fruit juice) made from ‘Pingguoli’ pear fruit were monitored. Solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS), principal component analysis (PCA) and relative odor activity value (ROAV) were used to further analyze the dynamic changes in aroma components during fermentation. The results showed that the contents of methanol and ethanol initially increased to their maximum values of (0.152 ± 0.017) and (3.927 ± 0.025) g/L on the 20th and 50th day, and then declined to (0.026 ± 0.000) and (2.623 ± 0.071) g/L on the 80th day, respectively. A total of 40 aroma components, mainly including esters, alcohols, phenolic compounds, acids and aldehydes, were detected during fermentation. PCA indicated that alcohols, aldehydes and phthalic acid esters (PAEs) were the main aroma components that resulted in the flavor changes during the early stage of fermentation, while fatty acid ethyl esters (FAEEs) and phenolic compounds became the main aroma components which led to the flavor differences during the middle and late periods of fermentation. ROAV evaluation demonstrated that some of the key aroma components changed from decanal and 1-nonanal to eugenol during fermentation. This study provides basic data for the high-value utilization and deep processing of ‘Pinguoli’ pear Jiaosu.
Characterization of Key Aroma Compounds in Chinese Zhiduwugu Light-flavor Baijiu (Chinese Liquor)
DU Jingyi, ZHU Tingting, HUANG Mingquan, WEI Jinwang, WU Jihong, ZHANG Jinglin, WANG Juan
2021, 42(2):  185-192.  doi:10.7506/spkx1002-6630-20200107-084
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Liquid-liquid extraction (LLE) and headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry-olfactometry (GC-MS-O) were used to investigate the volatile flavor components of Zhiduwugu light-flavor (ZD) Baijiu. A total of 40 aroma compounds were selected by aroma extract dilution analysis (AEDA), and among them, isobutyric acid, ethyl laurate, guaiacol, and 2-phenylethanol showed the highest flavor dilution (FD) factors (> 4 096). In total, 35 aroma-active compounds were identified by MS analysis as well as comparing their retention indexes and aroma characteristics and with those of reference standards, and 30 of these compounds were quantitatively determined using cinnamyl acetate, 4-octanol and 2-methylhexanoic acid as internal standards. An aroma model prepared by mixing 17 aroma compounds with odor activity values (OAVs) greater than 1 showed an aroma very similar to that of ZD Baijiu. Based on FD factors and OAVs, isoamyl acetate, ethyl 3-phenylpropionate, guaiacol, isobutyric acid, ethyl laurate, 3-(methylthio)-1-propanol, ethyl acetate, 2-phenylethanol and 3-methyl-1-butanol were identified as the greatest contributors to the aroma of ZD Baijiu.
Differences in Characteristic Volatile Substances in Different Cultivars of Flammulina filiformis
WANG Hetong, PAN Hongshan, WANG Chao, FANG Donglu, HU Qiuhui, MA Ning
2021, 42(2):  193-199.  doi:10.7506/spkx1002-6630-20200804-054
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In this study, by solid phase micro-extraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS) technology and electronic nose (E-nose) technology, we identified and quantitatively analyzed the volatile components in five cultivars of Flammulina filiformis (coded as 3, 11, 54, L4 and L7), and classified the cultivars by principal component analysis (PCA) combined with cluster analysis (CA). The results showed that a total of 53 volatile compounds belonging to eight chemical classes were detected in all cultivars, including alcohols, aldehydes and aromatic hydrocarbons, accounting for 60% to 95% of the total content. Only two volatile substances, 1-hexanol and 2-undecone, were common to all cultivars. E-nose could well distinguish these cultivars and assign samples of each cultivar to one cluster, indicating that the E-nose data are stable and repeatable, and can be used in combination with SPME-GC-MS data to analyze the volatile substances of different cultivars of F. filiformis. In addition, PCA and hierarchical CA distinguished the five cultivars clearly and effectively. This study can provide a reference for the breeding, processing and development of new cultivars of F. filiformis.
Dynamic Changes in the Physicochemical Properties and the Volatile Flavor Compounds in ‘Merlot’ and ‘Vidal’ Icewines during Storage
ZHOU Xueyan, CHEN Yu, LIN Ke, ZHANG Dandan, GE Zhengkai, SU Congyan, WANG Bin, SHI Xuewei
2021, 42(2):  200-211.  doi:10.7506/spkx1002-6630-20191115-184
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In our study, the quality changes of ‘Merlot’ and ‘Vidal’ icewines produced in Yili, Xinjiang Uygur Autonomous Region were monitored during two-year storage at intervals of one year. The physicochemical properties and aroma compounds of icewines were determined by high performance liquid chromatography (HPLC), titration, spectrophotometry and headspace solid phase microextraction combined gas chromatography (HS-SPME-GC-MS). According to the results of SPSS statistical analysis, good discrimination was achieved among icewines based on the physicochemical indexes. After two-year storage, the contents of total acid, volatile acids and malic acids in ‘Merlot’ icewine decreased by 5.9%, 13.2% and 39%, respectively, and lactate content increased by 57.2%. Browning index (BI), wine color (WC), color intensity (CI), color hue (CH), and the proportions of yellow (Ye) and red (Rd) changed significantly after one year of storage; CI, CH and Ye showed increasing trends with storage time, while Rd showed an opposite trend. After two-year storage, the contents of total acid and malic acid in ‘Vidal’ icewine increased by 9.8% and 33.1%, respectively, while the content of volatile acid decreased by 29.8%. WC, BI, CI, CH, and Ye changed significantly after one year of storage; WC and CI increased with storage time, whereas there was no significant difference in Rd or the proportion of blue (Bl). The results of principal component analysis (PCA) showed clear discrimination among icewine samples of different ages and between the two icewines. Two-year-old icewines contained the largest number of volatile compounds such as isopropyl alcohol, isoamyl acetate, ethyl hexanoate, hexyl acetate, decanal, citronellol, ethyl phenylacetate, phenethyl acetate, and β-damascenone in ‘Merlot’ icewine; and 1-pentanol, isoamyl alcohol, ethyl formate, isopropyl alcohol, furfural, 1-butanol, isoamyl acetate, acetaldol, hexyl acetate, octyl acetate, 3,4-dimethylbenzaldehyde, 5-hydroxymethylfurfural, phenethyl acetate, β-damascenone in ‘Vidal’ icewine. The above results showed that storage time had a significant impact the physicochemical properties and aroma compounds of icewines. Most importantly, we found that the volatile flavor composition of ‘Vidal’ icewine changed more significantly as compared to ‘Merlot’ icewine during storage.
Metabolic Profiling and Distribution of Bioactive Components of Different Parts of Tartary Buckwheat Seeds
CHAI Duo, WANG Meiting, JIANG Yumeng, LIU Longze, XIAO Lin, WANG Binchen, DONG Liang
2021, 42(2):  212-217.  doi:10.7506/spkx1002-6630-20191023-245
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In this paper, gas chromatography-mass spectrometry (GC-MS) was used to analyze the metabolite profiles of different parts of tartary buckwheat seeds, and the contents of amino acids, sugars, fatty acid methyl esters and free fatty acids in these parts were determined. Six bioactive ingredients in tartary buckwheat seeds were simultaneously determined by high performance liquid chromatography (HPLC), including rutin, quercetin, vanillic acid, p-coumaric acid, kaempferol and chlorogenic acid. The results showed that the number and amount of metabolites in the germ were higher than those in the other parts tested. The contents of the six bioactive ingredients were higher in the bran than those in the other parts, but the content of rutin in the hull was the highest.
Analysis of Major Flavor Components of Tuotuo Pork, a Traditional Meat Product in Liangshan, Southwest China’s Sichuan, by HS-SPME-GC-MS-O Combined with Electronic Nose
ZHOU Huimin, ZHANG Shunliang, HAO Yanfang, CHEN Song, PAN Xiaoqian, WU Qianrong, LI Su, ZHU Ning, QIAO Xiaoling, ZHAO Bing
2021, 42(2):  218-226.  doi:10.7506/spkx1002-6630-20191024-263
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The volatile flavor components of Tuotuo pork samples from four different producers were investigated by electronic nose (EN) and headspace solid-phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Furthermore, principal component analysis (PCA) was carried out on the major flavor components. The results showed that EN combined with linear discriminant analysis (LDA) was an effective method to distinguish the difference in volatile components of different Tuotuo pork samples. By HS-SPME-GC-MS-O, a total of 45 volatile compounds were detected from the samples, mainly aldehydes, alcohols, ketones, olefin, and heterocyclic compounds. Through odor activity value (OAV) calculation and olfactometry, 27 flavor substances with OAV ≥ 1 were identified, eight of which were found in all four samples, including hexanal, heptanal, octanal, (E)-2-heptenal, nonanal, (E)-2-octenal, (E,E)-2,4-decadienal, and 1-octene-3-ol, together contributing to 71.16%–93.12% of the total flavor as the major flavor components. In addition, volatile compounds derived from the seasonings used, including L-carvone, pinene, β-pinene, D- limonene, (E)-β-ocimene, citral, propyl mercaptan, eucalyptus alcohol, and linalool contributed to the remaining 12.71%–28.58%. Based on the 27 flavor components, PCA showed good separation among the samples. Samples C and D scored higher on PC1, while samples A and D scored higher on PC2. The contribution of (E,E)-2,4-decadienal to the flavor of Tuotuo pork was bigger on PC1, whereas the contribution of linalool was the highest on PC2, followed by limonene and 1-octene-3-ol. Therefore, (E,E)-2,4-decadienal, linalool, limonene, and 1-octene-3-ol were the important flavor substances to discriminate among Tuotuo pork from different producers.
Analysis of Volatile Compounds in the Surface and Inner Layers of Xinjiang Roast Lamb Leg at Different Roasting Times Using SPME-GC-MS
DING Dan, WANG Songlei, LUO Ruiming, WANG Yongrui, BAI Shuang, SHEN Fei, BAI He
2021, 42(2):  227-234.  doi:10.7506/spkx1002-6630-20200116-199
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In order to study the effect of roasting time on volatile compounds in the surface and inner layers of Xinjiang roast lamb leg, we applied solid phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS) to analyze the volatile compounds in the surface and inner layers of roast lamb leg with roasting times of 0.5, 1, 1.5, 2, 2.5, and 3 h. The results showed that a total of 64 and 55 volatile compounds, mainly hydrocarbons, alcohols, aldehydes, heterocycles, ketones, esters and acids, were respectively detected in the surface and inner layers of roast lamb leg. As roasting proceeded, the types and contents of hydrocarbons, alcohols, aldehydes, heterocycles, ketones and esters in the surface layer of roast lamb leg were higher than those in the inner layer. Roasting time of 1.5 h was the key point to produce volatile compounds in roast lamb leg. Based on their relative odor activity values (ROAVs), it was concluded that the characteristic volatile flavor compounds detected at 2.5 h contributed the most to the surface layer and inner layer of roast lamb leg. Among them, 1-octen-3-ol, nonanal, (E,E)-2,4-decadienal, octanal and hexanal contributed the most to the aroma of roast lamb leg.
Comparative Analysis of the Glyceride Compositions of Pressed and Extracted Camellia Seed Oil Using Ultra-high Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry
HU Qian, ZHANG Jiukai, HAN Jianxun, XING Ranran, LIU Han, CHEN Ying
2021, 42(2):  235-240.  doi:10.7506/spkx1002-6630-20200115-182
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The glycerides compositions of pressed and extracted camellia oil were comparatively investigated by lipidomic ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). A total of 55 glycerides, including 43 triacylglycerols (TAGs) and 12 diacylglycerols, were detected utilizing information dependent acquisition (IDA). TAG 54:3 was the most predominant triacylglycerol found in both pressed and extracted camellia oil, accounting for more than 30% of the total triacylglycerols, and its content differed but not significantly between the oils (P > 0.05). Unsupervised principal component analysis (PCA) was performed with 55 glycerides as variable parameters. The two-dimensional PCA score plot showed clear separation between the oils. Furthermore, a predictive model was established using supervised orthogonal partial least squares discriminant analysis (OPLS-DA) for distinguishing the oils. The validation results showed that model could accurately distinguish the pressed oil from the extracted camellia oil with good recognition ability and prediction ability. This study provides a new research idea for identifying high-value edible oils produced by different processing methods.
Analysis of Differential Whey Proteome between Bovine Colostrum and Mature Milk Using Isobaric Tags for Relative and Absolute Quantitation Technology
DENG Wei, LI Yuntong, LI Mohan, CAO Xueyan, ZHENG Yan, WU Junrui, YUE Xiqing, YANG Mei
2021, 42(2):  241-246.  doi:10.7506/spkx1002-6630-20191129-300
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In order to elucidate the differentially expressed whey proteins between bovine colostrum and mature milk, isobaric tags for relative and absolute quantitation (iTRAQ) technique was used for proteomic analysis of the two milks. A total of 599 whey proteins were identified quantitatively, 60 of which were differentially expressed proteins. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the biological processes of transport, subcellular localization, and single biological action, in molecular functions such as apical plasma membrane, extracellular region, and extracellular region part, and in cell components including protein binding and anion binding. Nine of the differentially expressed proteins were related to signal transduction and six of them were glycosylated whey proteins. In addition, protein-protein interaction network analysis revealed that some of these differentially expressed proteins were the key whey protein factors with high connectivity. This study provides a theoretical basis for improving bovine colostrum and mature milk quality and developing milk powder for infants and young children and functional dairy foods in the future.
Main Components of Stevia Residue Extract and Their Antioxidant Activities
ZHAO Lei, PAN Fei, LIN Wenxuan, XU Meili, BAO Xi, CHEN Yanlin, WANG Chengtao, LIAN Yunhe
2021, 42(2):  247-254.  doi:10.7506/spkx1002-6630-20191023-247
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The objectives of this study were (1) to analyze the main components of stevia residue extract and their antioxidant activities and (2) to elucidate their contributions to the total antioxidant capacity of stevia residue extract. Methods: The main components of stevia residue extract were qualitatively and quantitatively analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and HPLC. The antioxidant activities were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzthiozoline-6)-sulphonic acid (ABTS) and ferric reducing/antioxidant power (FRAP) methods, and comparatively evaluated using antioxidant potency composite (APC) index. The eight main components of stevia residue extract were 5-CQA, 4-CQA, caffeic acid, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, quercetin-3-rhamnoside and quercetin, among which, 4,5-diCQA was the most abundant, whose content was (126.7 ± 1.27) mg/g, followed by caffeic acid ((97.2 ± 0.36) mg/g) and 5-CQA ((46.5 ± 0.29) mg/g). The results of APC indexes showed that the antioxidant activities of the main components declined in the following order: caffeic acid (92.56%) > quercetin (78.31%) > 3,4-diCQA (62.09%) > 5-CQA (58.92%) > 3,5-diCQA (48.15%) > 4,5-diCQA (36.55%) > 4-CQA (35.5%) > quercetin-3-rhamnoside (34.24%). At concentrations of 50–400 μg/mL, the antioxidant activity of stevia residue extract was stronger than that of its simulant. Stevia residue extract contained high contents of chlorogenic acids and flavonoids and exhibited strong antioxidant activity, and caffeic acid contributed the most to its antioxidant. In addition to the eight main components, other components with antioxidant activities may also exist in stevia residue extract.
Determination of 17 Amino Acids in the Dried Rhizome of Lycopus lucidus Turcz. var. hirtus Regel from Different Habitats
HUANG Xiaolan, HE Xufeng, YANG Qin, GU Wenchao, ZHOU Xiangde, ZHANG Hua, ZHOU Nong
2021, 42(2):  255-261.  doi:10.7506/spkx1002-6630-20200108-090
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An ultra-high performance liquid chromatography (UPLC) method for the simultaneous determination of 17 amino acids in the dried rhizome of Lycopus lucidus Turcz. var. hirtus Regel collected from six different habitats in China was established using pre-column derivatization with phenyl isothiocyanate (PITC). The chromatographic separated was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm) within 19 minutes using a mobile phase composed of 0.1 mol/L sodium acetate solution and 80% acetonitrile solution (adjusted to pH 6.5 with acetic acid). The detection wavelength was set at 254 nm, the column temperature at 40 ℃, and the flow rate of sample loading at 0.2 mL/min. The results showed for all the 17 amino acids, a good linear relationship (r ≥ 0.999 0) was obtained within their respective concentration ranges. The average recoveries were 89.69%–106.02% with relative standard deviations (RSDs) ranging from 0.44% to 2.60%. The limits of detection (LODs) were 0.020–0.110 μg/mL, and the limits of quantitation (LOQs) were 0.067–0.368 μg/mL. The 17 amino acids were all detected in the hydrolysate of L. lucidus Turcz. var. hirtus Regel, including seven essential amino acids and nine medicinal amino acids. The ratios of essential amino acids (EAAs) to total amino acids (TAAs) were between 23.99% and 29.36%, and the ratios of medicinal amino acids (MAAs) to TAAs ranged between 68.44% and 74.16%. There existed a significant difference in the contents but not types of amino acid among rhizome samples from different geographical origins. Aspartic and glutamic acid were the most abundant amino acids in all the samples, and their average contents were 13.338 and 8.478 mg/g, accounting for 26.32% and 16.73% of TAAs, respectively. The habitats were divided into 3 categories by cluster analysis. Among the habitats, the samples collected from Heze, Shandong province and Wanzhou district, Chongqing had the highest and lowest amino acid contents (81.663 and only 19.463 mg/g), respectively. In conclusion, the dried rhizome of L. lucidus Turcz. var. hirtus Regel has high nutritional and medicinal value, and this study can provide basic data for the quality evaluation and utilization of this plant.
Comparative Analysis of Bioactive Ingredients, Hypoglycemic Activity and Volatile Components of Unfermented and Fermented Tea Made from Toona sinensis Buds at Different Harvest Periods
JIANG Pengfei, GAO Haidong, ZHAO Lili, WANG Zhaogai, SHI Guanying, ZHANG Le, WANG Xiaomin, WANG Xuzeng
2021, 42(2):  262-270.  doi:10.7506/spkx1002-6630-20200116-194
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Toona sinensis buds harvested in April through June of 2018 were separately used to prepare unfermented and fermented teas. The differences in the main bioactive ingredients, antioxidant activity, hypoglycemic activity and volatile components of the six tea samples were analyzed to reveal the quality change of fermented Toona sinensis tea with harvest time and to determine the optimal harvest time. Results demonstrated that significant variations were found among the six samples in the main bioactive ingredients, antioxidant activity and hypoglycemic activity and each had distinctive volatile components. The contents of total flavonoids, polysaccharides, tea polyphenols and tea pigments in both unfermented and fermented samples increased with later harvest date, the contents of saponin, free amino acid and caffeine showed fluctuating trends, and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and α-glucosidase inhibitory activity decreased gradually. For each harvest date, the contents of total flavonoids, polysaccharides, saponins, tea polyphenols and free amino acids in fermented tea samples were lower than those in the unfermented counterparts, while the contents of caffeine and tea pigment were higher. The DPPH radical scavenging activity decreased, whereas the α-glucosidase inhibitory activity increased after fermentation. Correlation analysis showed that total flavonoids were the main DPPH radical scavenger and α-glucosidase inhibitor in the six samples. A total of 7 classes of 90 volatile components were identified in these samples, among which, the number and amount of terpenes were the highest. A total of 42 and 49, 47 and 52, 50 and 51 volatile compounds were detected in unfermented and fermented T. sinensis teas in April, May and June, respectively. Principal component analysis (PCA) performed on the seven classes of volatile components generated 3 principal components, accounting for 88.01% of the total variance, which could reflect most of the information about these samples. Aldehydes, terpenes, alcohols, and terpene oxides were the main factors distinguishing the six samples. The fermented and unfermented T. sinensis teas in April and May gained higher scores. Comprehensive analysis showed that the quality of fermented T. sinensis tea made from T. sinensis buds in May was better.
Nutritional and Physicochemical Properties of Acer truncatum Seed Protein
LIU Yudi, LI Jiamei, WANG Kunhua, WANG Xiaojing, XU Huaide
2021, 42(2):  271-277.  doi:10.7506/spkx1002-6630-20191105-055
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Acer truncatum Bunge. seed protein (ABPI) and salt extractable protein (ABSPI) were extracted from the seeds of A. truncatum Bunge. by using alkali dissolution acid precipitation method and salt extraction method, respectively. Their nutritional and physicochemial properties were compared. The results showed that both ABPI and ABSPI contained the eight essential amino acids (EAAs) with glutamic acid being the most abundant amino acid, accounting for 37.86% and 38.45% of the total amino acid content, respectively. The surface hydrophobicity (664.86), total sulfhydryl content (35.79 μmol/g) and disulfide bond content (11.50 μmol/g) of ABPI were significantly higher than those of ABSPI (P < 0.05), and the thermal denaturation temperature was 128.05 ℃, which was also higher than that of ABSPI (118.33 ℃). The water-holding capacity (4.64 mL/g) of ABSPI was significantly higher than that of ABPI (2.46 mL/g) (P < 0.05), while the oil-holding capacity (3.58 mL/g) was lower than that of ABPI (4.82 mL/g). The foaming capacity, foam stability and emulsifying properties of ABPI showed similar trends to ABSPI with pH. Fourier transform infrared (FTIR) spectroscopy showed that both ABPI and ABSPI displayed typical protein absorption peaks, but the tertiary structure of ABSPI was incomplete. The results of scanning electron microscopy (SEM) showed that the microstructures of the two proteins were significantly different. ABSPI showed an ordered cluster of globular proteins, while the structure of ABPI was dense tight and the surface was irregularly ridged. ABPI had better physicochemical properties and functional characteristics than ABSPI.
Safety Detection
Simultaneous Detection of Four Veterinary Drugs Using Immunochip Assay Based on Smart Phone
WU Ying, FAN Congcong, SU Xiaona, SHEN Yudong, TAN Shu, ZHONG Cuili, XU Xiaoxuan, ZENG Daoping, YANG Jinyi
2021, 42(2):  278-282.  doi:10.7506/spkx1002-6630-20190927-334
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An immunochip based on smart phone for simultaneous detection of four veterinary drugs including ractopamine (RAC), clenbuterol (CL), chloramphenicol (CAP), florfenicol (FF) and its metabolite florfenicol amine (FFA) in pig urine was developed in this study. The immunochip test was conducted based on the competitive reactions with the antibodies on nitrocellulose (NC) membrane. After chromogenic reaction, a calibration curve for the concentration of each veterinary drug versus the intensity of spots on the immunochip was established. The limits of detection (LODs) of the immunochip assay for RAC, CL, CAP, and FF were 0.146, 0.137, 0.035, and 3.73 ng/mL respectively, and the linear working ranges were 0.353–1.017, 0.309–0.897, 0.082–0.249, and 9.424–35.594 ng/mL, respectively. The cross-reactivity rate with FFA was 81.5%. The results determined by this method were consistent with those determined by colloidal gold immunochromatography assay (GICA) and enzyme-linked immunosorbent assay (ELISA). To sum up, this immune chip is a cheap, simple and powerful tool for on-site and high throughput screening of veterinary drug residues.
Non-destructive Detection of Volatile Basic Nitrogen Content in Prawns (Fenneropenaeus chinensis) Based on Spectral and Image Information Infusion
WANG Ya, ZHANG Cuncun, FU Yuye, ZHANG Fan, WANG Jie, WANG Wenxiu
2021, 42(2):  283-290.  doi:10.7506/spkx1002-6630-20200103-027
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A support vector machine (SVM) model for rapidly predicting the total volatile basic nitrogen (TVB-N) content in prawns was established using near-infrared spectroscopy and machine vision technology based on spectral and image information infusion. Spectral and image information of 51 samples stored at 4 ℃ for 0 to 12 days was obtained, and their TVB-N content was measured according to the national standard method of China. The results showed that the characteristic dual-band spectral information in the range of 350–1 000 nm and 940–1 650 nm was fused and pre-processed by the first derivative method. Besides, the competitive adaptive reweighted sampling (CARS) algorithm was used to select the characteristic wavelengths. The developed model worked well with correlation coefficient in the prediction set (Rp) of 0.968 7, standard error of prediction (SEP) of the validation set of 10.56 mg/100 g, and relative percent deviation (RPD) of 3.38. On the other hand, the model constructed using the characteristic image information had poor performance, with Rp of 0.933 5, SEP of 19.79 mg/100 g and RPD of 1.74. Compared with the above two models, the model developed based on spectral and image information fusion had improved accuracy and stability with Rp of 0.988 4, SEP of 7.51 mg/100 g and RPD of 6.29. These results confirmed the potential near-infrared spectroscopy combined with machine vision to predict the TVB-N content in prawns, which could allow rapid detection of freshness changes of prawns during cold storage.
Determination of Nine Fluorescent Whiteners in Plastic Food Contact Materials by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
ZHANG Lini, ZOU Xiao, YU Wenqin, DAN Jiayu, TANG Jiali, GAO Yakai
2021, 42(2):  291-298.  doi:10.7506/spkx1002-6630-20191008-028
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A method for simultaneous determination of nine fluorescent brighteners (FWA52, FWA135, FWA184, FWA185, FWA199, FWA367, FWA368, FWA378 and FWA393) in plastic food contact materials was developed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were extracted ultrasonically and sequentially with trichloromethane and methanol at room temperature for 30 minutes either, and the combined extract was blown to near dry by nitrogen and redissolved in methanol. For chromatographic separation, gradient elution was performed with ammonium acetate solution-acetonitrile as the mobile phase. Detection was performed using an electrospray ionization source in the positive ion (ESI+) model with multiple reaction monitoring (MRM), and quantification by external standard method. The results showed that the calibration curves for the nine fluorescent brighteners were linear in the range of 0.5–1 000 ng/mL with correlation coefficients greater than 0.999. The limit of detection (LOD) was 0.05–3.2 μg/kg, the limit of quantitative (LOQ) was 0.2–10 μg/kg, and the recoveries for spiked samples ranged from 76.11% to 106.55%, with relative standard deviations (RSDs, n = 6) of 1.97%–8.31%. The method proved to be simple, accurate, and sensitive, and had good recovery and repeatability. It could suitable for the determination of fluorescent whitening agents in plastic contact products.
Simultaneous Determination of 16 Mycotoxin Residues in Aged Tea by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry
LIU Wenjing, HUANG Biao, FU Jianwei, WEI Hang, HUANG Caibiao
2021, 42(2):  299-305.  doi:10.7506/spkx1002-6630-20200611-157
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An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of 16 mycotoxins from different types of aged tea. The sample was extracted with formic acid-acetonitrile (10:90, V/V), added with a blend of QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction salts, shaken and centrifuged; the extract was cleaned up by passing through an OASIS PRIME HLB cartridge and a dispersive solid-phase extraction (dSPE) purification tube, separated on an ACQUITY UPLC HSS T3 C18 column, detected using an electrospray ionization source in the positive ion multiple reaction monitoring (MRM) mode, and quantified using tea matrix matching standard solution. The 16 mycotoxins showed a good linear relationship within their respective concentration ranges with correlation coefficients > 0.999. The average recoveries from tea matrix spiked at three different levels ranged between 63.4% and 109.7%, with relative standard deviations (RSDs) between 1.7% and 11.3%. The method limits of detection (LODs) ranged from 0.03 to 7.00 μg/kg. Among 121 aged tea samples selected for analysis, aflatoxin B1 was detected from one oolong tea sample at a concentration of 34.0 μg/kg, and ochratoxin A was also detected from one black tea sample at a concentration of 1.6 μg/kg. The method proved to be stable, accurate, sensitive and fast, and could meet the requirements for the analysis of multiple toxic residues in a variety of tea samples.
Development of a Fluorescence Method for Detection of Organophosphorus Pesticides Based on Oxidase-mimicking Activity of MnO2 Nanosheets
LUO Lin, DENG Chuyao, JIA Baozhu, LIN Haohong, LIAO Caixia, WU Zhuoyu, HE Zhenxi, WANG Hong, XU Zhenlin
2021, 42(2):  306-311.  doi:10.7506/spkx1002-6630-20200107-087
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In this study, a rapid, simple and highly sensitive fluorescence method for the detection of organophosphorus pesticides was developed based on the oxidase-like activity of manganese dioxide nanosheets and the principle of enzymatic inhibition of organophosphorus pesticides. The contents of organophosphorus pesticides in samples was determined by measuring the change in the fluorescence intensity of the reaction system. An acetylcholinesterase (AChE) concentration of 9.375 U/L, a substrate concentration of 10 mmol/L and a reaction time of 11 minutes were found to be the optimal conditions to achieve the best detection performance. The dynamic detection range of paraoxon was 1.56–200 ng/mL, and the limit of detection (LOD) was 0.5 ng/mL (RSN = 3). The recoveries from tap water, cabbage and oat samples spiked with varying concentrations of paraoxon were in the range of 83.4%–105.9%. These results indicated that this method had several remarkable advantages including rapidity, and high sensitivity and accuracy.
Rapid Detection of Shiga Toxin-producing Escherichia coli by Immunochromatography Combined with Asymmetric Polymerase Chain Reaction
SHAN Shan, HUANG Zhaohong, HUANG Yanmei, LIU Daofeng, LIU Chengwei, HUANG Yunhong, LONG Zhong’er
2021, 42(2):  312-318.  doi:10.7506/spkx1002-6630-20200113-129
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Shiga toxin-producing Escherichia coli (STEC) is strongly pathogenic. Establishing a simple and rapid method for STEC detection is of importance for controlling the occurrence and spread of foodborne outbreaks. In this paper, the biotinylated target single stranded DNA was obtained by asymmetric polymerase chain reaction (aPCR) with the biotinylated downstream primers of the typical virulence genes stx1 and stx2. The DNA amplification products were conjugated with the upstream primers which were labeled on polystyrene microspheres. The biotin from the conjugates could be captured by streptavidin sprayed on a lateral flow strip, forming a red line visible to the naked eye. STEC strains producing different types of Shiga toxin could be detected by this method. It displayed a high specificity and accuracy for detecting 23 strains.
Distribution Characteristics of Persistent Organic Pollutants in Bamboo Shoots Produced in Zhejiang Province
SHEN Danyu, YUAN Xinyue, ZHENG Yuewen, WU Shutian, LIU Yihua, MO Runhong, ZHONG Donglian, TANG Fubin
2021, 42(2):  319-324.  doi:10.7506/spkx1002-6630-20200103-032
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The distribution characteristics of three groups of persistent organic pollutants (POPs), 21 organochlorine pesticides (OCPs), 18 polychlorinated biphenyls (PCBs) and 16 polycyclic aromatic hydrocarbons (PAHs), in different parts of bamboo shoots and bamboo shoots from different harvest seasons and producing regions in Zhejiang province were studied. The results showed that the distribution of OCPs was more abundant in the shoot than in the shell, and in the upper part of the shoot than in the lower part, and it was four-fold higher in spring bamboo shoots than in winter bamboo shoots. The level of OCPs pollution was higher in bamboo shoots from Shangyu than in those from other regions. Toxaphene was the most abundant among these OCPs (about 105 μg/kg). The distribution of PCBs was higher than in the shoot that in the shell, in the bottom part than in the upper part, and in spring than in winter. The level of PCBs pollution in bamboo shoots from Wuyi was the highest, and PCB52 was the most abundant PCB (1.14 μg/kg). The distribution of PAHs in the shoot was close to that in the shell, slightly higher in the upper part than the bottom part, and 1.5 times higher in spring than in winter. The highest level of PAHs distribution was found in bamboo shoots from Yongjia, and the most abundant PAH was phenanthrene (5.85 μg/kg). None of the 55 POPs in bamboo shoots exceeded the limits specified in the existing standards and were at a relatively safe level. This study not only improves the theoretical basis for bamboo shoot quality and safety, but also provides reference and support for the remediation and management of environmental pollution.
Optimized QuEChERS Combined with UPLC-MS/MS for Determination of Fipronil and Its Metabolites in Poultry-Derived Foods
DAI Jinbo, SHEN Jie, HE Xiaofeng, NIE Rongrong, DONG Wenjing, LIANG Qinxian
2021, 42(2):  325-332.  doi:10.7506/spkx1002-6630-20200103-026
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A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) pretreatment coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of fipronil and its metabolites in poultry-derived foods. Plackett-Burman design and Box-Behnken design were sequentially used to select and optimize the QuEChERS parameters with a significant influence on the recovery of analytes as well as their levels. The optimum parameters were obtained as follows: samples were extracted using acetonitrile, and the extract was salted out with a mixture of anhydrous magnesium sulfate and sodium chloride, and then purified by Z-Sep+ combined with multi-walled carbon nanotube (MWCNT) before being detected by UPLC-MS/MS and quantified by matrix-matched calibration. The results indicated that the average recoveries of fipronil, fipronil desulfinyl, fipronil sulfone and fipronil sulfide from eggs, chicken meat, and chicken liver at three different spiked levels were 78.9%–113.5%, with relative standard deviations (RSDs) of 2.10%–7.60%. The limit of detection (LOD) was 0.1–0.2 μg/kg, and the limit of quantitation (LOQ) was 0.5 μg/kg for all analytes. In the developed method, the matrix effects were effectively reduced. This method was simple, sensitive and accurate and could be used for rapid and quantitative determination of fipronil and its metabolites in poultry-derived foods.