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25 January 2017, Volume 38 Issue 2
Bioengineering
Molecular Dynamics Simulation of Thermal Stability Improvement of Lipoxygenase from Anabaena sp. PCC 7160
QIAN Hui, LU Zhaoxin, ZHANG Chong, BIE Xiaomei, ZHAO Haizhen, Lü Fengxia
2017, 38(2):  1-6.  doi:10.7506/spkx1002-6630-201702001
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Homology modeling was used to predict the 3D structure of lipoxygenase from Anabaena sp. PCC 7160 (Ana-LOX) and to simulate its motion trajectory in aqueous solution by molecular dynamics simulation at 298 and 320 K,respectively. After root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values were calculated,thermal stability was evaluated based on the RMSF value. Force analysis was also performed to predict the unstable region.Results indicated that the T94, I96, and A325 to Q327 areas of Ana-LOX were flexible and they played a crucial role in thethermal instability of Ana-LOX. The half-life of I96V mutant constructed by site-directed mutagenesis increased 2.5 foldscompared with that of the wild-type enzyme, confirming the validity of the prediction. Our research can provide a theoreticalbasis for the improvement of the thermal stability of lipoxygenase.
Gene Cloning, Expression and Characterization of Protein Disulfide Isomerase from Wheat (Triticum aestivum L.)
LIU Guang, HU Songqing, ZHANG Tingting, WANG Jingjing, LI Lin, HOU Yi
2017, 38(2):  7-14.  doi:10.7506/spkx1002-6630-201702002
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Purpose: The gene encoding wheat protein disulfide isomerase (wPDI) was cloned and expressed in Escherichiacoli, and its enzymatic properties were investigated. Methods: The wpdi gene was obtained by reverse transcriptionpolymerase chain reaction amplification using the total RNA from wheat seeds as template. The recombinant plasmid pET-30b-wpdi was constructed and transformed into E. coli BL21 (DE3). After metal chelating chromatography, the enzymaticproperties of the purified wPDI were determined. Results: A 1 548 bp gene fragment was amplified and sequenced as wpdigene that had 99% identity with that of the wheat cultivar Wyuna. The optimized conditions for wPDI expression weredetermined as follows: induction at 22 ℃ using isopropyl β-D-1-thiogalactopyranoside at a concentration of 0.5 mmol/L for6 h. The recombinant wPDI consisted of four thioredoxin-like domains and had a molecular weight of 66.2 kD. The enzymeexhibited enzymatic activities (including reductase activity and isomerase activity of disulfide bonds) and chaperone activity.Conclusions: The expression of wPDI and its enzymatic properties can provide the foundation for its application in the flourprocessing industry.
Microbial Diversity of Traditional Fermented Jinzhou Pickles
SUN Huijun, ZHANG Ying, WANG Hongyu, YANG Mei, YUE Xiqing, WU Rina
2017, 38(2):  15-19.  doi:10.7506/spkx1002-6630-201702003
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In order to improving the quality of Jinzhou pickles, this experiment studied the microbial community compositionof Jinzhou pickles. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was adopted to analyzethe microbial diversity of ten traditional pickle samples in Jinzhou area, Liaoning province. The results showed that fivebacterial strains were identified namely Lactobacillus plantarum, Weissella cibaria, Lactobacillus fabifermentans, Weissellahellenic and Psychromonas arctica as well as three fungal species i.e., Candiada tropicalis, Campylobacter mucosalis andCandida albicans. The predominant bacteria were Lactobacillus plantarum, Weissella cibaria and the predominant funguswas Candiada tropicalis.
Improvement of the Thermal Activity and Stability of Hyperthermophilic α-Amylase ApkA by Site-Directed Mutagenesis
ZENG Jing, GUO Jianjun, YUAN Lin
2017, 38(2):  20-26.  doi:10.7506/spkx1002-6630-201702004
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Hyperthermophilic α-amylases, which are active and stable at high temperatures, are of great interest forresearchers studying starch liquefaction. Research into the molecular basis of thermal adaptation of hyperthermophilicα-amylase can provide theoretical guidance to improve the thermal activity and thermal stability of α-amylases. Based onthe sequence analysis of hyperthermophilic α-amylase ApkA from Thermococcus kodakarensis KOD1, a signal peptidedeleted mutant (ApkAds) and an A180K site mutant (ApkAdsA180K) were constructed. Compared with ApkAds, the mutantApkAdsA180K exhibited a sharp increase in thermal activity and stability. The optimal temperature of ApkA was 90 ℃ andthe corresponding specific activity was 2 946.75 U/mg, while the optimal temperature of the mutant was 100 ℃ and thecorresponding specific activity was 4 501.08 U/mg. When incubated at 90 ℃, ApkAds and the mutant exhibited half-livesof 5 h and 7 h, respectively. The tertiary structure of ApkAdsA180K obtained by homologous modeling indicated that K180and D212 are involved in salt bridge formation. These results suggest that the salt bridge between K180 and D212 plays animportant role in maintaining the thermal activity and stability of ApkAdsA180K.
Antibiotic Resistance Evaluation and Resistance Gene Profile of Epibiotic Lactic Acid Bacteria on Red Bell Peppers Used for Sichuan Pickle Fermentation
CAI Ting1, LU Qianwen, XIANG Wenliang, ZHANG Qing, ZHANG Qisheng, CHEN Gong, CAI Yimin
2017, 38(2):  27-33.  doi:10.7506/spkx1002-6630-201702005
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For formulating reasonable measures for the prevention and control of bacterial antibiotic resistance to ensurefood safety, penicillin (PEN), erythromycin (ERY), tetracycline (TET), streptomycin (STR) and chloramphenicol (CHL)resistance of lactic acid bacteria (LABs) and the corresponding resistance genes were evaluated, including Enterococcusmundtii (n = 5), Enterococcus faecalis (n = 2), Enterococcus hirae (n = 2), Lactococcus lactis (n = 7), Leuconostocmesenteroides (n = 2), Leuconostoc holzapfelii (n = 3) and Weissella cibaria (n = 79) from fresh red bell peppers usedfor Sichuan pickle fermentation. All of the isolated strains were susceptible to PEN or ERY, but they had solo, doubleor triplicate resistance to TET, STR and CHL. All the isolates of L. mesenteroides as well as some strains of E. hirae,E. faecalis and L. holzapfelii showed solo STR resistance. Some strains of E. faecalis, E. hirae, L. lactis and W. cibaria haddouble resistance to STR and TET, as well as STR and CHL. However, isolates with triplicate resistances to STR, TETand CHL were only found in W. cibaria. It was found that except norA, sepA, tet(A), tet(O) and aac(6’)-aph(2’) genes,all antibiotic resistance genes were harbored by the resistant isolates partly or completely. The multiple-drug resistanceefflux pump genes efrA, tolC, norC, sugE and mdfA showed higher positive rates (which were 49%, 41%, 48%, 41% and47%, respectively) than the ribosomal protection protein genes and the enzymatic modification genes in the correspondingpolymerase chain reaction (PCR). Even though the dissemination of these antibiotic resistances needs to be further studied,such results demonstrated that food safety concerns will be partly focused on antibiotic resistance of LABs on fresh red bellpeppers according to Qualified Presumption of Safety criteria.
Modeling the Effect of Combined Thermal and High Pressure Treatment on Inactivation of Lipoxygenase in Pork
HUANG Yechuan, LI Feng, WU Zhaomin
2017, 38(2):  34-39.  doi:10.7506/spkx1002-6630-201702006
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This study aimed to investigate the effect of high pressure processing on lipoxygenase (LOX) activity in pork. Pork Longissimus dorsi muscles were treated in the pressure range of 200–700 MPa and in the temperature range of 20–60 ℃. Changes in LOX inactivation rate constants were studied, and kinetic models of LOX inactivation based on Arrhenius and Eyring equation were established, respectively. The results showed that the inactivation rate constants increased with increasing pressure in the studied temperature range, and increased with increasing temperature in the studied pressure range. The Arrhenius and Eyring equations were suitable for the whole temperature and pressure ranges, respectively. Both the Arrhenius and Eyring kinetic models had a good fitting degree with high R2 values (0.989 and 0.965, respectively). Thus, the regression models could well predict changes in LOX activity in pork during combined thermal and high pressure processing in a certain range of pressure and temperature.
Thermal Inactivation Properties of Shigella in Smoked Cooked Ham
ZHANG Peipei, LI Miaoyun, ZHAO Gaiming, CUI Wenming, GAO Xiaoping, LIU Yanxia, ZHAO Lijun, WANG Yafei
2017, 38(2):  40-45.  doi:10.7506/spkx1002-6630-201702007
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This study aimed to examine the thermal inactivation pattern of Shigella in smoked cooked ham. Shigella (108 CFU/g) were inoculated into smoked cooked ham, cultured at constant temperature and constant humidity and then subjected to thermal treatment at 55 or 63 ℃. The number of surviving cells was counted on selective media. A Logistic model and a 3rd degree polynomial model were separately used to fit the thermal inactivation curve by CurveExpert software. Decimal reduction time (D values) represented the heat resistance of Shigella. The results showed that the curves for 11 Shigella strains fitted by the 3rd degree polynomial model were better, with correlation coefficients (R2) above 0.98. The D values of different Shigella strains were different, ranging from 4.46 to 10.66 min at 55 ℃ and from 0.48 to 3.22 min at 63 ℃ in the inoculated smoked cooked ham. The heat resistance of different Shigella strains were different, and strain S-5 had the strongest heat resistance while S-6 was the weakest.
Gene Cloning, Expression and Functional Charaterization of NAD(P)H Oxidases Gene (nox) from Listeria monoeytogenes
WU Man, LI Sen, CHEN Guowei, LUO Qin, LIU Wukang, DING Chengchao, DONG Qingli, LIU Qing
2017, 38(2):  46-51.  doi:10.7506/spkx1002-6630-201702008
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Nicotinamide adenine dinucleotide phosphate oxidases (Nox) is ubiquitous in higher animals and plants, and it is responsible for generating reactive oxygen species (ROS). The aim of this work was to explore whether the NOX mediates the generation of ROS in Listeria monocytogenes (Lm). The nox gene from the wild-type Listeria monocytogenes EGDe (GenBank ID: 986631) was tested. The gene was induced to express Nox in Escherichia coli BL21 and the molecular weight of the expressed enzyme was measured by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Then the over-expressed strain EGDe-nox was built to detect ROS production and examine the influence of nox gene over-expression on the expression of virulence genes by using quantitative real-time polymerase chain reaction (qRT-qPCR). The results showed that the molecular weight of the recombinant Nox was about 33 kD. Compared with EGDe, the ROS production of EGDe-nox was not changed significantly. The over-expression of nox resulted in up-regulated expression of the invasion-related genes actA, inlA and inlB and the virulence gene prfA. These results suggest that the Nox is unable to dominate ROS production independently but its over-expression can up-regulate the expression of virulence genes.
Fermentation Kinetics, Structure and Characteristics of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Bovine Bone
LIU Lili, LI Dan, YIN Guangjun, LIANG Yanyu, KANG Huaibin
2017, 38(2):  52-58.  doi:10.7506/spkx1002-6630-201702009
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In this work, we conducted a kinetic study on the mixed culture fermentation of bovine bone meal with Bacillus cereus MBL13-U, which was isolated in our previous study, Streptococcus thermophilus and Lactobacillus plantarum for preparing angiotensin converting enzyme (ACE) inhibitory peptides. Kinetic models with a determination coefficient greater than 0.97 for cell growth, product formation and substrate consumption were developed through nonlinear fitting using Logistic equation and Luecleking-Piret equation. The model predictions were in good agreement with the measured values. The structural properties of the ACE inhibitory peptides were characterized by scanning electron microscopy (SEM), ultraviolet (UV) spectroscopy and differential scanning calorimetry (DSC). The results showed that gully marks appeared on the surface of bovine bone-derived ACE inhibitory peptides, because they had a strong moisture absorption capacity with the maximum absorption at 222 nm as collagen-derived peptides do. Besides, the ACE inhibitory peptides had an increased thermal denaturation temperature and a better thermal stability than bovine bone collagen, apart from good solubility, acid and alkali resistance, and their salt tolerance was strong when the NaCl concentration was below 0.9 mol/L. Pepsin and trypsin had slight effects on bovine bone ACE inhibitory peptides.
Production of Optically Pure L-Phenyllactic Acid by Using Whole Cells of Recombinant Escherichia coli
ZHU Yibo, LU Rubin, CHENG Jun, WANG Ying, QI Bin, WANG Limei
2017, 38(2):  59-64.  doi:10.7506/spkx1002-6630-201702010
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The L-lactate dehydrogenase gene (ldhL) and glucose dehydrogenase gene (gdh) were respectively amplified from Bacillus megaterium Z2013513 by PCR and inserted into the plasmid pETDuet-1 to construct the recombinant vector pETDuet-ldhL-gdh. Then, the vector was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant strain E. coli BL21(DE3)/pETDuet-ldhL-gdh. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzymatic activity analysis showed that both ldhL and gdh were successfully co-expressed with biological functions in the recombinant strain. Using whole cells of recombinant E. coli BL21(DE3)/pETDuet-ldhL-gdh, 24.26 mmol/L L-phenyllactic acid was obtained from phenylpyruvic acid at 37 ℃ and 200 r/min after 60 min transformation. The product enantiomeric excess percent was over 99% with substrate molar conversion rate of 59.55%. The results showed the cofactor regeneration biotransformation system was capable of efficiently producing optically pure L-phenyllactic acid.
Construction of Engineered Strain Blocking Glucose Metabolism in Corynebacterium glutamicum
HAN Wuyang, LIU Jinlei, DU Hongyan, WANG Beichen, YI Hong, LI Tianming
2017, 38(2):  65-74.  doi:10.7506/spkx1002-6630-201702011
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In this study, we used the wild-type Corynebacterium glutamicum strain ATCC 13032 as a starting strain to obtain five mutants with knockout of the ptsG and ptsH-ptsI genes as well as the abc, abc2 and iolt1 genes by homologous recombination without resistance marker using reverse metabolic engineering strategies. Our experimental results showed that compared to the wild-type strain, the glucose consumption of the mutant CGΔptsG was 50% using glucose as the sole carbon source, giving an OD value of 1.473, the glucose consumption of the mutant CGΔptsH-ptsI was 39.5%, giving an OD value of 1.226, the glucose consumption of the mutant CGΔabc was 36%, giving an OD value of 1.09, and the glucose consumption of CGΔabc2 was 26.2%, giving an OD value of 0.486, while the mutant CGΔiolt1 could not utilize glucose to grow, suggesting that glucose metabolism of C. glutamicum was blocked. It turned out that the glucose transporter function was controlled by the ptsG, ptsH-ptsI, abc, abc2 and iolt1 genes, encoding transporter proteins.
Optimization of Self-Suction Semi-Continuous Vinegar Fermentation for Flavor Improvement
HONG Housheng, ZHAO Min, LUO Haiyan, DOU Bingran
2017, 38(2):  75-81.  doi:10.7506/spkx1002-6630-201702012
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Semi-continuous submerged vinegar fermentation in a self-suction fermentor has the advantages of high production efficiency and short production cycle. However, the taste of the produced vinegar is not as good as desired. For increased non-volatile acid content and improved vinegar flavor, the traditional liquid-state fermentation process for vinegar production was modified by raw material selection, addition of saccharification agent and mixed-culture alcoholic fermentation. The vinegar production process was determined as follows: After addition of water, ground wheat was liquefied and saccharified by addition of 6.5% (m/m) flavor-enhancing saccharification agent at 35 ℃ for 1 h; afterwards, the substrate was inoculated with a mixture of 5% (V/V) lactic acid bacteria and 0.28 g/100 mL yeast, fermented at 32 ℃ for 4 d, and finally inoculated with acetic acid bacteria. The liquid-state fermentation carried out in shaking flasks yielded a product containing 0.47 g/100 mL non-volatile acids. The total acidity of vinegar produced in a 5 L self-suction fermentor by the semi-continuous submerged fermentation process was no less than 6 g/100 mL, and the volatile acid content was no less than 0.5 g/100 mL, both of which reached the requirements of the Chinese national standard.
Effects of TCA Cycle Metabolism on the Acetic Acid Fermentation of Acetobacter pasteurianus
YIN Haisong, ZHANG Renkuan, CHANG Yangang, ZHENG Yu, WANG Min
2017, 38(2):  82-86.  doi:10.7506/spkx1002-6630-201702013
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The most commonly used microorganism for acetic acid fermentation is Acetobacter pasteurianus. In this investigaton, tricarboxylic acid cycle (TCA cycle) energy metabolism in Acetobacter pasteurianus was interfered by adding acetic acid, TCA cycle inhibitors, and intermediate metabolites. The effects of energy metabolism on the cell growth and acetic acid fermentation of Acetobacter pasteurianus were studied. Results showed that addition of 1% acetic acid obviously strengthened intracellular TCA cycle energy metabolism. The expression levels of the key enzymes involved in the intracellular TCA cycle were significantly up-regulated, and intracellular ATP concentration was increased by 125% compared with that without adding acetic acid. TCA cycle energy metabolism was inhibited by adding TCA cycle inhibitors, the cell growth and acetic acid concentration were significantly inhibited and bacterial biomass was reduced by 90% and 87%, respectively. Acetic acid concentration was reduced by 90% and 94%, respectively. Addition of 0.05% intermediates metabolites (oxaloacetic acid, malic acid, and succinic acid) increased intracellular ATP concentration by 202%, 185% and 165%, respectively, suggesting significantly enhanced TCA cycle-coupled respiratory chain energy metabolism. Bacterial biomass was increased by 92%, 106% and 104% respectively, and acetic acid concentration was increased by 30%, 33% and 31%, respectively, after fermentation for 72 h. Our findings show that TCA cycle energy metabolism has a significant impact on the bacterial growth and acetic acid production of Acetobacter pasteurianus and that strengthening TCA energy metabolism has a positive effect on acetic acid fermentation.
Phylogeny and Performance Assessment of γ-Aminobutyric Acid-Producing Microorganisms from Sichuan Pickles
ZENG Lin, LIU Bo, XU Xiaoyan, ZHANG Qing, YANG Ying, LU Qianwen, ZHAO Tingting, TANG Jie
2017, 38(2):  87-91.  doi:10.7506/spkx1002-6630-201702014
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In order to broaden and evaluate microbial resources for producing γ-aminobutyric acid (GABA), lactic acid bacteria and yeast strains with the ability to produce GABA were isolated, screened and identified from Sichuan pickles. A total of 338 strains of lactic acid bacteria and 67 strains of yeast were initially isolated from Sichuan pickles. Further, 12 strains of lactic acid bacteria (LAB) and 3 strains of yeast which could produce GABA were screened out by paper chromatography. Then the 12 LAB strains were identified as Lactobacillus acidophilus (8.3%), Lactobacillus fermentum (8.3%), Lactobacillus kimchii (8.3%), Lactobacillus suebicus (8.3%), Lactobacillus longer (16.7%), Lactobacillus parafarraginis (8.3%), Lactobacillus similis (8.3%), Lactobacillus plantarum (25.2%) and Lactobacillus pentosus (8.3%), while the 3 yeast strains were identified as two strains of Saccharomyces cerevisiae and one strain of Candida tanzawaensis using physiological and biochemical tests and phylogenetic analysis. The GABA-producing ability of these selected strains was further assessed by high-performance liquid chromatography (HPLC). Our results indicated that Lactobacillus plantarum BC114, Lactobacillus brevis BC237 and Saccharomyces cerevisiae JM037 showed higher GABA-producing capability, and the cultured broth supernatants contained 1 720, 1 080 and 670 mg/L GABA, respectively.
Effects of Fermentation Conditions on Quality and Flavor of Kombucha
YUAN Lei, ZHANG Guohua, Faizan Ahmed SADIQ, HE Guoqing
2017, 38(2):  92-97.  doi:10.7506/spkx1002-6630-201702015
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The objective of this study was to analyze how different fermentation methods can affect the fermentation profile of kombucha in terms of its flavor, fermentation yield (total acid/sugar consumption) and sensory evaluation. The variable factors considered included black tea type, tea extraction modes, concentrations of sugar and tea and inoculum size of starter culture. The optimized conditions for the best quality of kombucha were obtained after adding 0.5% mellow black tea to boiling water and keeping it at room temperature for 15 min, followed by the addition of 7% sugar and 8% komucha starter culture. Finally, the fermentation process was carried out at 30 ℃ for 10 days. After fermentation the broth was clear with pale yellow color, and sweet and moderately sour taste. A considerable change in flavor profile was observed before and after the fermentation under the optimized conditions. A total of 19 volatile aroma compounds were identified including 7 alcohols, 6 esters, 4 acids, 1 phenol and 1 hydrocarbon. The contents of alcohols, acids and esters were 25.09%, 46.40% and 15.91%, respectively.
Strain Screening and Quality Evaluation of Fermented Fresh Rice Noodles
MA Xia, LI Luyao, HE Yan, CHENG Zhaohui, YU Haiyan
2017, 38(2):  98-103.  doi:10.7506/spkx1002-6630-201702016
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A dominant bacterial strain named S2219 was isolated from naturally fermented rice. This strain was identified as Lactococcus lactis based on its morphological, physiological and biochemical characteristics and 16S rDNA sequences. The protein, fat and starch contents, texture properties and sensory quality attributes of unfermented fresh rice noodles, naturally fermented fresh rice noodles and fresh rice noodles fermented by the strain S2219 were measured and compared. Our results indicated that the pure culture fermented rice noodles were lower in protein and fat but higher in amylose compared with the naturally fermented and unfermented samples. Moreover, the pure culture fermented rice noodles had significantly improved texture properties such as chewiness, springiness and resilience as well as a smoother taste than traditional fermented rice noodles. Accordingly, pure culture fermentation was the best choice for the production of fermented rice noodles.
Analysis of Fungal Diversity of Different Commercial Brands of Pixian Broad Bean Paste Using PCR-DGGE Method
ZHU Yongqing, LI Zhihua, LI Huajia, WANG Xuetao, DONG Ling, HUANG Qiaolian
2017, 38(2):  104-108.  doi:10.7506/spkx1002-6630-201702017
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In this study, the fungal diversity and community structures of 13 brands of Pixian broad bean paste were analyzed using polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). The typical DGGE bands were sequenced, and a phylogenetic tree was constructed. The results showed that fungal diversity was abundant in the samples and both unique and common bands were identified. Candida sp., Aspergillus, Zygosaccharomyces rouxii and Kodamaea ohmeri were the predominant fungi. Especially, a total of 10 bands, accounting for 32.25% of the entire sequenced bands, were assigned to Aspergillus flavus. Thus, special attention should be paid to the prevention and control of A. flavus in the broad-bean paste industry.
UV-Induced Mutagenesis and Screening of Spirulina maxima for High Photosynthetic Rate
YANG Shenghui, ZU Tingxun, LUO Guanghong, WANG Danxia, CHEN Tianren, GAO Zicheng
2017, 38(2):  109-114.  doi:10.7506/spkx1002-6630-201702018
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Purpose: To screen a mutant strain of Spirulina maxima with high photosynthetic rate. Methods: The mutagenesis and screening were conducted by a combination of spontaneous isolation, ultrasonic treatment, UV irradiation and extremely high CO2 stress. Results: Three mutants, namely KYZ1, KYZ2 and KYZ3, were obtained. The filament morphology, biomass, CO2 availability, and protein and chlorophyll contents of these three mutants were compared. Finally, KYZ2 was screened as the best strain. Conclusions: Strain KYZ2 revealed longer and larger filaments, faster growth, higher photosynthetic rate, and higher contents of protein and chlorophyll compared with the starting strain. Thus, the mutant strain could have potential for industrial culture.
Correlation between β-Glycosidase Activity and Acid Stress Tolerance in Oenococcus oeni
CHEN Qiling, REN Xiaoning, WANG Ling, TIAN Yu, ZHAO Meijing, SONG Qiaozhi, LIU Shuwen1
2017, 38(2):  115-120.  doi:10.7506/spkx1002-6630-201702019
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Objective: β-Glycosidase plays a crucial role in the hydrolysis of aglycones and subsequent release of grapederived aroma compounds in wine. However, β-glycosidase activity is influenced by many factors. This study was designed taking into account the acid tolerance of Oenococcus oeni to assess and correlate their glucosidase activities with acid stress tolerance. Methods: The cells of O. oeni were implanted with N+ ion to obtain acid-tolerant (pH 3.0) and acid-sensitive (pH 9.0) mutants. Besides, β-glycosidase activities of all the tested strains were determined. Three groups of variant O. oeni were selected and their β-glycosidase genes were sequenced. Results: The acid-tolerant mutants harbored a significantly increased β-glucosidase activity that was 2–4 times higher than that of the original strains, and 2–7 times higher than that of the corresponding acid-sensitive mutants. Sequencing results of eight representative amplicons indicated that all the mutants and their parental strains were identical to each other, except a3 mutant, which formed two base transversions at 108 gene locus (G→C) and at 1 232 locus (A→T). Conclusion: Correlation analysis indicated that O. oeni β-glycosidase activity was significantly positively related to acid stress resistance (P < 0.05).
Component Analysis
Quality Analysis of Traditional Fermented Soybean Paste in Northeast China
TANG Xiaoyang, JIANG Jing, TAO Dongbing, ZHANG Ying, SUN Huijun, YUE Yuanyuan, WU Rina
2017, 38(2):  121-126.  doi:10.7506/spkx1002-6630-201702020
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The sensory and physicochemical properties of 43 traditional fermented soybean paste samples collected in Northeast China were determined in order to understand their quality characteristics. The physicochemical parameters including NaCl, acidity, water, amino nitrogen, reducing sugar, total acid, crude protein, and total free amino acids were correlated with sensory quality using SPSS 17.0 software. Principal component analysis and cluster analysis were carried out as well. The results showed that the best quality parameters of fermented soybean paste were 12–17.75 g/100 g NaCl, 70.35%– 78.56% moisture, 0.6–0.84 g/100 g amino nitrogen, 0.78–1.96 g/100 g total acid, and 6.06–9.83 g/100 g reducing sugar.
Correlation between the Formation of Volatile Compounds and Lipids in Hepatopancreas of Female Chinese Mitten Crab during Cooking
NI Yiqun, WU Na, WANG Xichang
2017, 38(2):  127-132.  doi:10.7506/spkx1002-6630-201702021
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The relationship between the formation of volatile compounds and changes in the fatty acid composition of female Chinese mitten crab (Eriocheir sinensis) during cooking was investigated. The volatile compounds were detected by head-space solid phase microextraction and gas chromatography-mass spectrometry (GC-MS). The total lipids (TLs) were separated by solid phase extraction for GC analysis of the fatty acid composition of polar (PLs) and neutral (NLs) lipids. Removal of total lipids had a marked effect on the profile of volatile compounds, especially aldehydes (hexanal, heptanal, (E)-2-octenal, (E,Z)-2,6-nonadienal, (Z)-2-decenal, undecanal, and pentadecanal) and ketone (2-nonanone). An increase in the NLs and a decrease in the PLs were observed during cooking, while the TLs did not change significantly. Further analysis showed reduced contents of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in the PLs during cooking. Furthermore, unsaturated fatty acids of the PLs, especially C18:1n-9c, C20:1n-9, C20:4n-6 and C22:6n-3, showed the same decreasing trend as those of the TLs. The findings demonstrated that the fatty acids in the PLs might be the flavor precursors of the aldehydes and ketone derived from lipid thermo-oxidation and degradation during cooking.
Analysis and Evaluation of the Nutrient Compositions in Different Tissues of Jellyfish (Rhopilema esculentum Kishinouye)
ZHANG Yuying, CHAI Yanping, QIN Lei, XUE Jia, XU Xianbing, DU Ming, DONG Xiuping
2017, 38(2):  133-138.  doi:10.7506/spkx1002-6630-201702022
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In order to achieve high value-added and comprehensive utilization of jellyfish, the proximate nutritional compositions, amino acid compositions and lipids and fatty acid compositions of different tissues of fresh jellyfish (umbrella, gastric column, scapulet, oral arm, tentacle, gonad and circular muscle) were analyzed and evaluated. The results showed that moisture contents in different tissues were over 93%, and gastric column was the highest in moisture (97.93%). Ash contents were high in all tissues investigated and the values in umbrella, tentacle, gastric column and oral arm were higher than 50% (on a dry weight basis). After deducting ash content, fat contents on a dry weight basis were higher in tentacle and circular muscle, which were 18.80% and 18.76%, respectively. Fat contents in other tissues were relatively lower. Crude proteins were high in all these tissues, especially in umbrella, scapulet and oral arm, which could reach 62.81%–80.94%. The total sugar contents in all the tissues were between 6.28% and 13.36%. Twenty amino acids were detected in gonad and circular muscle, in which essential amino acids were above 25%, which is higher than that of other tissues. Polar lipid contents were higher than cholesterol in different tissues. In umbrella and tentacle, saturated fatty acids (SFA) were more abundant than unsaturated fatty acids (UFA), while in other tissues SFA were lower than UFA. The present research showed that all the tissues in jellyfish were rich in nutrients, and different tissues in jellyfish had different features, which explicate great potential of jellyfish for development and utilization.
Effect of Drying Temperature on the Aroma Components and Physiochemical Properties of Jujube
Lü Shan, LING Min, DONG Haoshuang, MENG Mingjia, CAO Zhen, LIU Mengjun, AO Changwei
2017, 38(2):  139-145.  doi:10.7506/spkx1002-6630-201702023
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The volatile compounds in dried jujube obtained in the temperature range of 80–160 ℃ were characterized by gas chromatography-mass spectrometry-olfactometry (GC-MS-O). The total sugars, reducing sugars, amino acids and color parameters were determined as well to evaluate the effect of drying temperature on jujube quality. A total of 99 volatile compounds were identified by GC-MS, 61 compounds of which were verified as aroma-active compounds by olfactometry. The main compounds, furfural, 5-methyl furfural, 5-hydroxymethylfurfural, 2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran- 4-one, furfuryl alcohol, 2-acetylfuran, 3,5-dihydroxy-2-methyl-4H-pyran-4-one, and 1,3-dione-cyclopent-4-ene, were generated at high drying temperature in a temperature-dependent manner. Some acids, such as acetic acid glacial, 2-methyl butyric acid, hexanoic acid, heptanoic acid, octanoic acid, benzoic acid, benzoic acid, and lauric acid, as well as sugar and amino acid contents, were decreased as the temperature increased. Color parameters showed that the browning reaction was enhanced, leading to a burnt flavor when the temperature was over 140 ℃.
Analysis of Volatile Compounds in Roasted Peanut Shell and Peanut Cakes by HS-SPME-GC-MS
LIU Yunhua, HU Hui, LIU Hongzhi, LI Jun, YANG Ying, SHI Aimin, LIU Li, WANG Qiang
2017, 38(2):  146-153.  doi:10.7506/spkx1002-6630-201702024
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The volatile compounds in roasted peanut shell and peanut meals were investigated using headspace solid phase micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). Principal component analysis (PCA) was performed on the relative concentration data. The results showed that a total of 119 volatile compounds were identified in roasted peanut shell and peanut cakes. They were grouped into 9 categories including aldehydes, ketones, hydrocarbons, pyrazines, furans, pyrroles, pyridines, amines and other compounds. The main volatile compounds in peanut cakes were Nand O-heterocyclic compounds, including pyrazines, pyrroles, furans and pyridines while furans and aldehydes are the main volatile compounds found in peanut shells. By PCA, 2,5-dimethyl pyrazine, methyl pyrazine, 2-acetyl-3-methyl pyrazine, 2-ethyl-3-methyl-pyrazine and N-methyl pyrrole were found to be the main volatile compounds in roasted peanut cakes while 2-pentyl furan, nonanal, 5-methyl furan aldehydes, n-hexanal and decanal were the main volatile compounds in roasted peanut shell.
SiO2/diol Based Solid-phase Extraction and Multi-dimensional Mass Spectrometry for Lipidomics Study of Channa argus
SHEN Qing, JIN Renyao, FENG Junli, XUE Jing, CHEN Kang, DAI Zhiyuan
2017, 38(2):  154-158.  doi:10.7506/spkx1002-6630-201702025
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In this study, a method using SiO2/diol based solid-phase extraction (SPE) and multi-dimensional mass spectrometry-based shotgun lipidomics was developed for studying the phospholipids in muscle tissue of Channa argus. Crude lipids were extracted by the Folch method. Phospholipids were obtained by SiO2/diol-SPE, and it’s the extraction efficiency was compared with that of two other commercial SPE cartridges. The conditions for SiO2/diol-SPE were optimized as follows: loading volume, 100 μL; washing solvent pH 2, and 1 mL of 50% acetonitrile as eluent. The eluate was directly injected into the electrospray ionization (ESI) source. The phospholipid molecular species were identified and quantified using the precursor-ion scan and neutral loss scan modes, and the structures of two fatty acid chains in each species were confirmed by multi-dimensional mass spectrometry. The results showed that a total of 67 phospholipid molecular species were detected, including 18 phosphatidylcholines (PC), 21 phosphatidylethanoamines (PE), 12 phosphatidylinositols (PI), and 16 phosphatidylserines (PS). The phospholipids in C. argus contained abundant unsaturated fatty acyl chains. This analytical method proved to be simple, efficient, and reliable.
Comparison of the Volatile Compositions of Essential Oils from Different Organs of Sage (Salvia officinalis L.)
BAN Zhaojun, CHEN Cunkun, LI Guopeng, YU Linkai, WANG Hongpeng, SUN Rong, MAO Jianwei
2017, 38(2):  159-163.  doi:10.7506/spkx1002-6630-201702026
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The essential oils of different organs of sage (Salvia officinalis L.) including flowers, leaves and stems, were extracted using methyl tert-butyl ether (MTBE) and then analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 31 volatile compounds were identified, including 4 moneterpenes, 17 sesquiterpenes, 6 moneterpenes derivations, 2 sesquiterpenes derivations and 2 esters. However, the volatile compositions of the essential oils were different. The flower essential oil contained the highest number of volatiles (31) while the leaf essential oil contained the highest amount of volatiles (2 334.48 ng/g). Germacrene, caryophyllene, α-copaene and linalyl acetate were the dominant volatiles in the different essential oils, which made different contributions to them.
Separation of L-Epicatechin in Liubao Tea by Molecularly Imprinted Solid Phase Extraction
LIU Boyang, LI Lijun, CHENG Hao, HUANG Wenyi, FENG Jun, KONG Hongxing
2017, 38(2):  164-169.  doi:10.7506/spkx1002-6630-201702027
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Molecularly imprinted polymers (MIPs) were synthesized by in situ polymerization using L-epicatechin as the template molecule, acrylamide (AM) as the functional monomer, azobisisobutyronitrile (AIBN) as the initiator, and ethylene glycol dimethacrylate (EGDMA) as the cross-linking agent. Firstly, the specific adsorption capacity of L-epicatechin MIPs was evaluated as a function of the proportions of template molecule, functional monomer, and cross-linking agent. The results showed that when the molar ratio of L-epicatechin to acrylamide to ethylene glycol dimethacrylate was 1:6:40, MIPs exhibited the best adsorption capacity, with template molecule recovery (KMIPs) of 84.62% and specific recognition factor (Q) of 4.55. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the MIPs. Finally, we used capillary electrophoresis to detect the effluent and the eluate under optimum conditions. As a result, a molecularly imprinted solid phase extraction-capillary electrophoresis (MISPE-CE) method for the determination of L-epicatechin was developed. The experimental results showed that molecularly imprinted polymers were successfully synthesized with good morphology and specific adsorption characteristics. This method is feasible for the analysis of L-epicatechin in Liubao tea.
Volatile Components of Lemon Peel Essential Oils Extracted by An Improved Clevenger Apparatus
FU Manqin, XIAO Gengsheng, CHEN Yulong, WU Jijun, XU Yujuan, CHEN Weidong
2017, 38(2):  170-175.  doi:10.7506/spkx1002-6630-201702028
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In the present study, the yield and volatile components of essential oils from lemon peel (Eureka lemon) extracted by a Clevenger apparatus with and without improvement were investigated. The improved Clevenger apparatus was adopted to extract essential oils from peels of five lemon cultivars grown in Guangzhou (Guangdong Province, China). The volatile components were analyzed structurally characterized by using gas chromatography-mass spectrometry (GC-MS). The results indicated that the improved apparatus gave 63.37% higher essential oil yield than the unimproved one, while having no effects on the types of essential oil volatiles. A total of 51, 50, 41, 43 and 42 components were detected from Citrus hystrix, Eureka, Allen Eureka, Femminello and seedless Lisbon, respectively, consisting mainly of alkenes, alcohols, aldehydes and esters along with small amounts of acids and ketones. Alkenes were the most dominant compounds in all the essential oils with the characteristic component D-limonene being the most abundant, followed by β-pinene and γ-terpinene. There were significant differences in the volatile components of peel essential oils from different lemon varieties. Except for 32 common components, each cultivar contained unique volatile components.
Main Chemical Composition and Melanoidin Composition of Roasted Coffee Beans from Different Regions of Yunnan
LIU Yaling, TAN Chao, GONG Jiashun
2017, 38(2):  176-183.  doi:10.7506/spkx1002-6630-201702029
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The main chemical composition of mildly, moderately, and heavily roasted coffee beans from three major coffeeproducing areas (Pu’er, Nujiang, and Dehong) in Yunnan province was analyzed and melanoidins were extracted from them for analysis by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) technique. Results showed that the contents of protein and fat in roasted coffee beans increased with the degree of roasting, and total amino acids decreased. The protein content of heavily roasted coffee beans from Pu’er was the highest (16.3 g/100 g), and the highest amino acid content (9.41%) was detected in raw coffee beans from Pu’er. On the other hand, moderately roasted coffee beans from Dehong had the highest crude fat content (13.85 g/100 g). The dominant minerals were K, Mg, P and Ca. The K content of heavily roasted coffee beans from Pu’er was the highest (2.2 g/100 g). The Py-GC-MS analysis indicated that the melanoidin composition of coffee beans varied significantly depending on geographic origin and roasting degree. However, there were some common characteristics. The contents of caffeine and acid were the highest, followed by esters, amines, phenols, pyrroles, furans, aldehydes, alcohols, ketones.
GC-MS Detection Combined with Principal Component Analysis to Study the Aroma Compounds of Seven Small Berries
ZHOU Lihua, MOU Dehua, LI Yan
2017, 38(2):  184-190.  doi:10.7506/spkx1002-6630-201702030
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Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to detect and analyze the aroma components of seven small berries that were introduced from Northeast China or Russia to Shijiazhuang, Xingtai and Zhangjiakou in Hebei province. The experimental data were analyzed by principal component analysis (PCA) using the SPSS 17.0 software. Results showed that a total of 150 aroma components were detected, with 31, 59, 47, 54, 39, 48 and 41 of them being present in blueberry, Cerasus humilis, Fengmanhong raspberry, Autumn Bliss raspberry, Harry Heritage raspberry, blackcurrant and goldenberry (Physalis peruviana), respectively. The main aroma compounds of blueberry were alcohols and aldehydes, constituting 43.9% and 40.9% of the total amount, respectively. The main aroma compounds of blackcurrant and Cerasus humilis were esters, accounting for 67.6% and 80.7% of the total amount, respectively. The main aroma compounds of Fengmanhong raspberry were alcohols and esters, representing 60.0% and 28.6% of the total amount, respectively. The main aroma compounds of Autumn Bliss raspberry and Harry Heritage raspberry were alcohols, accounting for 41.75% and 37.37% of the total amount, respectively. Ketones and esters were the main aroma compounds of goldenberry, accounting for 31.9% and 27.3% of the total amount, respectively. Our data on the aroma components of these seven berries can pave the foundation for their industrial development and utilization.
Identification of Flavor Components of Chicken Bouillon Flavor Components by Gas Chromatography-Mass Spectrometry and Electronic Nose
TIAN Huaixiang, ZHANG Yajing, WU Xuan, QIN Lan, CHEN Chen, XIAO Lizhong, YU Haiyan
2017, 38(2):  191-197.  doi:10.7506/spkx1002-6630-201702031
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The volatile components of fresh chicken bouillon, out-of-date chicken bouillon and rancid chicken bouillon were extracted by headspace solid-phase micro-extraction (SPME) and identified by gas chromatography-mass spectrometry (GC-MS). Sensory evaluation and an electronic nose were used to evaluate the flavor attributes of chicken bouillons. The ability of the electronic nose to differentiate chicken bouillons was evaluated by principal component analysis (PCA) and cluster analysis (CA). The correlation between flavor components and electronic nose was established by partial least squares (PLS). The results showed that a total of 53 flavor compounds were identified in chicken bouillons, including 3 alkenes, 6 alcohols, 4 ketones, 12 aldehydes, 6 esters, 3 aromatic compounds, 13 sulfides, 4 heterocyclic compounds and 2 other compounds. The results of sensory evaluation agreed with the electronic nose results. The flavor of fresh chicken bouillon was regarded to be more similar to that of out-of-date chicken bouillon, and they were also similar in terms of volatile composition. PLS showed that 30 flavor components exhibited good correlation with the electronic nose sensors. These findings indicated that combination of GC-MS and electronic nose with chemometrics could evaluate the olfactory flavor of chicken bouillons.
Characteristic Components of Aqueous Extracts of Raw Pu-erh Tea with Different Storage Times
ZENG Liang, TIAN Xiaojun, LUO Liyong, GUAN Xingli, GAO Linrui
2017, 38(2):  198-205.  doi:10.7506/spkx1002-6630-201702032
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Aqueous extracts of raw Pu-erh tea (7542) samples from the same batch obtained from the same producer with different storage times were prepared by vacuum freeze drying in this study and their characteristic components were detected by spectrophotometry and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The experimental data were analyzed with principal component analysis (PCA) and cluster analysis (CA) to establish the characteristic components of Pu-erh tea aqueous extracts and a dendrogram for cluster analysis of raw Pu-erh tea samples stored for different times. The results showed that the contents of tea polyphenols, catechins and free amino acid of raw Puerh tea aqueous extracts decreased gradually with storage time of Pu-erh tea while flavones increased; the contents of total soluble sugars and caffeine showed an irregular fluctuation. A total of 107 substances were detected from raw Puerh tea aqueous extracts with different storage times by UPLC-MS/MS. Using PCA, the 26 characteristic components of raw Pu-erh tea aqueous extracts were determined to include citric acid, gallic acid, epicatechin, theanine, caffeine, and epigallocatechin gallate. Cluster analysis results indicated that the clustering of raw Pu-erh tea samples changed significantly after storage for 3 years and 8 years and that aqueous extracts of raw Pu-erh tea samples with similar storage times were more likely to be clustered.
Processing Technology
Separation of Anthocyanin Monomers from Blueberry Fruits through Chromatographic Techniques
LIU Jingbo, CHEN Jingjing, WANG Erlei, LIU Yanjun
2017, 38(2):  206-213.  doi:10.7506/spkx1002-6630-201702033
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To fully develop the potential application of blueberry fruits, this paper is focused on the preparation of anthocyanin monomers from blueberry fruits using column chromatography and semi-preparative high performance liquid chromatography (HPLC). Crude blueberry anthocyanins were ultrasonically extracted with acidified 70% ethanol and further subjected to two cycles of ethyl acetate extraction for the purpose of facilitating the dissolution of anthocyanins and removing non-anthocyanin flavonoids. The purity of crude blueberry anthocyanins reached 62.49% after Amberlite XAD-7HP column chromatography separationand Sep-Pak C18 solid-phase extraction. Three anthocyanin fractions were obtained by Sephadex LH-20 column chromatography with purities ranging from 65% to 75%. Using semi-preparative HPLC, two pure anthocyanin monomers were successfully isolated from the three purified anthocyanin fractions, which were identified as delphinidin-3-O-galactoside and malvidin-3-O-galactoside by analytical HPLC, with purities of 96.98% and 95.63% respectively. The present study can provide technical references for large-scale production of anthocyanin glycoside monomers, and also provide a good theoretical basis for the production of high value-added products of blueberry anthocyanins.
Optimization of Production of Structured Lipids Containing Conjugated Linoleic Acid by Lipozyme TLIM Catalysis
WANG Xiaoya, LIU Wenqiang, ZHU Wenting, ZENG Kai, ZHU Xuemei, XIONG Hua, HU Jiangning
2017, 38(2):  214-219.  doi:10.7506/spkx1002-6630-201702034
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In this study, structured lipids containing conjugated linoleic acid (CLA) were produced from the reaction between camellia seed oil and CLA ethyl ester catalyzed by Lipozyme TLIM, where Sn-2 positional CLA was obtained by enhancing acyl migration. Four reaction parameters, namely temperature, water activity, reaction time and molar ratio of substrates, were optimized by response surface methodology (RSM) for improved total CLA incorporation and acyl migration. In the synthesized structured lipids, total and Sn-2 positional CLA were up to 36.97% and 12.54% when acyl migration was maximized. The incorporation ratios of total CLA and Sn-2 positional CLA were both higher than the published data.
Synthesis and Mass Spectrometry Analysis of Docosahexaenoic Acid Astaxanthin Ester
YANG Lu, LI Xuemin, ZHOU Qingxin, LI Zhaojie, XU Jie, XUE Changhu
2017, 38(2):  220-226.  doi:10.7506/spkx1002-6630-201702035
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In this study, docosahexaenoic acid (DHA) and astaxanthin were applied as reaction substrates to synthesize DHA astaxanthin ester The optimal reaction conditions were determined as follows: 250 mg of astaxanthin 282 mg of DHA, 5 mL of anhydrous acetone, 100 mg of 4-dimethylaminopyridine (DMAP) and, 100 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), were reacted in a nitrogen atmosphere under dark conditions with magnetic stirring at 25 ℃ for 3 h. As identified by liquid chromatography-mass spectrometry (LC-MS), the synthesized products were composed of free astaxanthin, DHA astaxanthin monoester and DHA astaxanthin diester. Each of these components was analyzed by HPLC-DAD and quantified by peak area normalization method. The results showed that under optimum conditions, DHA astaxanthin esters accounted for 95.67% of the total products, mainly DHA astaxanthin diester (77.46%), DHA astaxanthin monoester (18.21%) and small amounts of free astaxanthin (3.24%). DHA astaxanthin diesters and DHA astaxanthin monoesters were separated by silica gel column chromatography with increasing solvent polarity from 100:0 to 92:8 (petroleum ether:acetone, V/V). Finally, the purity of the purified DHA astaxanthin diesters and DHA astaxanthin monoesters were (98.2 ± 0.5)% and (94.0 ± 0.6)%, respectively.
Optimization of Enzymatic Reaction between Chitosan and Gallic Acid for Enhanced Antioxidative Activity and Reduced Cytotoxicity
WU Hao, WANG Yiying, ZHEN Tianyuan, LUO Dan, ZHANG Xiao, YANG Shaolan, WANG Chengrong
2017, 38(2):  227-232.  doi:10.7506/spkx1002-6630-201702036
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In this paper, chitosan (CTS) grafted copolymer with gallic acid (GA) was synthesized by laccase and horse radish peroxidase to enhance the antioxidant activity of chitosan. The effects of enzyme type and dosage, initial reaction pH, temperature, reaction time, and enzyme-to-substrate ratio on the grafting ratio were studied. The maximum grafting ratio (65.2%) was obtained when the reaction took place at 25 ℃ for 5 h in 100 mL of acetate buffer solution (100 mmol/L) at pH 4.5 with a mass ratio of GA to CTS of 3 in the presence of 4 U of laccase under constant stirring. At the same dosage, the CTSGA copolymer showed significantly higher antioxidant activity than chitosan and had no cytotoxicity.
Optimization of Fluidized Bed Drying Process of Bleached Shellac
TU Xinghao, LI Kun, ZHANG Hong, ZHENG Hua, DU Liqing, ZHANG Wenwen, LI Kai
2017, 38(2):  233-239.  doi:10.7506/spkx1002-6630-201702037
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In this study, bleached shellac was dried with fluidized bed for the purpose of raid drying at lower temperatures. The two main factors, including inlet air temperature and air flow rate, which influence the product color index and drying time, were investigated using one-factor-at-a-time method and response surface methodology (RSM) with central composite experimental design. A regression model was established for each response. The results showed that the predicted value agreed well with the measured one, with an error of only 1.12% and that the two models (R2 = 0.949 1 for color index, R2 = 0.937 7 for drying time) could be used for describing and predicting the fluidized bed drying of bleached shellac. The optimal operating conditions were determined as follows: drying at 36 ℃ at an air flow rate of 45 m3/h for 28 min, resulting in a color index of 0.90. The dried product reached the Chinese national standard for the highest quality grade of refined bleached shellac.
Optimization of Modification Process for Millet Bran Dietary Fiber and Structural Elucidation
KANG Lijun, KOU Fang, SHEN Meng, WANG Weihao, CAO Longkui
2017, 38(2):  240-247.  doi:10.7506/spkx1002-6630-201702038
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Millet bran was pretreated by steam explosion and then subjected to combined ultrasonic-microwave treatment in order to modify millet bran dietary fiber (MBDF), namely to increase soluble dietary fiber (SDF) yield. The process conditions were optimized using response surface methodology. In addition, the structures of modified and unmodified MBDF were investigated by means of gel permeation chromatography-refractive index-multi-angle light scattering (GPCRI- MALS), infrared spectroscopy and X-ray diffraction. The steam explosion conditions were set as 1.0 MPa and 90 s for pressure and time, respectively. The optimal conditions for combined ultrasonic-microwave treatment were determined as microwave power of 535 W, solid-to-solvent ratio of 1:50 (g/mL) and time of 57 min, giving an SDF yield of as high as 10.841%. The results of GPC-RI-MALS showed that the molecular weight of modified millet bran SDF was smaller that of the unmodified one, due to the shorter molecular chain and the reduced degree of polymerization resulted from the modification. The infrared spectroscopic analysis illustrated that there were no significant changes in the chemical functional groups between the modified SDF and IDF, and both of them possessed the characteristic absorption peaks of carbohydrates. The X-ray diffraction analysis revealed that the crystallinity of the modified IDF increased, indicating that its non-crystalline region was partially degraded and converted to SDF. Scanning electron microscopic (SEM) observation showed that the surface of modified millet bran SDF became rough, loose and porous and was composed of aggregated small particles.
Optimization of Two-Step Lipase-Catalyzed Synthesis of Human Milk Fat Substitutes by Response Surface Methodology and Fatty Acid Composition Analysis
XIONG Zhiqin, PAN Lijun, JIANG Shaotong, SHEN Hong, CAO Lili
2017, 38(2):  248-254.  doi:10.7506/spkx1002-6630-201702039
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Human milk fat substitutes (HMFS) were prepared by a two-step enzymatic procedure. Firstly, triacylglycerols (TAGs) rich in palmitic acid (PA) at Sn-2 position were synthesized by Novozym 435 lipase-catalyzed acidolysis of camellia seed oil with palmitic acid in organic system. The optimal alcoholysis conditions were determined using one-factor-ata- time experiments and response surface methodology as follows: n-hexane as reaction solvent at a dosage of 10 mL/g; palmitic acid/camellia seed oil mass ratio, 1.1:1; lipase dosage (on the basis of substrate mass), 10.1%; reaction time, 16.5 h; and temperature, 50 ℃. Under these conditions, TAGs with 66.08% PA at Sn-2 position were obtained. Then HMFS were synthesized by transesterification of these TAGs with fish oil catalyzed by the Sn-1,3 specific lipase Lipozyme TL IM. The final product was tested, and the results showed that the content and distribution of palmitic acid in the HMFS were similar to those of human milk fat. Besides, it had high levels of eicosapentaenoic acid (EPA), and docosahexenoic acid (DHA). Therefore, the HMFS could be used as a fat source for infant formula.
Optimization of Extraction and Antioxidant Activity of Polysaccharides from Epimedium Leaves
TAN Li, CHEN Ruizhan, CHANG Qingquan, LU Juan, JIN Chenguang, YIN Wei
2017, 38(2):  255-263.  doi:10.7506/spkx1002-6630-201702040
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In this study, an ultrasonic-assisted enzymatic extraction (UAEE) method was proposed and optimized for the extraction of polysaccharides (CEPs) from Epimedium leaves. In the first step, the optimization of enzyme mixtures for the hydrolysis of Epimedium leaves was carried out by the combined use of one-factor-at-a-time method and orthogonal array design. Subsequently, the optimization of extraction parameters was done using Box-Behnken design with response surface methodology. The results showed that the influence of the dosage of enzymes on the extraction yield of CEPs was in the following order: cellulose > pectinase > papain > α-amylase, and the optimal combination found were papain 50 U/g, pectinase 250 U/g, cellulase 200 U/g and α-amylase 100 U/g. The optimal extraction parameters were determined as 46.8 ℃, 42.3 min, 4.3 and 311 W for temperature, time, pH, and ultrasonic power, respectively. Under these conditions, the experimental yield of CEPs was 5.98%, which was well in close agreement with the value (6.2%) predicted by the proposed model. Three major fractions (EPs-1, EPs-2 and EPs-3) from the CEPs were purified by DEAE-Sepharose fast-flow and Sephadex G-100 column chromatography. The antioxidant activities of the three fractions were evaluated by 1,1-diphenyl- 2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide anion racial scavenging capacity assays, and ferricreducing antioxidant power (FRAP) assay in vitro. It was indicated that UCEE could be an effective and environmentfriendly technique for extracting active ingredients from plant materials. All the three polysaccharides exhibited significant antioxidant activities in a dose-dependent manner. These results suggested that Epimedium polysaccharides could be explored as potential antioxidants for use in functional foods or medicines.
Optimization of Fermentation Process of A Mixture of Highland Barley Bran and Coix Seed by Monascus purpureus Using Response Surface Methodology
PU Lining, CHEN Guangjing, KAN Jianquan
2017, 38(2):  264-270.  doi:10.7506/spkx1002-6630-201702041
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This study aimed to optimize the solid-state fermentation process of a mixture of highland barley bran and coix seed (1:1, m/m) by Monascus purpureus (CICC.5046) for improved production of Monacolin K as a hypolipidemic agent by the combined use of one-factor-at-a-time method and response surface methodology. The changes in the main substances with physiological activities were investigated during the fermentation process. The one-factor-at-a-time experiments selected fermentation temperature, inoculum size and filling quantity as the main factors that affect the production of Monacolin K, and these factors were optimized by response surface methodology to be 29 ℃, 8% and 40 g of substrates with 60% water added contained in a 250 mL beaker, respectively. After fermentation for 12 d under these conditions, the Monacolin K yield was 110.556 mg/kg. During the fermentation, Monacolin K, pigments and soluble polyphenols were significantly increased whereas soluble polysaccharides, total flavonoids and β-glucan were decreased significantly. In general, the substance with hypolipidemic activity was increased more significantly.
Comparative Effects of Ultra-High Pressure and Ultrasonic Treatment on the Extraction and Antioxidant Activity of Polyphenols from Lonicera caerulea Fruits
LI Xinyuan, LI Bin, YAN Tingcai, LIU Sunwen, SUN Xiyun, SHI Yimo, ZHANG Qi, MENG Xianjun
2017, 38(2):  271-277.  doi:10.7506/spkx1002-6630-201702042
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In the present study, response surface methodology was used to optimize the conditions for ultra-high pressure (UHP) extraction of polyphenols from Lonicera caerulea fruits and a comparison was performed with ultrasonic-assisted extraction (UAE) with respect to extraction efficiency, processing conditions and antioxidant activity of polyphenols. The results showed that the optimal conditions for UHP-assisted extraction that provided the maximum yield of polyphenols of (778.23 ± 3.45) mg/100 g berries were determined as follows: 50% ethanol as the extraction solvent, a solid-to-liquid ratio of 1:19 (g/mL), an extraction temperature of 30 ℃, an extraction pressure of 406 MPa, and an extraction time of 11.5 min. The optimal conditions for ultrasonic-assisted extraction that gave the maximum yield of polyphenols of (785.74 ± 3.89) mg/100 g berries were determined to be 50% ethanol as the extraction solvent, a solid-to-liquid ratio of 1:25 (g/mL), an extraction temperature of 40 ℃, an ultrasonic power of 500 W, and an extraction time of 90 min. Antioxidant test results showed that the antioxidant activity of polyphenols from ultra-high pressure extraction was significantly higher in terms of 2,2’-azinobis( 3-ehtylbenzothiazoline-6-sulfonic acid) diammonium salt radical (ABTS+·) scavenging capacity, ferric reducing antioxidant power (FRAP), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity when compared with those from ultrasonic extraction and VC at the same concentration levels. Although the ultra-high pressure extraction took a shorter time and yielded polyphenols with higher antioxidant activity, its efficiency in large-scale extraction of polyphenols was not as good as that of the ultrasonic-assisted extraction due to the limitation in the size of the container used. Therefore, considering extraction yield and extraction efficiency, the ultrasonic-assisted extraction was better than the ultra-high pressure extraction for Lonicera caerulea fruit polyphenols.
Extraction of Polysaccharides from Mixed Fructus Schisandrae and Radix Astragali and Their Protective Effects on Acute Liver Injury Induced by Alcohol in Mice
SUN Hui, YUAN Rongshuang, LI He, ZHANG Yuhang, SHI Jichao, CHEN Jianguang, WANG Chunmei*
2017, 38(2):  278-282.  doi:10.7506/spkx1002-6630-201702043
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Objective: This study aimed to optimize the extraction of polysaccharides from mixed Fructus Schisandrae and Radix Astragali (SAP) and to examine their protective effects on alcohol-induced acute liver injury in mice. Methods: An orthogonal array design was applied to optimize SAP extraction by water extraction and alcohol precipitation. A total of 40 mice were randomly divided equally into normal control (CON) group, CON + SAP group, liver injury model (MOD) group and MOD + SAP group. The mice in the SAP-treated groups were intragastrically administered with 100 mg/kg SAP, and those in the CON and MOD groups were administered with an equal volume of distilled water for 30 days in the same way. Fifty percent ethanol (12 mL/kg) was orally given to the mice in the MOD and MOD + SAP groups at 1 hour after the last administration, and those in the CON and CON+SAP group were given an equal volume of distilled water intragastrically. Sixteen hours later, blood and liver samples were collected for the calculation of liver indexes of the mice in each group and the detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum as well as the contents of reduced glutathione (GSH), malondialdehyde (MDA) and triglyceride (TG) in liver tissues. Hematoxylin-eosin staining (HE) was performed to observe pathological changes of liver tissues. Results: The optimum conditions for SAP extraction were determined as follows: solid-to-liquid ratio, 1:45; extraction time, 3 h; and temperature, 100 ℃. Compared with the alcohol-induced liver injury model, SAP significantly reduced the liver index in mice (P < 0.05) and the levels of ALT and AST in serum (P < 0.05), elevated the content of GSH (P < 0.01), significantly reduced the contents of TG and MDA in liver tissues (P < 0.05), and improved the pathological changes of liver tissues. Conclusions: SAP could have a protective effect on alcohol-induced acute liver injury in mice.
Optimization of Ultrasonic-Assisted Extraction by Response Surface Methodology and Antioxidant Activities of Pectic Polysaccharide from Okra Flowers
SONG Siyuan, SU Ping, WANG Lijuan, ZOU Mingming, SUN Xin
2017, 38(2):  283-289.  doi:10.7506/spkx1002-6630-201702044
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Based on one-factor-at-a-time experiments, response surface methodology was employed to optimize the ultrasound-assisted extraction of pectic polysaccharide from okra flowers. The optimal conditions were determined as follows: extraction temperature, 55 ℃; extraction time, 30 min; ultrasonic power, 85.5 W; and raw material-to-water ratio, 1:40 (g/mL). Under these conditions, the experimental yield of pectic polysaccharide was 12.62%, which was well matched with the value predicted by the developed model. Furthermore, the pectic polysaccharide from okra flowers exhibited significant antioxidant activities in vitro and could be developed as a potential natural antioxidant ingredient in the food industry.
Safety Detection
Authentication of Three Monofloral Honeys by High Performance Liquid Chromatography with Electrochemical Detection
HE Qiong, HE Liangliang, KANG Yuxin, CHENG Ni, Lü Xingang, CAO Wei
2017, 38(2):  290-295.  doi:10.7506/spkx1002-6630-201702045
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A novel method for the identification of the floral origins of honeys was established by high performance liquid chromatography with electrochemical detection (HPLC-ECD). The HPLC-ECD fingerprints of three monofloral honeys collected from different areas of China were established. From the HPLC-ECD fingerprints, the areas of the common peaks were obtained and principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed to classify 45 honey samples (15 medlar honey samples, 15 vitex honey samples, and 15 litchi honey samples) according to their floral origins. These samples were successfully classified by PCA and HCA with 100% correct classification rates. To evaluate the reliability of the model based on 45 honey samples, some medlar honey, vitex honey and litchi honey samples which were not included in the modeling sample set were validated with correct prediction rates of 100%, 80% and 100%, respectively. The results indicated that HPLC-ECD combined with PCA and HCA may be used as a fast, accurate and environmentally safe method to differentiate honeys according to their floral origins.
Development of Immunoagnetic Bead-Based ELISA for the Detection of Carbaryl
ZHANG Can, CUI Hanyu, HAN Yufeng, WANG Xianping, HE Ming, CHEN Bo, LIU Yuan
2017, 38(2):  296-300.  doi:10.7506/spkx1002-6630-201702046
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This paper develops an immunomagnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the detection of carbaryl. Under optimum conditions, the method exhibited a good linear relationship in the range of 1 × 10-3-10 mg/L, with a correlation coefficient (R2) of 0.994. The maximum inhibition rate reached 90.6%, and the halfmaximum inhibitory concentration (IC50) was 0.077 mg/L. The detection limit (LOD) of this method was 1.48 × 10-3 mg/L. The precision for three replicate detections of carbaryl (1 mg/L) was 1.67% (relative standard deviation, RSD). The recovery of rice and Chinese cabbage spiked with carbaryl was between 70.5% and 123.1%. Also, it was found that the detection results were highly correlated with those obtained by high performance liquid chromatography (R2 = 0.91). In conclusion, the immunomagnetic bead-based ELISA can be used for rapid detection of carbaryl.
Early Detection of Mechanical Damage in Chinese Winter Jujube (Zizyphus jujuba Mill. cv. Dongzao) Using NIR Hyperspectral Images
SUN Shipeng, PENG Jun, LI Rui, ZHU Zhaolong, Vázquez-Arellano MANUEL, FU Longsheng
2017, 38(2):  301-305.  doi:10.7506/spkx1002-6630-201702047
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Fruits of Chinese winter jujube (Zizyphus jujuba Mill. cv. Dongzao) are sensitive to mechanical stress and can easily develop brown spots after suffering mechanical stress during mechanical harvesting and postharvest handling. The damage cannot be detected easily by machine vision at very early stages of maturity. Thus, a near-infrared (NIR) hyperspectral imaging system was used to detect mechanical damage in Chinese winter jujubes. For reducing the dimensionality of hyperspectral data, three feature selection methods, successive projections algorithm, (SPA), correlation-based feature selection (CFS), and consistency, were used. In addition, three classifiers, i.e., k-nearest neighbor (k-NN), naive bayes (NB), and support vector machine (SVM), were evaluated to segment the pixels of the jujubes into two regions: damaged and nondamaged. Results revealed that two consistent wavebands, i.e., 1 353 nm and 1 691 nm, were established by all the feature selection methods. Besides, SVM offered the best performance with a correction recognition rate of 95.16% using the selected features by the consistency method. NB offered similar performance with a correction recognition rate of 84.26% in the selected wavebands. Hence, this work can pave the foundation for early on-line detecting Chinese winter jujube damage caused by mechanical stress.
Gas Chromatography-Mass Spectrometry Method for Rapid Detection of Geraniol and α-Terpilenol in Ethylene-Vinyl Alcohol Copolymer Active Food Packaging Film
WANG Haili, LI Li, CHEN Mo, YANG Chunxiang, WANG Jing, YU Chen
2017, 38(2):  306-310.  doi:10.7506/spkx1002-6630-201702048
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A method for detecting geraniol and α-terpilenol incorporated into ethylene-vinyl alcohol (EVOH) active food packaging film was established by using gas chromatography-mass spectrometry (GC-MS). The extraction and detection conditions were optimized. The external standard method was used to conduct quantitative analysis. The results showed that the GC-MS method had a good linearity over the concentration range of 0.02–2.0 mg/L for geraniol and α-terpilenol with correlation coefficients of 0.999 2 and 0.999 0, respectively. The limits of detection (LOD) for the analytes were 0.003 and 0.007 mg/kg and limits of quantification (LOQ) were 0.010 and 0.020 mg/kg, respectively. The recoveries of the preservatives were in the range of 82.5%–98.5% at spiked levels of 0.1, 0.2 and 1.0 mg/kg with relative standard deviations (RSDs) ranging from 3.6% to 10.4%. The method is rapid, simple and highly sensitive. It can provide a foundation for further insight into the release mechanism of active substances from active food packaging materials and offer a detection method for ensuring the safety of active food packaging film for application.
A Precise Quantitative Assay for Measuring Pork Incorporated into Mutton Products by Droplet Digital PCR
REN Jun’an, DENG Tingting, HUANG Wensheng, GE Yiqiang, CHEN Ying
2017, 38(2):  311-316.  doi:10.7506/spkx1002-6630-201702049
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The adulteration of meat products happened frequently in recent years, and authentication of meat products is necessary to protect consumers from an inferior product with a false description. Although at present, there are numerous qualitative methods for meat species identification, fewer quantitative detection methods have been reported. Herein, a droplet digital PCR method for the quantitative determination of pork incorporated in mutton products was developed. The single copy house-keeping gene encoding replication protein A1 (PRA1) was chosen as the target to design species-specific primers and probes for mutton and pig, respetively. Each assay was proved specific to the target species, respectively. The ratio constants between copy numbers and unit mass of pork and mutton were obtained by theoretical analysis and then verified by experiments. Consequently, the relative mass fractions of pork and mutton in the sample were measured based on the DNA copy numbers. The limit of quantitation (LOQ) of the method was confirmed to be 1%. The results showed that the absolute error was less than ±1.3%, and the relative error was less than ±10% in the range of pork proportion from 5% to 80%. The method developed in this paper was successfully applied to quantitate pork content incorporated into commercial mutton products and it may be useful for food administration laboratories to carry out meat species quantification in raw and processed foods.