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Table of Content

25 March 2019, Volume 40 Issue 6
Food Chemistry
Characterization and Simulated Gastrointestinal Digestion of Paramyosin from Perna viridis
ZOU Rui, ZHANG Lingjing, ZHONG Chan, WENG Ling, LIN Liyun, LI Yujin, LIU Guangming, CAO Minjie
2019, 40(6):  1-8.  doi:10.7506/spkx1002-6630-20180326-353
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In order to investigate the thermal stability, pH stability and digestion characteristics of paramyosin (PM) in simulated gastrointestinal fluids, we purified PM to homogeneity from the muscle of Perna viridis by consecutive ammonium sulfate fractionation and hydroxyapatite chromatography, and we further identified it by peptide mass fingerprinting (PMF). Circular dichroism (CD) spectroscopy was employed to measure its secondary structure and thermal denaturation temperature. SDS-PAGE was carried out to explore its thermal stability, pH stability and digestion characteristics in simulated gastrointestinal fluids. PM showed a single band corresponding to 99.5 kDa on SDS-PAGE. Using peptide mass fingerprinting, a total of 26 peptide fragments, including 330 amino acid residues, were obtained from purified PM, which revealed 100% identity to PM from Mytilus galloprovincialis, indicating that the purified protein is PM. CD spectral analysis demonstrated that PM had a typical α-helix structure with thermal denaturation temperature (Td) of (56.3 ± 0.2) ℃. No obvious precipitate was observed when PM was heated in the temperature range from 30 to 100 ℃ for 30 min. PM was stable between pH 6.0–11.0. However, it was unstable below pH 5.0 and aggregated. PM could only be partially hydrolyzed by pepsin, trypsin or chymotrypsin individually, while it was effectively degraded by continuous hydrolysis with the three proteinases. However, protein bands with molecular mass of approximately 30 kDa remained undigested. In conclusion, PM from Perna viridis is thermally stable and resistant to gastrointestinal digestion. This work provides a valuable theoretical basis for mussel processing and further study on PM.
Effects of Foliar Application of Lanthanum and Cerium on Protein Content and Amino Acid Composition of Soybeans and Nutritional Evaluation
REN Hongyu, LI Haoyang, CHEN Haiyan, BAI Lu, MIAO Yanli, TAO Pei, LIU Wenlong, ZHANG Xingwen
2019, 40(6):  9-15.  doi:10.7506/spkx1002-6630-20180129-408
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In this work, we aimed to study the effect of foliar application of rare earth elements on protein content and amino acid composition of soybeans and to evaluate the nutritional quality of soybean protein. In this pot experiment, LaCl3, CeCl3 and LaCl3 + CeCl3 solutions of three different concentrations were sprayed on soybean leaves in the seedling and early flowering stages. Ripe seeds were harvested for the determination of protein content using a near-infrared grain analyzer. An automatic amino acid analyzer was used to determine amino acid compositions. The protein nutritional quality of soybean seeds was evaluated by calculating amino acid score (AAS), chemical score (CS), score of ratio coefficient of amino acid (SRCAA) and essential amino acid index (EAAI). The results showed that foliar application of rare earth elements could significantly increase the content of protein, with a maximum increment of 5.17% as compared with control group. The ratio of essential to total amino acids in the different experimental groups was between 32.89% and 35.17%, which was significantly higher in all the experimental groups except for La10 than in the control group. According to AAS values, the first limiting amino acid in the La10 group was valine, while the first limiting amino acids in all other groups were methionine and cystine. The AAS, SRCAA, and EAAI in the 60 mg/L CeCl3 treatment group were 94.05, 84.08 and 82.46, respectively, higher than those in all other groups. On the other hand, the nutritional quality of soybean protein decreased at higher concentration of rare earth elements. The above results show that in the seedling and early flowering stages, foliar application of rare earth elements at the appropriate concentrations can improve the nutritional quality of soybean seeds.
Effect of Regenerated Cellulose Gel on the Stability of Pre-Emulsion with Whey Protein Isolate
ZHAO Yinyu, TIAN Xiaona, ZHOU Guanghong, ZHANG Wangang
2019, 40(6):  16-21.  doi:10.7506/spkx1002-6630-20180109-108
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The objective of this study was to improve the stability of oil-in-water (O/W) pre-emulsion stabilized by whey protein isolate (WPI) using regenerated cellulose (RC) gel. Emulsions were prepared by adding 0%, 0.4%, 0.8%, 1.2% and 1.6% RC, respectively. The stability was examined by measuring stability index, emulsifying activity index (EAI), droplet size, microstructure and rheological properties. The results illustrated that emulsion stability and EAI were significantly improved by RC (P < 0.05) and creaming was effectively inhibited by the addition of 0.8% RC. With the increase of RC, the droplet size and flocculation of emulsions showed a decreasing trend (P < 0.05). The rheological properties showed that RC had a great influence on the stability of WPI-stabilized emulsions. Addition of 1.2% RC enhanced the apparent viscosity of emulsions, resulted in the formation of a strong gel network structure, significantly reduced the droplet size and improved the stability.
Effect of Transglutaminase on Freeze-Thaw Stability of Soybean Protein Isolate Emulsion
YU Guoping, YUE Chonghui, CHEN Yuan, LIU Peng, LIU Yanqiu, DONG Liangwei
2019, 40(6):  22-27.  doi:10.7506/spkx1002-6630-20171207-081
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In this study, transglutaminase (TGase) was used to improve the freeze-thaw stability of oil-in-water emulsion stabilized by soybean protein isolate (SPI). We evaluated the influence of TGase reaction time, enzyme dosage and the number of freeze-thaw cycles on the freeze-thaw stability of SPI-stabilized emulsion as reflected by oiling off and creaming index. Meanwhile, the microstructure and thermal characteristics of the emulsions containing SPI and TGase-modified SPI as emulsifier were compared, and the effect of TGase modification on SPI composition was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, the relationship between enzymatic modification and emulsion stability was analyzed. The results show that after three freeze-thaw cycles, the emulsion containing TGasemodified SPI maintained good stability. A crosslinking time of 3 h and an enzyme dosage of 1.5% were found to be optimal conditions to obtain better freeze-thaw stability. The emulsion remained stable as indicated by its microstructure. The changes in thermal properties during freeze-thaw showed that the crystallization and melting behavior of the emulsion altered. SDS-PAGE revealed that SPI composition changed after enzymatic modification, thus affecting the freeze-thaw stability of the emulsion. This study provides a theoretical foundation for applying TGase-modified SPI in frozen food.
Influence of Enzymatic Extraction on the Composition and Molecular Mass Distribution of Arabinoxylans in Maize Bran
GU Chunmei, YIN Jiayu, JIANG Lei
2019, 40(6):  28-34.  doi:10.7506/spkx1002-6630-20171105-051
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Arabinoxylans (AXs) were extracted from fresh maize barn with four xylanases commonly used in the food industry, Grindmyl Powerbake 950, Pentopan mono BG, Viscozyme L and Amano HC 90. The monosaccharide composition and molecular mass distribution of the extracts were analyzed by gas chromatography (GC) and high-performance sizeexclusion chromatography (HPSEC). The optimum xylanase was selected and its optimum dosage was determined. Monosaccharide composition analysis showed that the four AXs were mainly composed of arabinose and xylose. The purity of the AX extracted with Amano HC 90 for 24 h was 70.51%, and the extraction yield was (17.37 ± 2.74)%. The weight average molecular mass of the AX was (67.87 ± 1.46) × 104 g/mol, the number average molecular mass was (15.06 ± 0.09) × 104 g/mol, and the relative molecular mass dispensability was 4.51 ± 0.13. Amano HC 90 gave a higher purity and yield of AX with higher molecular mass and more homogenous molecular mass distribution. Thus, this xylanase was more suitable for the extraction of AX from maize bran in the food industry, and its optimum dosage was 1 500 U/g.
Effect of pH Value on Rheological and Structural Properties ofL ow Methoxyl Pectin/Sodium Caseinate Composite System
PENG Yuanyuan, WANG Yugui, TANG Xueqian, ZHAO Xin, ZHANG Fusheng, ZHENG Jiong
2019, 40(6):  35-40.  doi:10.7506/spkx1002-6630-20180327-365
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We studied the effect of pH on the rheological and structural properties of low methoxyl pectin/sodium caseinate composite system. At different acidic pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5), static shear and dynamic viscoelastic rheological properties, zeta potential, particle size, turbidity, and microstructure were measured. The results showed that the rheological properties of the composite system changed in two stages. Fluid index n and tanδ increased whereas consistency coefficient K decreased with the increase of pH from 3.0 to 4.0. As the pH increased from 4.5 to 5.5, n and tanδ decreased rapidly and then increased to a level higher than the initial one while K increased steeply and then decreased. Zeta potential decreased with increasing pH; it was positive at pH 3.0 to 4.0 but was negative at pH 4.5 to 5.5. Particle size and turbidity increased to a maximum at pH 4.5 and then decreased with the increase of pH. As the pH value decreased, the microstructure of the composite system changed from a disordered state into a dense one. At pH 3.0–4.0, the structure of the casein surface was dominated by low methoxyl pectin, and the network structure of low methoxyl pectin was more obvious at pH 4.5–5.5. Therefore, changes in pH can cause changes in the rheological and structural properties of the composite system, thereby affect the stability of the system.
Effect of Extruded and Steam Exploded Wheat Bran on Pasting Properties of Wheat Flour
LI Zhi, WU Tao, LIU Rui, SUI Wenjie, ZHANG Min
2019, 40(6):  41-47.  doi:10.7506/spkx1002-6630-20171129-362
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In this work, the effects of the amount and particle size of natural, extruded, and steam exploded wheat bran on the gelatinization characteristics of wheat flour with different gluten contents were investigated. The results showed that the peak viscosity, final viscosity and setback of wheat flour significantly decreased with the increase of wheat bran content (P < 0.05), while the gelatinization temperature and onset time increased quadratically, more significantly with the addition of extruded and steam exploded wheat bran relative to the natural one. The peak viscosity, final viscosity and setback of natural wheat flour with high and medium contents of gluten declined first and then rose with decreasing particle size of wheat bran, whereas the gelatinization temperature and onset time decreased continuously. The setback of low-gluten flour significantly changed with particle size of wheat bran in the range of 0.150–0.420 mm. There was no significant difference in the effect of the particle size of extruded and steam exploded wheat bran on the peak viscosity of medium-gluten flour (P > 0.05), while the peak viscosity of high-gluten flour was significantly influenced by the particle size of natural and steam exploded wheat bran (P < 0.05).
Bioengineering
Screening of Trichoderma reesei Mutants and Optimization of Fermentation Conditions for Higher Cellulase Production
ZOU Zongsheng, WANG Jingya, ZHAO Yunying, MAO Yin, DENG Yu
2019, 40(6):  48-54.  doi:10.7506/spkx1002-6630-20180403-032
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Mutants of Trichoderma reesei RUT-C30 induced by atmospheric and room temperature plasma (ARTP) were screened for cellulase production. Whole-genome sequencing was performed to analyze the mutation types of the selected mutant. Also, we investigated the optimization of fermentation medium and conditions for improved production of cellulase. Mutant JNDY-13 was found to produce the highest cellulase activity. Through shake-flask fermentation, the filter paper activity (FPA) of JNDY-13 was 2.21 IU/mL, which was 2.21 times higher than that of T. reesei RUT-C30. The highest FPA of 5.40 IU/mL was achieved through fed-batch fermentation in a 5-L fermentor under optimized conditions. A total of 752 mutations were identified in JNDY-13, and the 18-base pair insertion in the galactokinase gene may account for the increased cellulase activity.
Heterologous Expression of Recombinant Cystathionine β-Lyase from Saccharomyces cerevisiae and Catalytic Synthesis of 2-Furfurylthiol
ZHA Musu, YANG Huaqing, WANG Xin, SUN Baoguo, WANG Chengtao, LU Song
2019, 40(6):  55-61.  doi:10.7506/spkx1002-6630-20180131-430
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In order to clarify the bioconversion mechanism of 2-furfurylthiol, we cloned the cystathionine β-lyase gene (Str3) from Saccharomyces cerevisiae G20 and expressed it in Escherichia coli BL21, and we further evaluated the catalytic activity of the recombinant enzyme. With the use of orthogonal array design, the optimized induction conditions for the recombinant strain were determined as follows: temperature 20 ℃, IPTG concentration 0.5 mmol/L, and time 13 h. The recombinant Str3p was harvested after ultrasonic disruption of the cells and purified by Ni column affinity chromatography and its molecular mass was measured to be about 52 kDa. The content of soluble Str3p was up to 1.26 mg/mL, the expression level and the enzymatic activity were increased by about 41.7% and 38.6%, respectively, compared with those before optimization. The recombinant Str3p was capable of catalyzing the degradation of cysteine-furfural conjugate into 2-furfurylthiol. The process was greatly influenced by pH value, and the maximum yield of 2-furfurylthiol of 47 μmol/L was obtained at pH 8.0. This study can provide useful information on biosynthesis of 2-furfurylthiol.
Bacterial Diversity Analysis Using Illumina HiSeq Sequencing of Jiangshui, a Chinese Fermented Vegetable Food, and Its Physicochemical Properties
WEI Benliang, LIU Zhanggen, XIAO Yangsheng, PENG Zhen, HUANG Tao, GUAN Qianqian, XIONG Tao
2019, 40(6):  62-68.  doi:10.7506/spkx1002-6630-20180312-156
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In this study, the bacterial community structure and diversity in Jiangshui samples from Shaanxi and Gansu provinces was analyzed through Illumina HiSeq sequencing of the V3-V4 region of the 16S rRNA gene. The results showed that the bacterial community structure in all samples was similar with high diversity. The relative abundance of Lactobacillus was high and Lactobacillus, L. fermentum, Pediococcus ethanolidurans and L. parafarraginis were the dominant species, whose relative abundance was 33.39%, 21.13% and 3.28%, respectively.The physicochemical properties showed that salinity ranged from 0.09% to 0.34%, pH values from 3.32 to 4.00,acidity from 0.194 to 0.872 g/L and nitrite content from 5.123 to 10.246 mg/kg (below the maximum limit of the national standard for preserved vegetables). In all Jiangshui samples, the most abundant organic acid was lactic acid followed by short-chain fatty acids such as formic acid, acetic acid, propionic acid and butyric acid. Lesser amounts of sucrose, glucose and fructose were found in the samples. This study can provide a theoretical basis for research on traditional fermented foods.
Process Optimization for the Production of Fermented Sausages Inoculated with Probiotic Starter Cultures Using Response Surface Analysis
CAO Chenchen, FENG Meiqin, SUN Jian, XU Xinglian, ZHOU Guanghong
2019, 40(6):  69-76.  doi:10.7506/spkx1002-6630-20181009-069
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Fermented sausages were made with a mixed starter culture of Lactobacillus plantarum and Staphylococcus simulans. We aimed to study the effect of inoculum amount and composition and fermentation temperature as well as interactions among them on quality attributes such as pH value, color, textural properties and sensory evaluation using onefactor- at-a-time and response surface methodology. The results indicated that the optimal fermentation conditions were as follows: ratio between L. plantarum and S. simulans 1:1, inoculum amount 107 CFU/g, and temperature 30 ℃. Under these conditions, a higher sensory score of 85.28 was obtained, agreeing with the model predicted value. The fermented sausages had good quality with moderate texture, good taste, color, and rich and pure aroma.
Biotransformation of Valeneene into Nootkatone
LI Xiao, FAN Gang, REN Jingnan, ZHANG Lulu, PAN Siyi
2019, 40(6):  77-84.  doi:10.7506/spkx1002-6630-20180309-117
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The purpose of this study was to investigate the effect of Aspergillus nigerus, Mucor sp. and Yarrowia lipolytica on the conversion of valeneene to nootkatone using solid phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS). The results showed that all these three strains were capable of transforming valencene to nootkatone. Other products, such as α-terpinol and D-carvone, were also found during the biotransformation. The highest amount of nootkatone was found to be produced by Yarrowia lipolytica with a yield of (252.52 ± 15.79) mg/L and conversion rate of (27.45 ± 1.72)% followed by A. niger and Mucor sp. The amounts generated by the latter two strains were (140.94 ± 30.26) and (137.67 ± 14.02) mg/L, and the conversion rates were (15.32 ± 3.29)% and (15.45 ± 3.36)%, respectively. Using an orthogonal array design the optimal fermentation medium for Y. lipolytica was determined to consist of lactose 40 g/L, peptone 15 g/L, and FeSO4·7H2O 0.6 g/L. Using the optimized culture medium, the yield of nootkatone was up to (457.32 ± 76.11) mg/L, and the conversion rate was 49%, which increased by 21.55% compared to that before optimization.
Application of Rennet from Bacillus amyloliquefaciens GSBa-1 in Mozzarella Cheese
TENG Junwei, LIU Zhenmin, SU Miya, GAO Hongyan, ZHENG Yuanrong, LI Liu, ZHENG Zhe, ZHANG Jian,YANG Zhennai
2019, 40(6):  85-92.  doi:10.7506/spkx1002-6630-20180128-383
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The rennet produced by Bacillus amyloliquefaciens GSBa-1, isolated from Jiuqu, a traditional Chinese liquor fermentation starter, was found to possess high milk-clotting activity (up to 1.46 × 106 SU/g) and low proteolytic activity. Mozzarella cheeses were prepared using the purified enzyme, and a commercial rennet was used as a control. During cheese ripening, the physicochemical properties, pH, microbial parameters, texture properties, free fatty acid composition, soluble protein content, flavor properties and other properties were studied. Results showed no significant difference (P < 0.05) in chemical composition was seen between the two cheeses. During cheese ripening, the viable count of Lactococcus lactis increased first and then decreased; however, the difference was not significant. The pH 4.6 soluble protein content and total free amino acid content (76 versus 55.3 mg/100 g) of cheese made with the microbial rennet were higher than those of the control cheese. The cheese prepared using the microbial rennet also had better textural properties than the control. Morphological observation of cheese using scanning electron microscopy showed that the internal network structure of the cheese prepared using the microbial rennet was more compact. Moreover, it contained more volatile flavor compounds due to the relatively strong proteolytic activity of the rennet. There were no significant differences in conformality, stringiness or sensory evaluation between the two cheeses (P > 0.05). All these results showed that the rennet produced by B. amyloliquefaciens GSBa-1 is a promising alternative to calf rennet in processing of Mozzarella cheese.
Screening and Functional Characteristics of Probiotics from Zuolajiao, a Traditional Fermented Hot Pepper Product
XIA Haiyan, ZHOU Siduo, ZHANG Mingzhe, FANG Han, HUANG Yi, DONG Mingsheng
2019, 40(6):  93-99.  doi:10.7506/spkx1002-6630-20180315-199
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The present study was designed to screen 16 strains of lactic acid bacteria isolated from Zuolajiao for their probiotic potential for the development of functional foods. The acid production rates and resistance to artificial gastric juice of these isolates were comparatively evaluated. They were identified by biochemical tests and 16S rDNA sequence analysis. In addition, their probiotic properties were evaluated with respect to stress tolerance, surface hydrophobicity, auto-aggregation ability, antibacterial activity, antioxidant capacity, nitrite degradation ability and safety. Three potential probiotic strains, namely Lactobacillus fermentum 17-1 and 18-2 and Lactobacillus brevis L3-5 were found to be tolerant to 0.4% phenol, lysozyme and heat treatment and have high surface hydrophobicity and auto-aggregation ability. They could produce broad-spectrum bacteriocins against pathogenic bacteria. L3-5 and 17-1 had stronger antioxidant potential. All strains exhibited bile salt hydrolase activity and γ-aminobutyric acid producing ability and were safe. In conclusion, Zhalajiao is a new natural source of probiotics, and L. fermentum 17-1 and 18-2 and L. brevis L3-5 can be used as potential probiotic strains.
Screening, Identification and Fermentation Characteristics of Yeast Strains for Low Concentrations of Urea and Fusel Alcohol in Chinese Rice Wine
WU Danli, LI Gaolei, WANG Mingyue, CHEN Xiaohan, LI Yudong
2019, 40(6):  100-105.  doi:10.7506/spkx1002-6630-20180302-019
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Five yeast isolates were compared for their capacities of producing urea and fusel alcohol. Strains AW001 and J-16-2 were selected for reduced production of urea and fusel alcohol. Urea production was positively correlated to the copy number of the CAR2 gene related to arginine assimilation as determined by real-time quantitative PCR. All strains were identified as Saccharomyces cerevisiae based on physiological and biochemical characteristics and rDNA ITS sequence analysis. Total sugar and total acid contents in the Chinese rice wines fermented with AW001 and J-16-2 met the national standard. These strains have great potential for development and utilization.
Transcriptomic Sequence Analysis of the Effect of Three Fatty Acids with Different Chain Length on Gene Transcription in Saccharomyces cerevisiae
HAN Li, LI Lei, YANG Hourong, HE Peixin, HUANG Shen
2019, 40(6):  106-112.  doi:10.7506/spkx1002-6630-20180316-221
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In this study, we focused on the influence of fatty acids with different chain lengths (hexanoic, lauric and hexadecanoic acid) on gene expression at the transcriptomic level in Saccharomyces cerevisiae at the logarithmic phase. The results demonstrated that the number of transcribed genes and transcription levels changed during logarithmic growth in the presence of each of the three fatty acids; hexanoic acid had the greatest impact on gene transcription levels while the impact of hexadecanoic was the lowest. Furthermore, by analysis of the transcriptomic sequencing data, we predicted that there might be 40 transporter protein genes and 17 transcript factors. Finally, we analyzed the transcription levels of the key enzyme genes involved in the fatty acid biosynthesis pathway that were affected by the fatty acids with different chain lengths. This research provides theoretical support for a better understanding of gene expression and the underlying regulatory mechanism in fatty acid biosynthesis by S. cerevisiae.
Dextranase from Arthrobacter oxydans KQ11: Identification of Key Residues in Catalysis
LIU Le, DING Yi, WANG Zixuan, FANG Yaowei, WANG Shujun, Lü Mingsheng
2019, 40(6):  113-120.  doi:10.7506/spkx1002-6630-20180305-031
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The catalytic domain and key amino acid residues of dextranase from Arthrobacter oxydans KQ11 (AoDex) were predicted by homologous sequence alignment. The mutants Gln418→Gly, Asp420→Gly, Glu423→Gly, Asp439→Gly, and Asp440→Gly were obtained from the catalytic domain 418-QTDGIELYKGSTMKNTFFNANDD-440 by site-directed mutagenesis. The mutant dextranases Q418GDex, D420GDex, E423GDex, and D439GDex had almost no enzymatic activity, while D440GDex had equal enzymatic activity to AoDex. However, at temperatures between 25 and 40 ℃, D440GDex activity increased by 2–3 folds compared to AoDex activity. The optimum pH for D440GDex was 6.5 while that for AoDex was 5.5. Q418, D420, E423, D439 were the key amino acid residues in the catalytic domain of AoDex. Mutation of D440 had a great impact on the enzymatic properties, suggesting it to be not the general base of the AoDex catalytic domain. These findings suggest that AoDex and the GH family 49 share a similar catalytic mechanism, which will provide theoretical support for improving the enzymatic properties of AoDex.
Prokaryotic Expression of Peanut Allergen Ara h 1 and Its Allergenicity
SHI Yunfeng, ZHANG Tong, CHEN Qin
2019, 40(6):  121-127.  doi:10.7506/spkx1002-6630-20180213-154
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An Ara h 1 gene was synthesized from total RNA extracted from peanut using oligo primers by reverse transcription polymerase chain reaction (RT-PCR). The gene was cloned into pET-28a vector and a recombinant expression vector was constructed. Then the recombinant vector was transformed into the bacterial strain Rosetta (DE3) for protein expression, and the target protein was purified by Ni affinity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular mass of the expressed protein was about 75 kDa, which matched with the theoretical value. Analysis by mass spectrometry (MS) confirmed that the protein was Ara h 1. The allergenicity of recombinant Ara h 1 protein was evaluated using an animal model of BALB/c mice. The results showed that recombinant Ara h 1 protein had a similar allergenicity to the native one. The contents of specific antibody, Th2 type cytokines and histamine in the serum were increased, and inflammation occurred in the lung and jejunum of mice sensitized to recombinant Ara h 1. Meanwhile, recombinant Ara h 1 could trigger degranulation of RBL cell, resulting in release of β-hexosaminidase. Accordingly, recombinant Ara h 1 could cause Th2 type allergic reaction.
Quorum Sensing Signal Molecule Production and Biofilm Formation in Serratia marcescens When Co-cultured with Hafnia alvei
LI Tingting, GAO Nana, YU Haifeng, GUO Jingwen, WANG Dangfeng, LI Jianrong, HUANG Jianlian
2019, 40(6):  128-135.  doi:10.7506/spkx1002-6630-20180117-232
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The two bacterial strains with quorum sensing isolated from spoiled sea bass were identified based on 16S rRNA gene sequence analysis as Hafnia alvei-14 and Serratia marcescens-01 with the ability to produce prodigiosin, respectively. The production of the quorum-sensing signal molecules N-acyl homoserine lactones (AHLs) and biofilm formation in S. marcescens-01 were investigated when co-cultured with H. alvei-14 under different culture conditions. The results showed that co-culture with H. alvei could promote AHLs secretion and biofilm production in S. marcescens, which might be related to quorum sensing. Changes in environmental conditions were the important factors that could affect the secretion of AHLs and biofilm formation. Higher salt concentration and too low or too high pH values were unfavorable to microbial secretion of AHLs and biofilm formation, while restrictive carbon and nitrogen sources could stimulate the quorum sensing system and promote the release of AHLs and biofilm formation.
Genomic and Phenotypic Characterization of the Biosynthesis Pathway of Exopolysaccharides in Streptococcus thermophilus KLDS3.1012
LI Bailiang, ZHAO Li, WANG Chengfeng, JIN Da, DING Xiuyun, LIU Fei, HUO Guicheng
2019, 40(6):  136-142.  doi:10.7506/spkx1002-6630-20180122-290
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The purpose of this study was to explore the biosynthesis pathway of exopolysaccharides (EPS) in Streptococcus thermophilus KLDS3.1012 at the molecular level and to verify it by the phenotypic traits. Firstly, genomic sequencing was performed using Illumina HiSeq and MiSeq sequencing platforms and a circular genomic map was skeletonized; subsequently, glycometabolism, sugar nucleotides synthesis and EPS gene clusters were analyzed by bioinformatics; finally, carbohydrate utilization was measured by API 50CH and the monosaccharide composition of the EPS produced by S. thermophilus KLDS3.1012 was analyzed using high performance anion exchange chromatography (HPAEC). In silico bioinformatic analysis unveiled that the genome of S. thermophilus KLDS3.1012 was related to sucrose, fructose, mannose, glucose, galactose and lactose uptake system, and biosynthesis of four sugar nucleotides (UDP-gluctose, dTDPrhamnose, UDP-galactose and UDP-N-acetylglucosamine) as well as an EPS gene cluster. The phenotypic traits revealed that S. thermophilus KLDS3.1012 could utilize these six sugars and the EPS produced it was composed of glucose, rhamnose and galactose. These results provide a theoretical basis for better understanding the relationship between the genetic elements and the structure of EPS in S. thermophilus KLDS3.1012 and for facilitating its potential applications as starter culture in the dairy industry.
Effects of Fermented Liquid Egg on Nutrition and Flavor of Custard Tart
XU Yaxin, XIAO Yishan, GUO Shengnan, MA Meihu, CAI Zhaoxia, HUANG Xi, FU Xing
2019, 40(6):  143-150.  doi:10.7506/spkx1002-6630-20180411-140
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Liquid egg was fermented with a mixed starter culture of Lactococcus lactis, Streptococcus thermophilus and Moniliella mellis before being used to make custard tart. The effects of different fermentation conditions (inoculum amount, fermentation time and temperature) on the flavor and texture of custard tart were studied by one-factor-at-atime and orthogonal array design methods. The results showed that the optimum fermentation conditions were as follows: inoculum size 2%, temperature 42 ℃, and time 42 h. The prepared custard tarts were intact in shape without any crack and the custard was solidified with bright yellow color, moderate sweetness and the best organoleptic quality. The textural properties were determined as follows: firmness 87.8 g, adhesion force 18.3 g, cohesiveness 74.3 g·mm, and gel strength 730.6 g·mm. Nutrient composition analysis showed that the crude protein in fresh liquid egg displayed a decreasing trend after fermentation while the contents of total amino acids, essential amino acids and umami amino acids were increased from 7.55%, 3.26%, and 3.08% to 9.13%, 4.01%, and 5.15%, respectively. In addition, the pH value changed from neutral (7.6) to acidic (5.1) condition. Analysis by GC-MS showed that a total of 24 and 46 volatile components were identified in the fermented egg liquid and the prepared egg tarts, which significantly increased compared to the unfermented egg (15) and the corresponding egg tarts (31), respectively. The characteristic flavor components of the fermented egg tarts were mainly hexanal, furfural, acetoin, butyl-butyryllactate and 2,5-dimethylpyrazine, exhibiting a unique aromatic flavor. In conclusion, the egg tarts prepared with fermented liquid egg were richer in flavor and nutrients. This study provides a theoretical basis for the application of fermented liquid egg in baked foods.
Gene Deletion in Saccharomyces cerevisiae Using CRISPR-Cas9 System
NIU Xiaodi, LI Tianming, LIU Jinlei, YANG Tianyong, LI Ziyi, FENG Huiyong
2019, 40(6):  151-158.  doi:10.7506/spkx1002-6630-20180125-317
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In this study, CRISPR-Cas9-mediated gene knockout in diploid cells of Saccharomyces cerevisiae was investigated. The effectiveness of the CRISPR-Cas9 system was tested using the can1 knockout phenotype. The experimental results showed that the inactivation efficiency of can1 gene reached 4%. The pdc, adh3, adh2, adh1 and pdh genes were knocked out by this system, respectively, and the efficiency of gene editing was 4/48, 3/48, 1/48, 3/28 and 1/16, respectively. This study also developed a procedure for continuous gene knockout, and it took 17 days to knock out pdc, adh3, and adh2 using this procedure. It took 6 days to simultaneously disrupt adh5 and lip with 9/32 and 10/32 efficiencies, respectively.
Screening for Broad-Spectrum Antagonistic Lactic Acid Bacteria from Intestine of Turbot and Identification of Bacteriocin Produced by It
MA Guohan, MA Huanhuan, Lü Xinran, LIU Jiayi, SUN Yue, BAI Fengling, LI Jianrong
2019, 40(6):  159-165.  doi:10.7506/spkx1002-6630-20180325-332
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Strain LP1-4, with strong antagonistic activity against both Gram-negative bacteria and Gram-positive bacteria, was screened out of lactic acid bacteria (LAB) isolated from the turbot intestine using the Oxford cup method. LP1-4 was identified as Lactobacillus plantarum according to physiological and biochemical characteristics and 16S rRNA sequence analysis. The antimicrobial substances in the cell-free supernatant (CFS) of LP1-4 were heat stable and effective in the pH range of 2.5–5.0, but were sensitive to protease. These substances were identified as bacteriocins. The antibacterial activity of LP1-4 reached the peak after 24 h of culture, and the diameter of inhibition zone of the ethyl acetate extract was 25.42 mm. A bacteriocin with molecular mass less than 1.0 ku was purified from the extract by ultrafiltration using Labscale TFF system and Sephadex G-25 column chromatography. The retention time of the target peak in high performance liquid chromatography (HPLC) was 5.0 min. The molecular mass of the bacteriocin was estimated to be 0.854 ku by matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). The amino acid sequence of this bacteriocin was Glu-Asn-Lys-Pro-Ala-Ala-Pro-Lys. In conclusion, LP1-4 has potential applications in controlling spoilage and pathogenic bacteria in aquatic products as a biological preservative.
Changes of Microbial Community during Pile Fermentation of Qingzhuan Tea
ZHANG Dandan, WANG Jiajia, ZHU Wen, YU Yun, MA Yuming, GAO Yupei, HUANG Youyi
2019, 40(6):  166-172.  doi:10.7506/spkx1002-6630-20180428-367
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This study explored changes in the microbial community in samples from the upper, middle and lower layers and mixed samples as well as pile temperature, water content and pH during the pile fermentation of Qingzhuan tea. The results showed that microbial quantities in different pile layers were quite different. The number of fungi in the upper layer before the first pile turning was the highest, where microbial activity mainly occurred, while the numbers of microbes in the middle and lower layers were extremely low, where hygrothermal reaction mainly occurred. The number of molds and yeasts in the upper layer reached its maximum in the early stage of pile fermentation while the number of bacteria and actinomycetes reached its maximum in the late stage. Overall, during the whole fermentation process, bacteria were the most dominant species, followed in turn by actinomycetes, mold and yeast. These results are beneficial to regulating the pile fermentation process of Qingzhuan tea.
Improved ε-Poly-lysine Production by Intergeneric Hybridization between Streptomyces griseofuscus and Brebvibacterium flavum
LI Shu, DU Zongjun, CHEN Guanjun
2019, 40(6):  173-179.  doi:10.7506/spkx1002-6630-20180117-231
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In order to improve the ε-poly-lysine (ε-PL) production of Streptomyces griseofuscus, intergeneric hybridization between S. griseofuscus and Brebvibacterium flavum for enhanced level of the endogenous precursor lysine in the cells was investigated for the first time in this study. One hybrid designated LS-32 was selected by using melanin as genetic marker, which produced 2.82 g/L of ε-PL in a shake flask, 1.73 folds as high as that of S. griseofuscus. In fed-batch fermentation, the ε-PL production of LS-32 was 56.4 g/L, which was increased by 38.9% as compared to that of S. griseofuscus. Randomly amplified polymorphic DNA (RAPD) analysis confirmed the intergeneric hybridization between S. griseofuscus and B. flavum. The activities of several key enzymes in LS-32 were greater than in S. griseofuscus. Furthermore, the concentrations of intracellular amino acids such as Asp, Glu and Arg in LS-32 were much higher than in S. griseofuscus. This research proves the importance of enhancing the endogenous lysine for improving ε-PL production and that intergeneric hybridization is a new breeding strategy for ε-PL production.
Biosynthesis of Trehalose from Cellobiose by Recombinant Escherichia coli
LI Xin, ZHENG Zhaojuan, YUE Taiwen, OUYANG Jia
2019, 40(6):  180-186.  doi:10.7506/spkx1002-6630-20180411-132
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This study aimed to explore the feasibility of synthesizing trehalose using cellobiose as substrate. Firstly, the otsA/otsB gene from Pseudomonas stutzeri A1501 was cloned and exogenously introduced into Escherichia coli BL21(DE3) to construct an OtsAB pathway to synthesize trehalose using glucose as the substrate. Subsequently, the content of uridine diphosphate (UDP)-glucose, the key precursor for biosynthesis of trehalose was increased by overexpressing the galU gene of E. coli itself, resulting in a 4-fold increase in trehalose production. By adding validamycin, the degradation of trehalose was suppressed to increase its yield. Furthermore, by overexpressing the cellobiose phosphorylase gene (cepA) from the naturally occurring cellulolytic bacterium Saccharophagus degradans, the recombinant E. coli was rendered capable of producing trehalose from cellobiose. After 48 h culture with the addition of 0.05 mmol/L validamycin against trehalose degradation, whole recombinant cells catalyzed the transformation of 20 g/L cellobiose to 1.3 g/L trehalose. This study confirms the feasibility of synthesizing trehalose using cellobiose as substrate and provides new ideas for producing fine chemicals from cellobiose.
Probiotic Properties of Lactic Acid Bacteria from Pickled Chinese Cabbage and Its Application in Fermented Milk
ZHAO Shengming, ZHAO Yanyan, MA Hanjun, PAN Runshu
2019, 40(6):  187-194.  doi:10.7506/spkx1002-6630-20180305-050
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A total of 49 strains of lactic acid bacteria isolated from pickled Chinese cabbage were screened for their probiotic properties by acid and bile salt tolerance tests. Four strains of Lactobacillus plantarum, A44, B51, B54 and C53, and two strains of Lactobacillus pentosus, A16 and B72, were selected as identified by 16S rRNA sequence analysis. The probiotic properties of the six strains were evaluated with respect to hydrophobicity, adhesiveness, auto-aggregation and hemolytic activity. The hydrophobicity of L. plantarum A44 against chloroform and xylene was more than 80%, the adhesion rate to Caco-2 cells was 13.57%, and the auto-aggregation rate was more than 60% after standing for 5 h. Our findings indicated that L. plantarum A44 had better prebiotic characteristics without hemolytic activity than other strains. Therefore, the functional characteristics of L. plantarum A44 in fermented milk were further studied. The results indicated that there were no statistically significant differences (P > 0.05) in pH, titrable acidity or water-holding capacity of fermented milk produced with a commercial starter culture with the addition of L. plantarum A44 as auxiliary starter. However, total viable count of fermented milk was significantly increased to 8.45 (lg (CFU/mL)) (P < 0.05) and viscosity was also significantly increased. Thus, L. plantarum A44 has great potential for application as an auxiliary probiotic starter due to its excellent probiotic properties.
Isolation and Identification of Lactobacillus plantarum Phage Lpla and Screening for Phage-Resistant Mutants
QI Ruiming, YU Meiling, JIANG Yanping, CUI Wen, ZHANG Xi, WANG Li, XU Yigang, TANG Lijie, LI Yijing, QIAO Xinyuan
2019, 40(6):  195-202.  doi:10.7506/spkx1002-6630-20180118-241
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In this study, one virulent phage of Lactobacillus plantarum WCFS1, named Lpla, was isolated from kimchi. Its characteristics were identified and its spontaneous mutants were screened for resistance to Lpla. The one-step growth curve showed the latency and burst period of Lpla were 15 min and 180 min, respectively and the burst size was 43 PFU/cell. According to its morphology as observed by electron microscopy, Lpla belonged to the Siphoviridae family (morphotype B1). Phage Lpla was highly tolerant to acid and alkali but not to ethanol, heat or UV. The highest adsorption rate of Lpla on the host cell surface was observed at 4 ℃ and the adsorption was improved by the addition of Ca2+ and/or Mg2+. Phage Lpla could adsorb to thermally killed cells, but the adsorption rate was decreased by 20%. Six non-lysogenic Lpla-resistant mutants with genetic stability were obtained. This study provides a theoretical basis for preventing and control phage infection and for developing phage-resistant strains.
Cloning and Expression Profile Analysis of the Phloridzin 2’-O-Glycosyltransferase Gene in Apple
RAN Junjian, LIANG Xinhong, CHEN Xiaojing, LI Xiangyang, JIAO Lingxia, ZHAO Ruixiang
2019, 40(6):  203-208.  doi:10.7506/spkx1002-6630-20180125-328
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Objective: Phloridzin 2-O-glucosyltransferase (P2’-GT) is the key enzyme involved in the final step in the phloridzin biosynthetic pathway, catalyzing transformation of phloretin into phlorizin. The purpose of this study was to clone a P2’-GT gene from Fuji apple and to investigate bioinformatics analysis of the protein encoded by the gene and its expression pattern. Methods: RNA was extracted from apple peel and was used to synthesize a cDNA template by reverse transcription. Specific primers were designed based on the P2’-GT reporter gene for PCR amplification and sequencing. Bioinformatic analyses were performed using online software (Pro Param tool and TMHMM). The differential transcript levels of P2’-GT in different organs of apple varieties at different growth stages were detected using real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Results: The cDNA was 1 452 bp long, encoding 483 amino acid residues. The encoded protein was unstable with a molecular mass of 53.6 kDa and a theoretical PI of 5.76. In addition, the protein had no obvious transmembrane structure. Its secondary structure consisted of α-helix, random coil and extended strand. The protein tertiary structure exhibited high similarity to hydroquinone glucosyltransferase (41.32%). Phylogenetic analysis indicated that P2’-GT had the highest homology to Pyrus bretschneideri glucosyltransferase. The expression level of this gene was high in apple peel but extremely low in leaves and roots and zero in apple pulp. P2’-GT gene expression was related to the growth period; transcript level was rarely detectable in the early stage, but increased rapidly to a maximum in the middle stage and then decreased by 50% in the maturation stage. Significant differences in the transcript level of this gene were found among three different varieties, with the highest and lowest level being observed in Granny Smith and Gala, respectively. This research provides theoretical support for the role of P2’-GT genes in regulating the synthesis of phloridzin.
Effect of Fermentation Temperature on the Sensory Quality of Citrus Wine
BI Jingying, LI Hua, WANG Hua
2019, 40(6):  209-216.  doi:10.7506/spkx1002-6630-20180307-089
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Purpose: To analyze the physicochemical properties, aroma composition and sensory attributes including delayed bitterness (DB) of citrus wines produced at six different fermentation temperatures (10, 14, 18, 22, 26 and 30 ℃) for the purpose of determining the optimum fermentation temperature. Methods: The fermentation was carried out in a smallscale fermentor and the physicochemical and sensory quality of citrus wine were measured. Results: As the temperature of fermentation increased, the color of citrus wine deepened gradually, and the wines fermented at 14 and 18 ℃ exhibited better color properties. The wine fermented at 18 ℃ had the highest aroma compound concentration and contained a wide variety of aroma compounds. Of the compounds responsible for DB taste, only limonin was detected and the lowest content of limonin was found at 18 ℃ followed by 14 ℃. The total acidity in the wine fermented at 14 ℃ was the lowest followed by 10 ℃. Conclusion: The wine fermented at 18 ℃ had better color properties and aroma quality and contained lower levels of DB components. Overall, it had the best sensory quality.
Identification of 2-Phenethyl Alcohol-Producing Yeast and Its Application in Soy Sauce Fermentation
ZOU Mouyong, ZHU Xingui, LIU Dan, LI Qiaolian
2019, 40(6):  217-222.  doi:10.7506/spkx1002-6630-20180420-264
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This investigation aimed to study the synthesis pathway of 2-phenethyl alcohol, one of the characteristic aroma components of soy sauce for the purpose of enhancing the production of 2-phenethyl alcohol during soy sauce fermentation. We isolated a 2-phenethyl alcohol-producing yeast strain, designated 922-1, from fermented mash for soy sauce. This strain was identified as Candida oceani by morphologic observation, physiological and biochemical test, ITS rDNA sequencing and phylogenetic tree analysis. C. oceani 922-1 was able to grow in a high salt medium (18% NaCl). Sole nitrogen source experiments showed that C. oceani 922-1 synthesized 2-phenethyl alcohol using phenylalanine as substrate by the Ehrlich pathway. C. oceani 922-1 was capable of producing a high content of 2-phenethyl alcohol (44.70 mg/L) in the fermented mash, which was increased by 17.3 times with 1% phenylalanine addition compared with the control group. Meanwhile, both the mellow aroma and strong flavor were improved. This study highlights the promising potential of C. oceani 922-1 for flavor improvement of high-salt fermented seasonings.
Composition Analysis
A Comparative Analysis of Fatty Acid Profiles of Camel, Bovine and Caprine Milk
LI Lei, WANG Kun, HE Jing, JI Rimutu
2019, 40(6):  223-227.  doi:10.7506/spkx1002-6630-20180409-105
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The aim of this study was to characterize the fat acid profiles of camel, bovine and caprine milk by gas chromatography. The results showed that there were significant differences in the types and contents of fatty acids among the three milks. A total of 33 fatty acids were detected in camel milk, with palmitic acid being the most abundant ((23.73 ± 2.17)%); the polyunsaturated fatty acid (PUFA) content of camel milk was (4.09 ± 0.54)%, which was significantly higher than that of bovine and caprine milk. A total of 29 fatty acids were detected in bovine milk, with palmitic acid being the most dominant ((27.44 ± 2.45)%), and 34 fatty acids were detected in caprine milk, dominated by oleic acid ((30.89 ± 3.57)%). Among these milks, the highest content of monounsaturated fatty acids (MUFA) was found in caprine milk ((35.26 ± 3.86)%). In addition, the fatty acid profile of camel milk was further analyzed by principal component analysis and correlation analysis. The results showed that the ratio of saturated fatty acids to MUFA to PUFA was 1:0.60:0.08, which was closer to the FAO/ WHO recommended value 1:1:1. There was a negative correlation between PUFA and short-chain fatty acids and long-chain fatty acids, with a maximum correlation with n-6 FA (0.978); there was a positive correlation between SCFA and LCFA, and between n-6 FA and LCFA.
Evaluation of Beer Crispness Using Gas Chromatography-Mass Spectrometry and Partial Least Squares-Discriminant Analysis
CHEN Hualei, YANG Zhaoxia, WANG Chenghong, YAN Peng, ZHANG Yuxin, LI Mei
2019, 40(6):  228-232.  doi:10.7506/spkx1002-6630-20180125-343
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Objective: This study aimed to establish a new method for the evaluation of beer crispness using partial least squares-discriminant analysis (PLS-DA). Methods: The concentrations of the chemical constituents associated with beer crispness were determined by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), and the obtained data were transformed to a matrix and analyzed by PLS-DA method using SIMCA-P software. Results: Beers with different crispness showed clear discrimination in the score plot of PLS-DA. According to the loading plot, ethyl caprylate, ethyl caprate and isoamyl acetate were the sensitive characteristic variables for the DA (1) set of samples with poor crispness. Fifty samples were selected as a validation set to establish a PLS-DA discrimination model. The correlation coefficients of the model for four beer samples with different crispness were 0.861, 0.798, 0.765 and 0.812, respectively, with a recognition rate of 92%. A total of 27 unknown samples were predicted by the PLS-DA model with root mean square errors of prediction (RMSEP) of 0.183, 0.321, 0.523 and 0.323 and a correct recognition rate of 74.07%. Conclusion: HS-SPME-GC-MS combined with PLS-DA is a simple and useful method for the evaluation of beer crispness.
Changes in Taste Compounds during Processing of Vinasse-Cured Duck as Studied by 1H NMR Combined with Multivariate Data Analysis
ZHOU Nannan, LOU Xiaowei, WANG Ying, PAN Daodong, SUN Yangying, DANG Yali, CAO Jinxuan
2019, 40(6):  233-239.  doi:10.7506/spkx1002-6630-20180416-208
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The metabonomes of fresh duck, vinasse-cured duck and vinasse were found to be dominated by 34 metabolites, including amino acids, sugars, organic acids, alkaloids, alcohols, nucleic acid and their derivatives. These compounds were not only the important metabolites during processing, but also were the main low molecular mass taste compounds in vinasse-cured duck. Our results showed that amino acids, alcohols, alkaloids and sugars decreased during boiling and salting, and then increased during vinasse-curing. Formate and hypoxanthine increased throughout all processing steps while lactate, creatine, 5’-IMP and inosine decreased. The most affluent metabolites in vinasse-cured duck were lactate (4.74 and 3.30 mg/g in dry and wet vinasse-cured duck), aspartate (2.06 and 3.15 mg/g in dry and wet vinasse-cured duck), creatin (2.78 and 1.30 mg/g in dry and wet vinasse-cured duck) and glucose (2.12 and 3.51 mg/g in dry and wet vinasse-cured duck). 1H nuclear magnetic resonance (1H NMR) spectroscopy is suitable to analyze the non-volatile compounds during the processing of vinasse-cured duck. The effects of wet vinasse-curing on the contents of metabolites were more significant than those of dry vinasse-curing.
Comparison of Main Chemical Constituents in Ganoderma lucidum Fruiting Bodies Collected from Three Producing Regions
YAN Zheng, HAO Limin, ZHANG Liming, KANG Caicai, MA Tianjiao, CUI Yan, ZHENG Zhiqiang
2019, 40(6):  240-246.  doi:10.7506/spkx1002-6630-20180620-396
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In order to explore the difference in the main chemical constituents of Ganoderma lucidum fruiting bodies from different geographical regions (Anhui 1, Jilin 1 and Jilin 2), various analytical methods were used to determine the proximate chemical composition, microelement composition, polysaccharide composition, ganoderic acid content, amino acid composition and fatty acid composition. The results showed that the contents of protein, amino acids, fatty acids, microelements and triterpene compounds in G. lucidum from different regions were quite different. The contents of three microelements (Ca, Mg and Fe), essential amino acids and unsaturated fatty acids were high in all three samples. Both the total contents of amino acids and fatty acids were in the decreasing order: Anhui 1 > Jilin 2 > Jilin 1. The contents of crude water-soluble polysaccharides in these samples were similar, whereas the content of alkali soluble polysaccharides and the monosaccharide composition of purified polysaccharides were considerably different. Among the three samples, the total content of ganoderic acid A and ganoderic acid B in Anhui 1 was the highest (5.62 mg/g). These conclusions provide technical support for further improving the existing quality standard for G. lucidum fruiting bodies.
Effects of Degumming and Deacidification on Volatile Composition of Rapeseed Oil
YAO Yingzheng, DONG Ling, LIANG Qiang, LI Jian, LI Pu, ZHU Yu, ZHOU Qi
2019, 40(6):  247-252.  doi:10.7506/spkx1002-6630-20180115-186
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In this experiment, gas chromatography-mass spectrometry (GC-MS) was used to study the effects of traditional degumming and deacidification on the volatile compositions of cold- and hot-pressed rapeseed oils. It was found that both volatile compositions and flavors of cold- and hot-pressed oils varied greatly and the volatile composition of hot-pressed oil was more complicated than that of cold-pressed oil. Degumming and deacidification could reduce the complexity of volatile composition in rapeseed oil. Principal component analysis (PCA) indicated that deacidification had a great influence on the volatile compositions of both oils, while degumming had a lesser influence. The degumming process had no significant effect on the acid value of rapeseed oil. Degumming and deacidification had significant effects on the peroxide value of coldpressed oil but not on hot-pressed oil.
Development of a Validated UPLC Method for Simultaneous Analyses of 20 Ginsenosides in Various Processed Ginseng Products
WANG Yanhong, WU Xiaomin, ZHU Yanping, ZHAO Dan, LI Yueru
2019, 40(6):  253-260.  doi:10.7506/spkx1002-6630-20180103-040
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A cost effective, simple, precise and accurate ultra-high performance liquid chromatography (UPLC) method was established and validated for simultaneous determination of 20 ginsenosides in various processed ginseng products, including Rg1, Re, Rf, 20(S)-Rg2, 20(R)-Rg2, Rb1, Rc, Ra1, Rb2, Rb3, Rd, Rk3, F2, 20(S)-Rg3, 20(R)-Rg3, compound K (CK), Rg5, 20(S)-Rh2, 20(R)-Rh2 and protopanaxadiol (PPD). The developed method was carried out on a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using a photodiode array detector (PDA). Good chromatographic separation was performed using a mixture of acetonitrile (B) and water (A) as the mobile phase with gradient elution at a flow rate of 0.3 mL/min and column temperature of 30 ℃ within 31 min. The method was well validated with respect to linearity (R2 > 0.998), precision (intra-day RSD ≤ 4.65% and inter-day RSD ≤ 4.88%) and accuracy (recovery rate between 85.71% and 108.50%), and the limit of detection (LOD) and limit of quantification (LOQ) were in the range of 0.81–3.10 μg/mL and 2.88–10.00 μg/mL, respectively. This method was rapid and reliable and was successfully used for the analysis of various processed ginseng products, including freshly stored ginseng, red ginseng and white ginseng and the results revealed significant variations in the ginsenoside levels. This method can be suitable for the analysis of active compounds and the quality control of fresh and processed ginseng.
Comprehensive Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry Analysis of Flavor Components in Pixian Bean Paste
LI Weili, YUAN Xu, LIU Yushu, WU Xiaoyu, TANG Yong, LIN Hongbin, LIU Ping, DING Wenwu, CHE Zhenming, WU Tao
2019, 40(6):  261-265.  doi:10.7506/spkx1002-6630-20180330-419
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Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF MS) was used to identify and quantify the volatile flavor composition of special grade Pixian bean paste. A total of 987 compounds were detected 218 compounds of which including organic acids, amino acid their derivatives, sugars and their derivatives, alcohols, esters, phenols, ketones and aldehydes. The most abundant compounds were organic acids (72, 30.36%), followed by amino acids and their derivatives (40, 26.58%). This study provides a scientific basis for volatile flavor composition analysis, processing optimization and quality criteria improvement of Pixian bean paste.
Analysis and Evaluation of Volatile Compounds in Different Strains of Industrially Cultivated Pleurotus eryngii
LI Qiaozhen, LI Xiaobei, WU Di, LI Zhengpeng, LI Yu, ZHOU Feng, YANG Yan
2019, 40(6):  266-271.  doi:10.7506/spkx1002-6630-20180125-348
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The volatile flavor compounds in the fruiting bodies of different strains of industrially cultivated Pleurotus eryngii were identified by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). An electronic nose system was used to discriminate among these strains by their flavor compounds. Principal component analysis (PCA) was used to identify the volatile components contributing primarily to the flavor. Based on general evaluation index (GEI) the aroma of the fruiting bodies of different P. eryngii strains was evaluated. The results showed that a total of 102 volatile components were detected in the fruiting bodies of seven strains of P. eryngii, including alcohols, alkenes and ketone ethers, and 36 volatile components were common to all the strains. The electronic nose system could differentiate among the seven strains. PCA showed that 2-butyl-2-octenal, 3-octanone, pentanol, acetone, hexanal, methyl isopentanoate, mercaptoethanol, 2,4-diclofenac, 3-methylbutyraldehyde and isoamyl alcohol were mainly responsible for the aroma of P. eryngii. The results of GEI showed that there were significant differences in the aroma of the industrially cultivated strains, among which, G.S. had the best aroma. This study provides a theoretical guideline for the development of flavor components and the breeding of P. eryngii.
Process and Technology
Optimization of Preparation of W/O/W Multiple Emulsion and Evaluation of Its Ability to Encapsulate Cyanidin-3-Glucoside
YUAN Li, SUN Chuchu, DANG Qingling, GAO Ruichang
2019, 40(6):  272-280.  doi:10.7506/spkx1002-6630-20180211-123
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In this experiment, the effects of stirring rate and emulsifier type on the stability of W/O/W double emulsion were studied in order to optimize the process parameters. The ability of W/O/W double emulsion to encapsulate cyanidin-3- glucoside (C-3-G) was evaluated as well. The optimum conditions were found to be as follows: stirring rate 15 500 r/min for the first emulsification, 7 000 r/min for the second emulsification, polyglycerol ricinoleate concentration (PGPR, oil phase emulsifier) 16%, hydrophile lipophile balance (HLB) of hydrophilic emulsifier 10, and hydrophilic emulsifier concentration 10%. Under these conditions, the retention rate after centrifugation reached 80.25%. C-3-G was well encapsulated by the optimized multiple emulsion. It was found that the drug-loading rate of the emulsion was 1 500 mg/L and the encapsulation efficiency was 98.08%. The encapsulation of C-3-G improved the emulsion stability by increasing the viscosity of the inner water phase.
Optimization of Preparation Process for Pinecone Polyphenol Microparticles by Response Surface Methodology
DIAO Yan, CHEN Bin, WANG Rui, LI Qiao, ZHAO Haitian, ZHANG Hua, WANG Lu, WEI Lijun, WANG Zhenyu
2019, 40(6):  281-288.  doi:10.7506/spkx1002-6630-20180307-081
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In order to minimize the degradation of pinecone polyphenols in the stomach, pinecone polyphenols from Pinus koraiensis (PPH) were encapsulated by acidic polysaccharide fragments from Auricularia auricula (AAP) and polylysine (PLL) according to the principle of polyelectrolyte self-assembly, and response surface methodology was used to optimize the preparation process. The effect of AAP, PLL and PPH concentration as well as interaction between them on the encapsulation efficiency was evaluated. As a result, a quadratic polynomial model was established describing the process. Scanning electron microscopy (SEM) was used to investigate the morphology of the microparticles prepared under optimized conditions, and the release rate was studied in simulated gastrointestinal environment. The results showed that the fitness of the model was good. All of the three variables had significant effects on the response. The encapsulation efficiency of polyphenol microparticles were (86.57 ± 1.07)% and the drug loading was (24.03 ± 0.81)% at 900 μg/mL AAP, 30 μg/mL PLL and 110 μg/mL PPH concentration. The diameter of the microparticles was 200–500 nm. They were released slowly in simulated gastric environment but rapidly in simulated intestinal environment. The measured encapsulation efficiency matched with the predicted value, indicating that response surface methodology was a suitable method for optimizing the preparation of pinecone polyphenol microparticles. The degradation of pinecone polyphenol microparticles in the stomach environment was decreased relative to free pine polyphenols.
Polysaccharides from Laminaria japonica: Optimization of Different Extraction Processes and Comparison of Physicochemical Properties and Antitumor Activity
LI Ying, HUANG Dechun, CHEN Guitang, AI Zonghua, JIANG Siqi, KANG Yixin, GAO Shijing
2019, 40(6):  289-295.  doi:10.7506/spkx1002-6630-20180312-147
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Three extraction methods, including hot water extraction (HWE), ultrasonic-assisted extraction (UAE) and multiple enzymatic extraction (MEE), were used to extract polysaccharides from Laminaria japonica. Extraction conditions were optimized using one-factor-at-a-time and orthogonal array design methods, and the physicochemical properties and antitumor activity of the polysaccharides were comparatively analyzed. The results showed that the optimized HWE conditions were as follows: temperature 85 ℃, extraction time 5 h, and solid to solvent ratio 1:80 (g/mL); the optimized UAE conditions were ultrasonic power 500 W, extraction time, 10 min, and solid to solvent ratio 1:90 (g/mL); the optimized MEE conditions were papain 75 U/g, pectinase 15 000 U/g, cellulase 108 000 U/g, pH 5, temperature 50 ℃, solid to solvent ratio 1:70 (g/mL), and extraction time 3 h. Under the above conditions, the average yield of polysaccharides was 5.97%, 5.81% and 14.29%, respectively. There were significant effects of different extraction methods on the polysaccharide composition. The solubility of the polysaccharides obtained by MEE was significantly better than those obtained with HWE and UAE. All the polysaccharides contained a small amount of protein or polypeptide, and had three-strand helical structures. MTT assay showed that the three polysaccharides exhibited different antitumor activities, among which the polysaccharides obtained by UAE showed the strongest inhibitory effect on human lung cancer cell A549 while the polysaccharides extracted by CEE displayed the strongest inhibitory effect on human breast cancer cell line MCF-7.
Optimization of Natural Antioxidant Combinations against Discoloration and Lipid Oxidation in Prepared Chicken Steak by Response Surface Methodology
LU Qing, HUANG Jichao, ZHU Zongshuai, LIU Dongmei, HUANG Ming
2019, 40(6):  296-303.  doi:10.7506/spkx1002-6630-20180324-315
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Objective: The aim of this study was to address the effect of rosemary extract and ascorbic acid separately and in combination on redness (a* value) and lipid oxidation in prepared chicken steak for the purpose of delaying discoloration and lipid oxidation during its storage. Methods: The concentrations of rosemary extract and ascorbic acid in the marinade were optimized using one-factor-at-a-time method and response surface methodology based on central composite design. The responses were a* value and thiobarbituric acid reactive substances (TBARS) value. Results: The addition of rosemary extract and ascorbic acid had significant influences on both a* and TBARS values. The coefficients of determination (R2) of the two quadratic polynomial regression models were found to be 0.932 0 and 0.886 3, respectively, and lack-of-fit test revealed a non-significant value for the models (P > 0.05), indicating that the models appropriately explain the variability of the observed responses. The linear term of rosemary extract (P < 0.05) and the linear and quadratic terms of ascorbic acid (P < 0.01) significantly influenced a* value, and the linear term of rosemary extract (P < 0.01) and the quadratic term of ascorbic acid (P < 0.05) significantly influenced TBARS value. Conclusion: The highest a* value and the lowest TBARS value were simultaneously obtained by the addition of rosemary extract at 0.198 g/kg and ascorbic acid at 0.255 g/kg. After 50 d storage, a* and TBARS values were experimentally measured as 21.88 and 0.59 mg/kg, which were consistent with the fitted results.
Ultrasonic-Assisted Enzymatic Hydrolysis of Blackberry Juice and Synergistic Effect
FAN Linlin, WANG Ying, CHENG Xianling, GAO Peng, ZHOU Jianzhong
2019, 40(6):  304-311.  doi:10.7506/spkx1002-6630-20180413-172
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In an effort to solve the problems of low yield and unstable quality during the processing of blackberry juice, response surface methodology was used to optimize the conditions for ultrasonic-assisted enzymatic processing of blackberry juice (with a mixture of pectinase and commercial enzyme preparation for juice processing). Moreover, the synergistic effect of ultrasonic and the enzymes on improving juice quality was analyzed. The results showed that the optimized process conditions were as follows: ultrasonic power, 200 W; enzyme concentration, 0.3%; hydrolysis time, 1.5 h; and temperature, 45 ℃. A higher juice yield of 80.89% and 68.21% transmittance were obtained under these conditions. The quality and stability analysis showed this juice had higher contents of anthocyanin and total phenols, and displayed a red color with better stability during storage as compared to enzymatic hydrolysis alone. Additionally, the results of scanning electron microscopy indicated that ultrasonic and the enzymes had a synergistic effect on destroying the cell structure of blackberries, thus increasing the efficiency of juice processing.
Safety Detection
Prediction of Total Viable Count in Sausage by Hyperspectral Imaging Technology Combined with Gradient Boosting Decision Tree (GBDT)
GUO Peiyuan, XU Pan, DONG Xiaodong, XU Jingjing
2019, 40(6):  312-317.  doi:10.7506/spkx1002-6630-20171128-341
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This experiment used a hyperspectral image system in the wavelength range of 400–1 000 nm to predict the total viable count in sausage. Spectral data of 450 sausage samples were selected as the training set, and another 50 samples as the test set. The spectra was preprocessed by multiplicative scatter correction (MSC) method and principal component analysis (PCA) was used to reduce the dimensionality of the spectral data. Support vector regression (SVR) and gradient boosting decision tree (GBDT) were separately used to establish quantitative analysis models for the training and test sets, and the optimal model was selected. The results showed that the GBDT models were better than the SVR models. The root mean square error (RMSE) of the GBDT models were 0.001 and 0.003 for the training and test sets, respectively, and the coefficients of determination (R2) were 0.998 and 0.996, respectively. This study proved that the GBDT method based on hyperspectral imaging technology was feasible and effective to predict the total viable count in sausage.
Canonical Correspondence Analysis of Growing Environment and Mineral Distribution Characteristics of Liuhe Rice
WANG Zhaohui, ZHENG Hui, ZHAO Qian, WANG Yanhui, CUI Hao, WANG Jinghui, ZHANG Dali
2019, 40(6):  318-324.  doi:10.7506/spkx1002-6630-20171218-227
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In order to screen for stable and reliable mineral elements to identify the geographical origin of Liuhe rice, two cycles of canonical correspondence analysis were performed to evaluate the environmental factors that affect the distribution of sampling locations. We analyzed the effectiveness at the regional scale of the selected elements and their stability in identifying the geographical origin of Liuhe rice. Results showed that the two environmental gradient axes explained 17.3% and 23.4% of the total variance, respectively. There was a strong spatial correlation between the mineral elements and the environmental variables. Se content was most influenced by the geographical environmental variables, while the least effect was observed on Ca, Zn and Mg contents. Mg, Ca, Na, Mn and Zn had good stability and spatial representativeness and could be used to identify the geographical origin of Liuhe rice. The results of this study can provide a useful theoretical foundation for modeling in geographical origin identification of Liuhe rice.
Comparison and Application of RNA Extraction Methods for Detection of Hepatitis A Virus in Food Samples
FENG Huawei, YAN Pingping, AI Haixin, LI Sijia, MA Lidan, LIU Hongsheng
2019, 40(6):  325-330.  doi:10.7506/spkx1002-6630-20180111-131
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Objective: Three RNA extraction kits were compared to select the best one for RNA extraction from hepatitis A virus (HAV) in foods, aiming to provide a technical basis for foodborne HAV RNA detection in laboratories. Methods: After artificial contamination of known negative samples, three commercial nucleic acid extraction kits, ABI AM1836, QIAGEN 74104 and ROCHE 11858882001, were used to extract HAV RNA and their performance was evaluated in terms of extraction efficiency, inhibitor removal efficiency and sensitivity, and the optimized extraction kit was applied to real samples. Results: ROCHE 11858882001 was the best extraction kit, followed by QIAGEN 74104 and ABI AM1836. Of 101 samples, one sample of blue honeysuckle was positive while the rest were negative. Conclusion: ROCHE 11858882001 is a rapid and effective method for extracting HAV RNA, and thus it is suitable for the routine detection of foodborne HAV.
Determination and Speciation Analysis of Heavy Metals in Ceramic Packaging of Baijiu Using the European Community Bureau of Reference (BCR) Extraction Method
LI Yangyang, DONG Liming, ZHOU Qi, LI Yingying
2019, 40(6):  331-337.  doi:10.7506/spkx1002-6630-20171128-338
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The dissolution and speciation distribution of Pb, Cd, Zn, Ni, and Co from ceramic packaging of Baijiu were investigated by the BCR extraction method, microwave digestion and acetic acid extraction. Results showed that the total sum of heavy metals in the ceramic packaging detected by the BCR method was 84.90%–109.38% as compared to microwave digestion, and heavy metal concentrations consistently decreased in the order of Zn > Ni > Pb > Co > Cd. Based on the BCR speciation distribution of heavy metals from the ceramic packaging, the dissolution risk of Ni and Zn was the most serious among all heavy metals, Pb and Co had medium risk of dissolution, and there was almost no risk of Cd dissolution. In addition, the total concentration of weakly acid extractable and reducing species with BCR extraction was 79.55%–411.75% as compared to 4% acetic acid extraction for 24 h. The results of correlation analysis showed that there was a more significant correlation between the weakly acid extractable species and heavy metal dissolution in 4% acetic acid. Correlation analysis of the BCR extractable species indicated that ceramic packaging may have increased risk of dissolution of heavy metals when it is used to store Baijiu containing acid and reducing and oxidizing substances for a long time. Therefore, the BCR extraction method is useful to further analyze the speciation characteristics of dissolved heavy metals from Baijiu ceramic packaging, which can provide a scientific basis for exploring the influence of heavy metal dissolution on Baijiu quality and human health risk.
Determination of Selenium Methionine in Rice Flour by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy
CHEN Meilin, DU Fenni, CHEN Ye, CHENG Chao, SHAN Changhai, MO Kaiju
2019, 40(6):  338-343.  doi:10.7506/spkx1002-6630-20181031-377
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Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to determine the selenium methionine content in rice flour in order to develop a fast and environmentally friendly method for the determination of selenium in food. IR spectra of samples with added selenomethionine were collected by an attenuated total reflection mid-infrared spectrometer. The partial least square method was used to develop a mathematical model and the model was validated by TQ Analyst software. The results showed that the infrared spectra in the range of 1 600–650 cm-1 were selected for analysis. After derivative preprocessing of the original infrared spectra, good prediction results were obtained by using either the leave-one-out method or the leave-more-out method. Comparatively, the leaveone- out model with first derivative pretreatment was simpler in calculation and gave more accurate prediction. The coefficient of determination for calibration (RC 2 ) of the left-one-out model was greater than 0.997, the number of factors was 6, the root mean square error of calibration (RMSEC) was 1.76–1.9, the coefficient of determination for prediction (RP 2) was 0.986 8, the root mean square error of prediction (RMSEP) was 3.27, and the relative prediction deviation (RPD) was 8.612 9. The predicted values with a relative deviation of less than 20% and 10% accounted for 92.91% and 82.27% of the total, respectively.