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15 May 2013, Volume 34 Issue 9
Structural Identification of Seleno-peptides and Seleno-amino Acids in Se-enriched Corn Protein Hydrolysates
WANG Zhen-zhen,ZHANG Jiu-liang,WANG Chi,HOU Tao,ZHANG Yan,HE Hui*
2013, 34(9):  1-4.  doi:10.7506/spkx1002-6630-201309001
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In order to clarify the combination form of selenium in corn proteins, the structures of seleno-peptides in Se-enriched
corn protein hydrolysates were identified by HPLC-MS/MS method coupled with mass spectrum database and seleno-amino
acids were identified by HPLC-ICP-MS method. Five seleno-peptides were confirmed as SeMet-MeSeCys-Glu, Met-MeSeCys-
Glu, MeSeCys-Glu-Asp, Ile-MeSeCys-Glu and γ-Glu-MeSeCys, and four seleno-amino acids were identified as selenomethionine
(SeMet), eleno-Ethionine (SeEt), Se-methylseleno-L-Cysteine (MeSeCys) and selenocystine (SeCys2), respectively. Seleno-amino
acids and dipeptide were been quantified using standard samples. Selenomethionine (SeMet) revealed the highest content (586.3 ±
49.5) ng/g, and Selenocystine (SeCys2) revealed the low content (102.6 ± 9.0) ng/g. Based on the above results, all five selenopeptides
contain MeSeCys, and only one contains SeMet. The major seleno-amino acid was SeMet.

Hydrolysis Characteristics of the proteins from Grass Carp Viscera
QING Yu-ting,CONG Yan-jun*
2013, 34(9):  5-9.  doi:10.7506/spkx1002-6630-201309002
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The effects of papain, alcalase, flavorzyme, pepsin, trypsin and neutral on the proteolytic activities of protein from the grass carp viscera were studied. Protein extracts were obtained by centrifugation. The molecular formula of protein was measured by SDS-PAGE.The results showed that the molecular formula of protein were 39.4KDa, 13.2KDa, 2.8KDa, respectively. The ammoniacal nitrogen contents in the hydrolyzates increased rapidly in the first 6 hours, then were stable. The peptide nitrogen contents reached the peak at the sixth hour for other protease except alcalase at the ninth hour. Ammonia nitrogen content of hydrolyzates were the highest for flavorzyme, and the differences were significant with each other except between alcalase and neutral. The peptide nitrogen contents of papain were higher than those by the other enzymes, while those by flavorzyme were the lowest. The taste of the hydrolyzates were constantly changing along with the enzymatic hydrolysis proceeded. As the release of amino acid and peptide, fresh flavor became notable. The umami of flavorzyme were significantly notable.

Influence of Natural Antioxidants on the Oxidation and Functionalities of Soy Protein Isolate during Storage
GUO Feng-xian,HUANG Xiao-lin,XIONG You-lingL.,CHEN Jie*
2013, 34(9):  10-13.  doi:10.7506/spkx1002-6630-201309003
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The effect of natural antioxidants (β-carotene and tocopherol) on the content of carbonyls and total sulfhydryl,
as well as solubility, emulsifying properties, and gel properties of soy protein isolate (SPI) during storage for 12 weeks
was studied. The results showed that in native SPI, carbonyl group content increased sharply and gel strength rose at the
end of the storage period, while total sulfhydryl group content fell and both solubility and emulsifying properties decreased
distinctly. Addition of β-carotene to SPI significantly supressed the increase in carbonyl group content after storage for 12
weeks (P < 0.05); meanwhile, when added together, β-carotene and tocopherol significantly inhibited the decrease in total
sulfhydryl group content (P < 0.05). Each antioxidant could block the decrease in solubility and emulsifying properties of
SPI after storage and partially surpress the increase in gel strenth.

A Two-dimensional Gel Electrophoresis Protocol for Proteomic Study of Royal Jelly
ZHAO Fang-yuan1,2,WU Ya-jun1,HAN Jian-xun1,CHEN Ying1,*,GE Yi-qiang2,3,*
2013, 34(9):  14-18.  doi:10.7506/spkx1002-6630-201309004
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In order to establish a two-dimensional gel electrophoresis (2-DE) protocol for proteomic study of royal jelly
(RJ), IPG strip with appropriate pH range, optimal loading amount for protein, focusing time of IEF, and separating gel
concentration was used. The results showed that the best 2-DE image of RJ proteins using IPG strip with pH 5—8, protein
loading amount of 150 μg, focusing time of 32000 V•h, and separating gel concentration of 10% was achieved. Moreover,
62 ± 4 protein spots were detected on the gel with high repeatability and resolution. The 2-DE system after optimization is
suitable for the analysis of RJ proteins, which provides theoretical basis for the proteomics of RJ.

Quantitative Structure Activity Relationship of Angiotensin Ⅰ-Converting Enzyme Inhibitory Tripeptides Derived from Food Proteins
CHEN Ji-wang1,LIU Shan-shan1,CAI Guang-xia1,WU Yong-ning1,2
2013, 34(9):  19-23.  doi:10.7506/spkx1002-6630-201309005
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Based on published IC50 values, a quantitative structure activity relationship (QSAR) model indicating
the relationship between amino acid sequences and inhibitory activity was developed for angiotensin Ⅰ-converting
enzyme (ACE) inhibitory tripeptides derived from food proteins. The following QSAR model for IC50 versus
hydrophilicity and steric and electronic properties (X3) of amino acid side chains was developed: Y = 1.952 +
0.1229X2 + 0.0924X3 + 0.0425X5 + 0.1777X7 + 0.136X8-0.0809X9-0.1763X10. According to this model, amino
acids with low hydrophilicity and low steric parameters such as Val, Leu and Ile preferably existed at the first site
near the N terminus, those with low hydrophilicity and low charge parameters such as Lys and Arg at the second
site, and those with low hydrophilicity and high steric parameters such as Pro and Phe at the third site. In the
prediction of the tripeptides VNP, VWP and VAP for IC50, an error varying from 0.06 to 0.23 was obtained between
the experimental and predicted values, which was within the range of model samples. Thus, the described mode has
good prediction capability.

Physico-chemical Characteristics of Type Ⅱ Collagen Isolated from Prionace glauca Cartilage
SONG Rui-rui1,2,BAO Bin3,BU Yong-shi1,WANG Yong-xian1,CHEN Li-juan1,WU Wen-hui2,3,*
2013, 34(9):  24-27.  doi:10.7506/spkx1002-6630-201309006
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Objective: To isolate type Ⅱ collagen from Prionace glauca cartilage using alkali treatment/pepsin hydrolysis
method, and to determine its biological activity. Methods: The freeze-dried Prionace glauca cartilage was crushed into 60-
200 mesh powder. Then, NaOH treatment and hydrolysis with pepsin were conducted. After salting and dialysis, the type Ⅱ
collagen was obtained. SDS-PAGE, amino acid composition analysis, UV spectroscopic scanning and differential scanning
calorimetry were applied to identify the type Ⅱ collagen. Results: Prionace glauca type Ⅱ collagen was composed of α1
chain and had the molecular weight of 130 kD. Glycine revealed the highest population in Prionace glauca type Ⅱ collagen
with the ratio of 356‰. Meanwhile, type Ⅱ collagen from Prionace glauca was rich in alanine, proline and hydroxyproline.
Moreover, type Ⅱ collagen from Prionace glauca had the characteristics of Gly-X-Y chain, UV absorption peak at
226 nm, thermal denaturation temperature of 41 ℃. Conclusion: Type Ⅱ collagen from Prionace glauca separated by alkali
treatment/enzyme hydrolysis has typical characteristics of type Ⅱ collagen.

Functional Properties of Seed Protein Isolate from Chinese Wolfberry (Lycium barbarum L.) Grown in Ningxia
WU Hua-yu,LIU Dun-hua*
2013, 34(9):  28-32.  doi:10.7506/spkx1002-6630-201309007
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Chinese wolfberry seed protein isolate (CWSPI) was extracted by the traditional method of alkali extraction and
acid precipitatio from Chinese wolfberry seeds. Amino acid analysis showed that the amino acid composition of CWSPI was
well-balanced and close to the FAO/WHO standards for composition pattern of essential amino acids, with high glutamic
acid content. CWSPI possessed excellent oil-adsorbing capacity and emulsifying properties, general solubitly and waterholding
capacity, and relatively poor foaming capacity and foam stability. The SDS-PAGE pattern of CWSPI displayed 6
subunits with molecular weight of 48, 35, 32, 20.4, 19 kD and 18.5 kD.

Different Heating Methods for the Reactivity between Beef Enzymatic Protein and Glucose
WU Xiao1,KONG Ling-hui1, YU Li-mei 2
2013, 34(9):  33-36.  doi:10.7506/spkx1002-6630-201309008
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Objective: To investigate the effect of different heating methods (microwave, high pressure, the flow of hot air,
water vapor) on reactivity between beef enzymatic protein and glucose. Methods: refined fresh beef as the base material, the
molecular weight distribution analysis and sensory evaluation analysis were applied. Results: the activities of beef enzymatic
protein changed in various ways among different treatment of heating methods, mainly including degradation, decomposition
and polymerization reactions. But the Maillard reaction was not significant. The beef flavor substances were distributed
between 1000 D and 4999 D, mainly in the range of 3000—4999 D. Conclusion: The beef flavor substances in the range of
3000—4999D were not significant in the roasted flavor, but showed good beef flavor and strong taste.

Effect of Transglutaminase on Heat-induced Gel Properties of Bighead Carp (Aristichthys nobilis) Surimi
JIA Dan1,2,LIU Ru1,2,LIU Ming-fei1,LI Yu-bin1,XIONG Shan-bai1,2,*
2013, 34(9):  37-41.  doi:10.7506/spkx1002-6630-201309009
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In order to explore the impact of TGase on heat-induced gel properties, the effect of transglutaminase (TGase) on
the dynamic rheological properties, gelation temperature (Tgel), gel activation energy (Ea), distribution of molecular weight,
gel strength and solubility from bighead carp (Aristichthys nobilis) surimi during heating processing were evaluated. Results
showed that Tgel and Ea decreased along with the increase of TGase concentration, while solubility and MHC intensity
decreased and tended to be stable afterwards. The weaken of energy barriers in thermal gelation cause more formation of
ε-(γ-Glu) lys bonds induced by TGase. While the breaking strength, gel strength, water-holding capacity and storage modulus
G′ revealed an initial increase, and then decreased along with the increase of TGase concentration. In contrast, when TGase
concentration was 28.08 U/100 g of surimi, the gel strength was the highest and the expressible moisture was the lowest.
Compared with the control group, the gel strength increased by 2.7 times; expressible moisture decreased by approximately
46%, and G′ exhibited a 2.5 fold enhancement. When the TGase was added more than 28.08 U/100 g of surimi, G′, the
breaking strength, the gel strength and water-holding capacity exhibited an obvious reduction. An appropriate concentration
of TGase could significantly improve the gel strength and water-holding capacity of bighead carp surimi.

Major Protein Fractions and Subunit Contents in Peanut from Different Cultivars
DU Yin,WANG Qiang*,LIU Hong-zhi,WANG Li,LIU Li
2013, 34(9):  42-46.  doi:10.7506/spkx1002-6630-201309010
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The composition of peanut protein from 170 different cultivars in China was analyzed by SDS-PAGE as well
as the relative content of major fractions. There were significant variations in protein composition and the relative content
of subunits among these peanut cultivars. Both arachin and conarachin were found in each cultivar, with a ratio between
0.80 and 1.68, and a coefficient of variation (CV) of 15.23%. This CV larger than 10% suggests great differences in protein
composition among different peanut cultivars. In addition, significant variations were also observed in the relative content
of subunits; in arachin, the 35.5 kD subunit presented the largest CV of 55.07%, which, however, was absent from 20.59%
(35/170, such as ShuangJi 35) of all the tested cultivars. Moreover there was an extremely significantly negative correlation
between arachin and conarachin(r = -0.998).

Polyphenol Oxidase Activity and Total Phenol Content in Red Wine Grape
WANG Yue-hui,XU Hong-yu,ZHANG Jing-fang*,HOU Li-xuan,CHENG Bing
2013, 34(9):  47-51.  doi:10.7506/spkx1002-6630-201309011
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Polyphenol oxidase (PPO) activity, total phenolic (TP) content and the browning degree (BD) of 27 cultivars of
red wine grape and the influence of this three factors on the value of L* of wine were analyzed. Variety selection was carried
out by system clustering using ratio η (PPO activity/TP content), and further divided using quadrant distribution between
the average of the two indicators. The results showed significant differences in PPO activity, total phenolic content and
browning degree among different red wine grape varieties. There was no significant correlation between the PPO activity and
total phenol content, but they were both significantly positively related to the browning degree. PPO activity, total phenolic
content and the browning degree were significantly negatively correlated with L* value of red wine. Cabernet Gernischet,
Seibel Noir, Cabernet Sauvignon, Merlot, Kapц Maт and Cabernet Franc were selected as fine red wine grape varieties with
low PPO activity and high total phenolic content.

Separation and Purification of Angiotensin Ⅰ-Converting Enzyme Inhibitor from Walnut Protein
GU Xin1,2,LI Di3,HOU Ya-kun1,2,CUI Jie1,2,WANG Jian-zhong1,2,*
2013, 34(9):  52-55.  doi:10.7506/spkx1002-6630-201309012
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An angiotensin Ⅰ-converting enzyme (ACE) inhibitor was separated and purified from pepsin hydrolysate of
walnut protein by ultrafiltration, gel permeation chromatography and HPLC. The inhibitory activity and in vitro digestive
stability of the ACE inhibitor were examined. Its structure was identified through N terminus amino acid sequencing as Tyr-
Glu-Pro. The IC50 of this inhibitor derived from walnut protein was 0.32 μg/mL. This activity was well maintained after
simulated digestion in vitro.

Antioxidant Properties of Maillard Reaction Products from Whey Protein Isolate
WANG Wen-qiong,BAO Yi-hong*,CHEN Ying
2013, 34(9):  56-60.  doi:10.7506/spkx1002-6630-201309013
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We undertook this study to examine the effects of different sugars (xylose, glucose, fructose, sucrose, maltose
and lactose) and protein:sugar ratio on browning in the Maillard reaction of WPI (whey protein isolate). The antioxidant
activity of the MRPs was also determined. The results showed that the MRPs of WPI-xylose system at a ratio of 2:1 had
the highest browning degree, thereby showing strong reducing capacity, lipid peroxidation inhibitory effect, DPPH radical
scavenging activity, and hydroxyl free radical scavenging activity.

Relationship between Hydrolysis Degree and Antibacterial Capability for Whey Protein
KE De-sen1,WANG Hong-xing2,WU Jin-xiong1,WANG Zheng-xun1,LIU Chun-mei1,2
2013, 34(9):  61-65.  doi:10.7506/spkx1002-6630-201309014
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Whey protein was hydrolyzed in different ways, the antibacterial ability and the degree of hydrolysis of
hydrolysates were measured, and their correlation was studied. The results showed that hydrolysates of whey protein at a
certain degree of hydrolysis had a significant antibacterial ability. The antibacterial ability of hydrolysates was closely related
to the degree of hydrolysis (r = 0.9740 when hydrolysis degree was less than 12 mg free amino acids/mL; r=-0.9468 when
hydrolysis degree was higher than 12 mg of free amino acids/mL). An optimal degree of hydrolysis was found under different
hydrolysis conditions when whey protein was hydrolyzed by trypsase. At this optimal degree of hydrolysis (10—12 mg of free
amino acids/mL), the hydrolysates of whey protein showed the highest antimicrobial activity. Hydrolysates prepared by
hydrochloric acid showed significantly lower antibacterial capacity and poorer stability when compared to those prepared
by trypsase. In summary, this study demonstrated that appropriate hydrolysis could significantly improve the antimicrobial
capacity of whey protein hydrolysates. The optimum hydrolysis degree was in the range of 10—12 mg of free amino acids/mL.
Whey protein could potentially be used for the development of new natural food preservatives.

Isolation, Purification and Antioxidant Activity of Sesame Peptide from Solid-state Fermentation
PENG Hui-hui1,LI Lü-mu1,2,3,*,QIAN Kun3,XU Fa-zhi2,WU Dong3,ZHOU Fen3,DING Xiao-ling2
2013, 34(9):  66-69.  doi:10.7506/spkx1002-6630-201309015
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The antioxidant activity of small peptide from sesame meal subjected to solid-state fermentation was studied.
Active small peptides were extracted from fermented sesame seed meal and three peptides with different molecular weights
were separated and purified by Sephadex G-15, named as Peptides 3, 4 and 6. The antioxidant activity of sesame peptide was
also systematically analyzed. DPPH scavenging activity was measured and salicylic acid method for the scavenging activity
and ferric ion reducing ability method for the determination of its total reduction capacity. DPPH radical scavenging activity
of 1 mg/mL crude extract and the peptide with 3 or 4 amino acid residues was 90%, but the DPPH radical scavenging
activity of the peptide with 6 amino acid residues was 80%. In addition, the hydroxyl radical scavenging rate of 0.6 mg/mL
peptide with 3 or 4 amino acid residues was 100%, while the hydroxyl radical scavenging rate of the crude extract and the
peptide with 6 amino acid residues was 90%. Total reducing capacity of 4 mg/mL crude extract and the peptide with 4 or 6
amino acid residues, and 2 mg/mL peptide with 3 amino acid residues was equivalent to 0.5 mg/mL glutathione. These results
showed that small peptides from solid-state fermentation of sesame meal have strong reducing power and strong scavenging effect on
DPPH and hydroxyl free radicals. The antioxidant activity can be enhanced when the molecular weight of sesame peptide is reduced.

Isolation and Activity of Antimicrobial Peptide from Ants
SU Xiang,CUI Jing-ai,CHEN Xiao-ping*
2013, 34(9):  70-73.  doi:10.7506/spkx1002-6630-201309016
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Objective: To isolate antimicrobial peptide from ants and explore its biological activity. Methods: Wild red ants
from Changbai Mountain were used as the raw material, ethanol-leaching method was used to extract the crude peptide and
heating-column chromatography was used for purification. The relative molecular mass and the bacteriostatic activity of
small molecule polypeptide with antibacterial activity were determined. Results: The antimicrobial peptide obtained from
this method revealed high purity. Relative molecular mass of the antimicrobial peptide was determined to be 3.746 kD. The
antimicrobial peptide from ants had bacteriostatic capacity to Escherichia coli, Staphylococcus aureus, Salmonella enteritidis
and Bacillus subtilis with minimum antibacterial concentrations of 0.325, 0.65, 0.1625 mg/mL and 0.325 mg/mL. Moreover,
the antimicrobial peptide from ants exhibited high thermal stability.

Screening and Evaluation of α-Amylase-inhibiting Fractions Extracted from Ya’an Tibetan Tea
NIE Kun-lun1,2,HE Li3,SU Xiao-juan1,2,BIAN Jin-lin1,2,GUO Jin-long1,DU Xiao1,2,*
2013, 34(9):  74-79.  doi:10.7506/spkx1002-6630-201309017
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Seven kinds of Ya’an Tibetan tea fractions were separated by liquid-liquid extraction by using 70% ethanol.
Major components of Ya’an Tibetan tea were analyzed. Furthermore, the inhibitory activity of the fractions on α-amylase
and corresponding active sites were determined. The results showed that the activity of α-amylase could be inhibited by
catechins, caffeine and TBs in Ya’an Tibetan tea. The decreasing order of the inhibitory activity of Ya’an Tibetan tea extract
fractions on α-amylase was catechin > TBs > caffeine.

Fast Separation and Purification of Superoxide Dismutase Using Magnetic Nanoparticles
YANG Lin1,SUN Shu-guo2,LUO Zhang1,*
2013, 34(9):  80-84.  doi:10.7506/spkx1002-6630-201309018
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The magnetic nanoparticles used to quickly separate and purify superoxide dismutase (SOD) from yak blood was
evaluated and a kinetic model of SOD adsorption was established in our experiments. The effects of pH of binding buffer (sodium
phosphate buffer) and the dissociation buffer (sodium acetate buffer), NaCl concentration and adsorption time on the specific
activity and recovery of SOD were investigated to determine the optimal conditions for separation and purification of the
enzyme. The specific activity of SOD revealed an initial increase and a final decrease with the increase of pH, but the enzyme
recovery revealed a stable increase, suggesting that optimal pH of binding buffer was 7.5. The specific activity and recovery of
SOD exhibited an initial increase and following decrease with the increase of NaCl concentration. Meanwhile, the maximum
SOD adsorption was observed in the presence of 0.5 mol/L NaCl. When pH of dissociation buffer was increased from 4.0 to
5.5, the specific activity of SOD revealed an improvement, but the recovery of enzyme revealed a reduction. So the optimal
pH of dissociation buffer was determined to be 5.0. The experimental results also showed that under the optimal adsorption
conditions, the optimal static adsorption time was 2 h. As the concentration of crude enzyme extract was increased, SOD adsorption could be
expressed by Langmuir adsorption model. The specific activity of SOD was always maintained at a high level (3000—3250 U/mg), which
showed that the best separation selectivity for SOD can be achieved by using magnetic nanoparticles.

Purification and Preliminary Identification of Total Flavonoids from Artemisia selengensis Turcz
DENG Rong-hua,GUO Yu-xing,LI Xiao-ming,Lü Li-shuang*
2013, 34(9):  85-89.  doi:10.7506/spkx1002-6630-201309019
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This study aimed at establishing the optimum conditions for purifying total flavonoids from Artemisia selengensis
Turcz with AB-8 macroporous resin. In addition, we preliminarily identified the resulting purified product by means of
chromogenic reaction and UV adsorption spectroscopy. AB-8 possessed excellent adsorption and desorption efficiencies for
total flavonoids. The optimum adsorption and desorption conditions were determined as follows: 2.26 BV of 1.476 mg/mL
sample solution at pH 4.0 was flowed through AB-8 column at a rate of 1 mL/min and then desorbed with 1.5—2.0 BV of
60% ethanol at a flow rate of 1 mL/min. The purity of the purified product obtained under these conditions was 30.26%,
compared to 5.80% for the crude total flavonoids extract. Chromogenic reaction and UV characteristic adsorption indicated
that total flavonoids from Artemisia selengensis Turcz might contain flavonols, isoflavones, flavonone and double hydrogen
flavonols with ortho-dihydroxyl groups in their structure.

Intein-mediated Activation of Transglutaminase from Streptomyces hygroscopicus
DU Kun,ZHOU Li,DU Guo-cheng,CHEN Jian,ZHOU Zhe-min
2013, 34(9):  90-94.  doi:10.7506/spkx1002-6630-201309020
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Microbial transglutaminase (MTG) from Streptomyces is secreted as an inactive form of pro-MTG, which can be activated by protease. However, protease has the potential for the hydrolysis of MTG as well as its substrates and can improve product separation costs. In this study, a pH-dependent mini-intein was inserted between the pro-region and MTG to mediate the cleavage of both parts by controlling pH. The recombinant protein (pro-Intein-MTG) was successfully overexpressed in Escherichia coli. The time required for converting recombinant protein to MTG was determined to be 24 h at 25 ℃ and pH 7.0 in vitro, resulting in an activity of 0.23 U/mL (cell concentration was diluted to OD600nm at 1.0). The specific activity and Km of the recombinant MTG was determined to be 14.3 U/mg and 69.4 mmol/L, respectively, which was consistent with that of wild type MTG from Streptomyces hygroscopicus. In addition, circular dichroism analysis showed that the recombinant and wild type MTG had similar secondary structure. These results indicated that the insertion of intein between the pro-region and MTG did not significantly affect the structure and function of MTG. This method is simple, economic and effective in the activation of pro-enzyme and has a great potential for industrial application.
Preparation of High-concentration Fermented Milk for Use in Foods and Effect of Simultanesou Enzymatic Hydrolysis and Fermentation on Fermentation Characteristics
BAO Zhi-ning,LIN Wei-feng*,YE Jun,XIONG Jian*
2013, 34(9):  95-98.  doi:10.7506/spkx1002-6630-201309021
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A high-concentration reconstituted milk with containing 7.8% protein was fermented by Lactobacillus casei to
produce fermented milk for use in foods. We examined the changes in fermentation characteristics including acidicty, the
viable cell number of the starter and amino nitrogen content. A complex enzymatic preparation of lipase and protease and
the inoculum were added together to simultaneously hdyrolyze and ferment the reconstituted milk. As a result, a fermented
milk higher in acidity and amino nitrogen was yield. Both indicators were greatly influenced by the addition of the complex
enzymatic preparation. The fermented milk obtaiend after 72 h of single fermentation exhibited a titrtable acidity of 268.5 °T
and an amino nitrogen content of 0.117 g/100 g, compared to 414.4 °T and 0.132 g/100 g for that obtained after 72 h of
simultaneous hydrlysis and fermentation. Furthermore, titratable acidity and amino nitrogen content of fermented milk
increased with increasing amount of the enzymatic preparation added to the reconstituted milk. However, neither pH nor the
viable cell number of Lactobacillus casei was affected by the dosage of the enzymatic preparation.

Preparation and Fusion of Yeast Protoplasts and Screening of Fusants with High Yield of β-Carotene
WANG Sui-lou,WANG Hai-xiang,YANG Zhi-ping,Lü Chun-hui,BU Fen
2013, 34(9):  99-103.  doi:10.7506/spkx1002-6630-201309022
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The protoplast fusion technique between R. glutinis NR-98 and Phaffia rhodozyma Ph-1 was studied. The strain
NR-98 could grow at 28 ℃, but had a lower yield of β-carotene. In contrast, the strain Ph-1 had high yield of β-carotene, but
could not grow at 28 ℃. The results of fusion experiments showed that the fusant R-5 revealed the parent strain properties
of thermotolerance and β-carotene yield. When using glucose as substrate at 28 ℃, the fusant R-5 had a high β-carotene
yield up to 9.20 mg/L, with the enhancement of 27.42% and 53.33% when compared with strain Ph-1 and strain NR-98,
respectively. The genetic quality of fusant R-5 was good as confirmed by stability experiments.

Effect of (–)-Epigallocatechin-3-gallate as an Inhibitor on the Structure-activity Relationship of Pancreatic Lipase
WANG Shi-hui,LIU Yun*
2013, 34(9):  104-107.  doi:10.7506/spkx1002-6630-201309023
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The effect of (–)-epigallocatechin-3-gallate (EGCG) on the structure-activity relationship of pancreatic lipase
was studied in this work. The secondary and tertiary structures of pancreatic lipase were measured by circular dichroism
(CD) and fluorescence spectroscopes (FS), respectively. Using olive oil as the substrate, the activity of pancreatic lipase as a
function of incubation time and EGCG concentration was also investigated. The results showed that the maximum inhibitory
rate was observed after 1 h mixing of EGCG with pancreatic lipase. The inhibitory rate rapidly increased with increasing
EGCG concentration up to 1 mmol/L and then remained basically unchanged as the EGCG concentration continued to
increase. The Lineweaver-Burk plot showed that EGCG non-competitively inhibited the activity of pancreatic lipase. CD
and FS analyses suggested that EGCG influenced both the secondary and tertiary structures of pancreatic lipase, and its
concentraiton was positively correlated with the secondary structure but had no significant correlation with the tertiary
structure. FS analyses also indicated that EGCG quenched the Trp fluorescence of pancreatic lipase statically or dynamically.

Preparation and Properties of Cross-linked Enzyme Aggregates of Trehalose Synthase
CHEN Ying1,XIAO Chen-peng2,CHEN Xiao-yun1,YANG Li-wei1,TANG Liu1,LI Ming-chun2,ZHANG Jun1,*
2013, 34(9):  108-113.  doi:10.7506/spkx1002-6630-201309024
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This paper describes the preparation and properties of cross-linked enzyme aggregates (CLEAs) of trehalose
synthase. The effects of different precipitants, enzyme-to-precipitant concentration ratio, cross-linking intensity and NaBH4
treatment on the activity of CLEAs were investigated as well as their structural features and reactivity. The optimum conditions
for CLEAs preparation were achieved when 10 mg/mL trehalose synthase solution was added with 30 mg/mL of PEG, crosslinked
with 0.5% glutaraldehyde at ambient temperature for 2 h, followed by NaBH4 reduction. CLEAs revealed optimum
reaction temperature of 70 ℃, 20 ℃ higher than that of the free enzyme, and optimum pH of 7.0. Compared to the free enzyme,
CLEAs indicated improved heat and pH stability. Moreover, the tolerance to some metal ions such as Zn2+, Cu2+, Fe2+ and Al3+
was remarkably enhanced. The microscopic morphology of CLEAs showed individual aggregates of 0.2—0.5 μm in particle
size and numerous clusters of different sizes in a “scaffolding” structure.

Expression and Purification of Putative Substrates of the Site-2 Protease Homologs from Synechocystis sp. PCC6803
QIN Chun-yan,CHEN Gu*
2013, 34(9):  114-120.  doi:10.7506/spkx1002-6630-201309025
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Nearly all cyanobacterial species contain genes encoding site-2-protease (S2P) homologs. Some S2P homologs
have been reported to play essential roles in regulating stress response through intramembrane proteolysis of membranebound
anti-sigma factors. However, the mechanism of action and substrates of S2P in Synechocystis sp. PCC6803, Sll0862,
Slr0643, Sll0528 and Slr1821 remain unsolved. In this study, we focused on the four sigma factor-anti sigma factor pairs, SigEChlH(
Slr1055), SigI(Sll0687)-Sll0688, SigG(Slr1545)-Slr1546 and SigH(Sll0856)-Sll0857. Using pET-30b(+) as vector, we
constructed recombinant plasmids, optimized expression and successfully purified the full length and truncated fragment of
putative substrates, including Slr1055, Slr1055Δ(1–232), Sll0688, Sll0688Δ(1–152), Slr1546, Slr1546Δ(1–174), Sll0857 and
Sll0857Δ(1–101) as well as RseA(1–148), the S2P substrate from E.coli. This work may lay the foundation for constructing in
vitro enzymatic digestion system to justify the relationship of various S2Ps with their putative substrates.

Cloning and Expression of D-Arabinose Isomerase from Geobacillus pallidus and Its Application in D-Tagatose Conversion
ZHANG E,ZHANG Liang,SHI Gui-yang
2013, 34(9):  121-126.  doi:10.7506/spkx1002-6630-201309026
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A new recombinant D-arabinose isomerase (D-AI) was used to convert D-galactose to D-tagatose. Subjected to strain screening in the laboratory, Geobacillus pallidus with strong enzyme production capacity was obtained. The D-arabinose isomerase gene (D-AI) was cloned into expression vector pET-28a, and transformed into E. coli BL21 (DE3). Results revealed that the recombinant strain could express a large number of soluble proteins by SDS-PAGE. Optimum pH and temperature of purified enzyme were 7.0 and 60 ℃, and the enzyme revealed good stability within the range of pH 6.0—8.0 and 30—70 ℃. The addition of Mn2+, Zn2+, Fe2+, Ca2+ and Ba2+ could improve the conversion rate of D-tagatose; in contrast, Mg2+ and Cu2+ could result in the different inhibition degrees of enzyme activity. The substrate D-galactose at the concentration of 18 g/L (containing 5 mmol/L Mn2+), was added to pH 7.0 crude enzyme solution from recombinant cells to react of 60 ℃. Under the optimal reaction time, the maximum conversion rate was 41.6% after 12 h reaction. Therefore, biological activity of the recombinant D-AI was successfully expressed in E. coli, and E. coli (BL21) has potential industrial production of D-tagatose.
Gene Cloning and Recombinant Expression of Chondroitinase AC from Pedobacter heparinus and Characterization of Recombinant Fusion Enzyme
WU Jing-jun1,LI Ye1,2,ZHANG Chong1,LI Mei1, XING Xin-hui1,*
2013, 34(9):  127-134.  doi:10.7506/spkx1002-6630-201309027
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Chondroitinase AC (ChonAC) is an important enzyme to reveal the biological function, structure and mechanism
of chondroitin sulfate, and it is important for low molecular weight chondroitin sulfate preparation and diseases treatment.
To achieve the efficient expression of ChonAC with high activity in recombinant Escherichia coli (E. coli), the ChonAC
gene (cslA) from Pedobacter heparinus was amplified by polymerase chain reaction (PCR), and the fusion expression vector
for expressing maltose-binding protein (MBP) fused ChonAC (MBP-ChonAC) was constructed. The results showed that the
fusion strategy using MBP was effective to enhance the solubility of the MBP-ChonAC when induced at low temperature of
15 ℃, the purity and the specific activity of MBP-ChonAC could reach 95% and 94.1 IU/mg-fusion protein (equivalent to
143.8 IU/mg ChonAC) by one-step MBPTrap HP purification, respectively. The enzyme characteristic study showed that the
optimum pH value, Ca2+concentration, NaCl concentration, and reaction temperature was 7.5—8.0 , 20 mmol/L, 50 mmol/L,
and 20—35 ℃, respectively. The half-life of MBP-ChonAC could reach 8.3 h at 30 ℃. The kinetic characterization showed
that the Km was higher while the kcat was a little bit lower than the reported native ChonAC for chondroitin sulfate A, while
the biocatalysis experiment for MBP-ChonAC indicated that the fusion of ChonAC with MBP did not affect the enzyme
function. Furthermore, the total activity of MBP-ChonAC by shake flask cultivation could reach 10800.5 IU/L, the highest
value reported so far, through the optimizations of host cells, IPTG induction concentration, and M9-based culture medium.

Preparation and isolation of food-based gelatin peptide and the ice crystal inhibition
WANG Shao-yun,ZHAO Li-na,ZHOU Yan-fu,RAO Ping-fan
2013, 34(9):  135-139.  doi:10.7506/spkx1002-6630-201309028
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Food-based gelatin peptides with antifreeze activity were obtained under controlled hydrolysis conditions by Alcalase. The specific antifreeze peptide was isolated by chromatography and identified by mass-spectrum, the full amino acid sequences were determined. The result showed that with E/S of 1:15, hydrolyzing time of 30min, hydrolyzing temperature of 45℃, gelatin peptides demonstrated the best antifreeze activity. The antifreeze peptide on ice crystal inhibition shows specific rules during cold-heat-stage cycles, the key approach is how to control hydrolysis conditions. The specific antifreeze peptide was fractionated by size exclusion (Sephadex G-50), ion exchange (sulfopropyl-Sephadex C-25) columns and C18-HPLC, respectively. The molecular mass of the specific antifreeze peptide was determined to be 2107 Da by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The purified gelatin peptide with the molecular mass of 2107 Da strongly inhibited ice crystal growth in ice cream mix. The full amino acid sequences were determined to be GERGFPGERGSPGAQGLQGPR by Edman degradation.

Cloning and Expression of Glutaminase Gene glsA2 in Prokaryotic System
LU Biao,WU Yong-jun*,WU Yu-jun,LUO Xi,LIU Yan-min
2013, 34(9):  140-142.  doi:10.7506/spkx1002-6630-201309029
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To test the activity of glutaminase from Bacillus subtilis BJ3-2, the gene glsA2 was cloned into prokaryotic
expression vector pET-32. The recombinant plasmid pET32a-glsA2 was then transformed into E.coli BL21 competent cells.
Optimum IPTG concentration and induction time for gene glsA2 expression were optimized. The result showed that the
expression was optimized with proper inducing conditions of 0.1 mmol/L IPTG for 6 h. Under these conditions, glutaminase
activity in supernatant of cell lysate reached 608.2 U/μg protein, which was 15 times higher than that of control.

Optimization of Fermentation Process for Recombinant Escherichia coli Expression of Phospholipase C
ZHAO Jin-xing,ZHANG Liang*,GU Zheng-hua,DING Zhong-yang,SHI Gui-yang
2013, 34(9):  143-149.  doi:10.7506/spkx1002-6630-201309030
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The culture condition of Escherichia coli BL21 (DE3)/pET28a-plcH expressing the phospholipase C gene from
Pseudomonas aeruginosa 41 was optimized. The expression vector was cultured in TB medium and important parameters
such as the type of bufffer and the type and concentration of nitrogen source were optimized. Furthermore, the inducive
influences of IPTG, lactose and glycine on phospholipase C production were comparatively discussed. The optimized
medium composition was determined as (g/L): trytone 12, yeast extrat 24, glycerol 5, and Tris-HCl (0.25 mol/L, pH 7.2—7.4).
After sterilization, 0.15 g/L kanamycin was supplemented. In conical flasks, strain E. coli BL21 (DE3)/pET28a-plcH was
incubated at 37 ℃ and 200 r/min for six hours, followed by addition of 5 g/L lactose, and then the culture was maintained
at 25 ℃ and 150 r/min for twenty more hours. Under the optimized conditions, the final activity of phospholipase C was
(1422.42±37.17) U/mL. In a 7 L fermentor, the total activity reached (11583.35±70.21) U/mL with an intracellular activity of
(10957.97±58.03) U/mL and an extracellular activity of (645.27±13.87) U/mL.

Optimal Chicken Enzymatic Hydrolysate for the Preparation of Chicken Flavoring
CHEN Hai-tao,XU Xiao-lan,ZHANG Ning,SUN Bao-guo,WANG He
2013, 34(9):  150-154.  doi:10.7506/spkx1002-6630-201309031
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The objective of this work was to select the optimal chicken enzymatic hydrolysate for the preparation of chicken
flavoring via thermal reaction. The enzymatic hydrolysis of chicken was optimized by one-factor-a-time method and orthogonal
array design. The degree of hydrolysis (DH) of chicken and sensory quality of chicken flavoring were investigated with respect
to hydrolysis parameters. The absence of pre-heat treatment was favorable for the hydrolysis of chicken. The optimal enzymatic
hydrolysis conditions were found to be sequential hydrolysis with protamex for 1 h followed by (without inactivating protamex)
animal-derived protease for 2 h at the same temperature of 50 ℃ with a total enzyme dosage of 1.100‰ and a mass ratio of 1:1
between both enzymes at a chicken-to-water mass ratio of 1:1. The maximum DH obtained under these conditions was 20.47%.
Moreover, chicken flavoring with strong aroma and high similarity to chicken was prepared with the resulting hydrolysate.

Cellulose Acetate-polypropylene Composite Membrane Immobilized Phospholipase
SHI Min,WANG Xue,MA Li-na,YU Dian-yu*
2013, 34(9):  155-158.  doi:10.7506/spkx1002-6630-201309032
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The transglutaminase was immobilized on the cellulose acetate-polypropylene composite membrane in this study.
The activity of the enzyme was used as the index. Based on the single-factor tests, the effects of enzyme addition amount,
absorption time, glutaraldehyde concentration and glutaraldehyde cross-linking time on the activity of enzyme were studied.
The optimal immobilization conditions required enzyme concentration of 15 mg/mL, adsorption time of 3 h, glutaraldehyde
concentration of 0.3 g/100 mL, glutaraldehyde cross-linking time of 4 h. The activity of immobilized enzyme under the
optimal immobilization conditions was up to 16.1 U/g. The optimal temperature for the free enzyme was 35-40 ℃,
compared to 45-50 ℃ for the immobilized enzyme. The optimal pH for the free enzyme was 6-7, compared to pH 5-6
for the immobilized enzyme. Compared with the free enzyme, the immobilized enzyme was shifted to the acidity side.

Purification and Characterization of Recombinant Glucose Oxidase from Pichia pastoris
HAO Jie-qing,WANG Shuai-kun,SHI Hui,WANG Zhen-wei,MENG Yan-fa*
2013, 34(9):  159-163.  doi:10.7506/spkx1002-6630-201309033
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Purpose: To explore the purification and properties of glucose oxidase (GOD) from recombinant Pichia
pastoris. Methods: The crude glucose oxidase was purified by Q-Sepharose Fast Flow chromatography. Polyacrylamide gel
electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine
its molecular weight. Ultraviolet and fluorescence absorption spectroscopy were used to observe its spectral characteristics.
Results: Purified GOD with a purification factor of 1.35 and a recovery of 92.7% was obtained. This enzyme exhibited a
dimeric form with a molecular mass of 150 kD. The GOD showed the highest activity at pH 6.0 and 40 ℃ and was stable
in a broad pH range of 5.0-8.0 and at 55 ℃ or below. The Km of the GOD enzyme was 21.06 mmol/L with glucose as
the substrate. The enzyme was inhibited intensively by Hg2+, Fe2+, Ag+ and Cu2+ and showed a maximum ultraviolet and
fluorescence absorption peak at 275 nm and 344 nm, respectively. The lyophilized enzyme was stable at 4 ℃ over a period of
6 months and could be stored for 3-5 d at normal temperature. Conclusion: The recombinant GOD has simple purification
procedure, high recovery, good stability and promising application potential.

Immobilization of Cells Producing Glucose Isomerases
DENG Hui1,2,CHEN Sheng1,2,CHEN Jian1,2,WU Jing1,2,*
2013, 34(9):  164-169.  doi:10.7506/spkx1002-6630-201309034
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In the present study, immobilization technology of recombinant Escherichia coli cells producing
Thermobifida fusca glucose isomerase was studied using chitosan flocculation and glutaraldehyde cross-linking
methods. In order to obtain the immobilization method for GIase with high performance and low cost, various factors
for affecting flocculation and crosslinking were investigated, and the physical and chemical properties, dynamic
constants and operation stability of immobilized enzyme preparation were determined. The results showed that the
optimal immobilization conditions were diatomaceous earth addition of 0.6% and 5 mmol/L Mg2+ in high-density
fermentation broth. After heat treatment at 60 ℃ for 30 min, cells and debris were flocculated by 0.1‰ chitosan
solution at pH 5.5 under fully rabbling, and recovery rate of GIase reached up to 98%. Flocculation was cross-linked
for 3 h in 0.25% glutaraldehyde solution at pH 6.5 under slight rabbling, and the enzyme activity reached up to 80%
of flocculation. Compared to free GIase, the optimal temperature was 80 ℃, and the optimal pH reduced from 10 to 9.
The initial enzyme activity 356 U/g, and its half-life reached up to 61 d.

Structural Characterization and Structure-activity Relationship of ACE Inhibitory Peptides from Peanut
WANG Qiang1,WANG Chun-yan1,2,HU Hui1,LIU Hong-zhi1,LIU Li1
2013, 34(9):  170-174.  doi:10.7506/spkx1002-6630-201309035
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Peanut short-chain peptides were prepared by hydrolyzing peanut protein isolate with both alcalase and protease
N120P and separated by gel filtration chromatography and preparatve RP-HPLC. As a result, 27 components were obtained,
and P8 was found to have the highest ACE inhibitory activiy among them, with an inhbitory rate of 85.77% at 0.010 mg/mL
and an IC50 of 0.0052 mg/mL (6.42 μmol/L). The relativel molecular mass of the component P8 was 808.80 with an amino
acid sequence of Lys-Leu-Tyr-Met-Arg-Pro(KLYMRP) as determined by MALDI-TOF-TOF MS. The short-chain peptide
KLYMRP prepared with P8 by solid-phase synthesis was confirmed to have potent ACE inhibitory activity with an IC50 of
0.0038 mg/mL (4.69 μmol/L). Analysis of structure-activity relationship indicated that the short-chain peptide Lys-Leu-Tyr-
Met-Arg-Pro consisted of Pro at the C terminus and Lys at the N terminus and had strong hydrophobicity, thus facilitating
active site docking with ACE. In addition, KLYMRP formed five hydrogen bonds and one Pi-Cation interaction with residual
groups at the ACE active positions and formed coordination bonds with Zn ions. All these contributed greatly to stabilize the
conformation of the ACE-KLYMRP complex and therefore benefited ACE inhibition.

Purification and Enzyme Properties of Pectinase from Penicillium oxalicu
ZHANG Ming-ai1,2,WANG Bao-wei1,2,*,YUE Bin2,JING Li-zhen2,3,GE Wen-hua2
2013, 34(9):  175-179.  doi:10.7506/spkx1002-6630-201309036
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Penicillium oxalicum separated from intestine of Wulong goose was used as fermentation microbe. Two major
components (PG and PE) of pectinase were purified from fermented products through salting out by ammonium sulfate,
Sephacryl S-200 gel filtration, and DEAE-Sephadex A-50 ion-exchange chromatography. The enzyme properties such as
optimal pH, temperature, and molecular weight were also studied. The results showed that the purification folds of PG and
PE were 32.21 and 23.98, respectively, and both of them reached up to electrophoretic purity and the molecular weights
of PG and PE were 61.4 kD and 42.2 kD, respectively. The optimal pH and temperature of PG was 5.0 and 40 ℃. PG was
stable in the range of pH 4.0-6.0. The enzyme activity remained stable between 30-40 ℃ with the extension of process
time. The optimal pH and temperature of PE was 6.0 and 50 ℃. PG was stable in the range of pH 5.0-7.0. The enzyme
activity was stable at 30 ℃ and the enzyme activity did not obvious change during 60 min treatment at 40 ℃.

Expression, Purification and Polyclonal Antibody Preparation of the Flagellin Gene fliC of Salmonella typhimurium
CHEN Ming1,2,XU Xing-lian2,ZHOU Guang-hong2,TANG Xiao-yan1,*,YUAN Fei3,CHEN Ai-liang1
2013, 34(9):  180-184.  doi:10.7506/spkx1002-6630-201309037
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In this study, the flagellar gene fliC of Salmonella typhimurium was amplified by PCR from the Salmonella
genome, and cloned into the prokaryotic expression vector pET28a(+). After being sequenced, the recombinant plasmid
pET28a-fliC was transformed into strain BL21(DE3)pLysS, and FliC protein was expressed by the recombinant strain after
IPTG(1 mmol/L) induction. By Ni-NTA purification, the 6×His-tagged proteins were extracted. The molecular weight of
the induced protein was about 54.6 kD as expected. And most of the expressed products were soluble. Western-blotting
analysis indicated that the expressed proteins had a specific reactivity with the monoclonal anti-His-Tag antibody. Polyclonal
antibodies were obtained via immunizing BALB/c mice with the purified protein, and the results showed that the polyclonal
antibodies had high titer and good specificity.

Enzymatic Proteolysis of Chicken Bone Residue and Application of Its Hydrolysates to Form Flavorings
LI Miao-Yun1,ZHAO Gai-ming1,*,XU Xiong1,WANG Yu-fen2,XIE Hua2,LIU Yan-xia1,TIAN Wei1
2013, 34(9):  185-188.  doi:10.7506/spkx1002-6630-201309038
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Chicken bone residue was hydrolyzed by papain, alcalase, neutral or protamex for preparing concentrated
hydrolysates and chicken flavorings. The results showed that the contents of remains obtained from the hydrolysis by four
proteases under the optimal enzymatic hydrolysis conditions were 26.37%, 32.47%, 43.25% and 30.36%, respectively.
Consistent results were obtained in small-mass production. The papain hydrolysis exhibited the lowest residue level. The contents of
total amino acids (TAA) and essential amino acids (EAA) in hydrolysates revealed a significant difference (P < 0.05), especially in
papain hydrolysates, with 6.505 mg/g and 14.965 mg/g, respectively. Similarly, chicken flavorings produced with different
protease hydrolysates were also significantly different (P < 0.05) and the one prepared with papain hydrolysate possessed an
outstanding flavor of chicken fat. Therefore, papain is suitable for in-depth development of chicken flavoring products from
chicken bone residue.

Optimization of Enzymatic Hydrolysis of Hazelnut Kernel Protein and Antioxidant Capacity of Its Hydrolysate
GUO Qing-qi1,2,ZHANG Na2,JIANG Yuan-song1,ZOU Chuan-shan1
2013, 34(9):  189-193.  doi:10.7506/spkx1002-6630-201309039
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The hydrolysis of defatted kernels of Corylus mandshurica grown in Northeast China with alcalase was
optimized by response surface methodology. A regression model was created. The optimal hydrolysis conditions were found
to be 3720 U/g, 50.6 ℃ and 8.4 for enzyme/substrate ratio, temperature and pH, respectively. The maximum predicted degree of
hydrolysis under the optimal conditions was 14.41%, compared to the actual value of (14.38 ± 0.06)%. The total reducing power
of the hydrolysate obtained was 70% of that of VC. In DPPH radical scavenging assays, its IC50 was 0.7311 mg/mL, about 138
times higher than that observed for VC.

Purification and Characterization of Recombinant EGCG-O-transferase from Tea Plant
Lü Hai-peng,ZHANG Yue,FEI Dong-mei,LIN Zhi*
2013, 34(9):  194-197.  doi:10.7506/spkx1002-6630-201309040
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Recombinant EGCG-O-transferase from tea plant was expressed in E.coli. The enzyme was then purified
from cell lysate using HisTMtrap affinity column and its enzymatic properties were investigated. After purification, a
pure protein with purity over 95%, recovery of 34.54% and specific enzyme activity of 0.0186 U/mg was obtained.
Further analysis showed that the recombinant protein had a molecular mass of 27.6 kD. It exhibited the highest
activity under the conditions of pH 7.5 and 35 ℃. Kinetic studies showed that Km and Vmax of EGCG-O-transferase
using EGCG as substrate were 0.100 mmol/L and 7.485 mg/(L·min), respectively.

Identification and Characterization of Lactic Acid Bacteria from Chinese Yak Milk Cheeses
YANG Ji-xia1,2,CHEN Zhi-lan3,YANG Hai-yan4,HUANG Ai-xiang5,CHEN Zong-dao1,2,HE Zhi-fei1,2,*
2013, 34(9):  198-204.  doi:10.7506/spkx1002-6630-201309041
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This study examined the species diversity of lactic acid bacteria from Chinese yak milk cheeses and their
fermentation performance. Totally 17 samples were collected from Tibet, Yunnan and Xinjiang province, colony
enumeration with MRS agar plate and pH were tested to obtain basic information of samples. Lactic acid bacteria were
isolated and investigated by phenotypic and genotypic methods. Phenotypic analysis included morphology, physiological
and biochemical characteristics, carbohydrate fermentation pattern. Genotypic analysis included 16S rRNA sequencing
and species specific PCR. The technological characterization included acid-producing capability, citrate utilization,
proteinase and exopolysaccharide. The average colony enumeration of 17 samples was 4.08±0.60 (lg(CFU/g)), average pH was
4.52 ±0.29. A total of 39 strains were isolated and identified, 35 strains were Lactobacilli including L. buchneri
(2), L. casei (16), L. diolivorans (3), L. fermentum (3), L. helveticus (3), L. kefiri (3) and L. plantarum (5), 4 strains were
pediococcus, including P. acidilactici (1) and P. pentosaceus (3). The strains with good acid-producing capability were X24,
X38, X29, X25, X21, X22, X30 and X35. Strain T7 revealed positive result for all tests of citrate utilization, proteinase
and exopolysaccharide-producing capability. The above findings imply the species diversity of lactic acid bacteria and the
potential presence of high-performance strains in Chinese yak milk cheeses.

Enzymatic Characterization of Alanine Aminopeptidase from Turkey Skeletal Muscle
TIAN Tian,WEI Yan-ling,HE Li-chao,RUAN Gui-ping,ZHANG Ying-yang,ZHANG Jian-hao*
2013, 34(9):  205-209.  doi:10.7506/spkx1002-6630-201309042
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An alanine aminopeptidase (AAP, EC 3.4.11.14) was purified from turkey skeletal muscle by using 45%—65%
ammonium sulphate fractionation, its enzymatic properties and the effect of salt and its hydrolysis products on enzyme activity
was studied. The results showed that optimal reaction temperature of AAP was 37—39 ℃, and the optimal pH was approximately
6.8—7.5. In addition, 0.8 mol/L NaCl revealed the inhibition on the activity of AAP by 10% so that salt had a weakly inhibitory
function to AAP activity. Alanine (Ala), as the major hydrolysis product, almost had no inhibitory effect on AAP, while the other
amino acids had significant feedback inhibition (P < 0.05) on AAP. We found that 100 mmol/L phenylalanine (Phe), lysine (Lys),
leucine (Leu) and methionine (Met) could inhibit the activity of AAP, and the sequence of inhibitory is Phe > Lys > Leu > Met.

Immobilization of Candida sp. Lipase on Nylon Net and Its Enzymatic Characteristics
ZHAO Lei,TANG Jing,WANG Cheng-tao*
2013, 34(9):  210-215.  doi:10.7506/spkx1002-6630-201309043
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Lipase (Candida sp. 99-125) was immobilized on nylon net by glutaraldehyde as cross-linking agent. Effects of
various parameters on the activity of immobilized lipase and characteristics of immobilized lipase were investigated. The
maximum enzyme activity achieved at the following conditions: glutaraldehyde concentration of 3%, cross-linking time of
60 min, enzyme concentration of 10 mg/mL, and immobilized time of 6 h. The optimal temperature of immobilized lipase
shifted from 45 ℃ to 50 ℃, compared with free lipase. The immobilized lipase maintained high activity in a broad pH range
of 5.0 to 7.0, with optimum pH at 7.0, which was identical to that of the free lipase. Thermal, pH, and operational stabilities
of lipase were greatly improved after immobilization onto nylon net. Immobilized lipase retained about 80% of the initial
activity after fifth repeated use. The Km (0.57 mol/L) and Vmax (0.29 × 10-3 mol/(L•s)) of immobilized lipase using olive oil as
substrate was significantly higher as compared to free lipase. The immobilization of lipase decreased its affinity to substrate
as compared to free lipase.

Optimization of solid-state fermentation medium of α-galactosidase produced by Aspergillus niger ZJUQH with response surface methodology
XU Teng-yang,FANG Ruo-si,DONG Ya-chen,CHEN Qi-he*
2013, 34(9):  216-219.  doi:10.7506/spkx1002-6630-201309044
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In order to enhance the production of α-galactosidase by Aspergillus niger ZJUQH, response surface
methodology was applied to optimize the solid culture medium. A fractional factorial design was used to investigate the
main factors affecting α-galactosidase yield. The results showed that two factors played important roles in the production of
α-galactosidase. Central composite design and response surface analysis were applied to establish a second-order regression
equation model for the yield of α-galactosidase. At last, the optimal fementation condition were obtained by response surface
analysis as follows: peptone 0.6 g, Na2HPO4·12H2O 0.08 g, ratio of solid to water 0.41 and inoculum quantity 1.5 mL/5g . Under
the optimal conditions, the activity of α-galactosidase reached (77.21±2.01) U/g, which was increase by 57.41% compared
with (49.05 ± 2.11) U/g before the oprimization, that is, peptone 0.4 g, Na2HPO4·12H2O 0.08 g, ratio of solid to water 0.33
and inoculation quantity 1.5 mL/5 g.

Optimization of Glucose Oxidase Production by Fermentation of Aspergillus niger H1-9b
TANG Jing,ZHAO Xin,SU Mo,TANG Yun-ming*
2013, 34(9):  220-223.  doi:10.7506/spkx1002-6630-201309045
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The fermentation technology for glucose oxidase (GOD) production by Aspergillus niger H1-9b containing GOD
at high activity level was optimized through single-factor and orthogonal tests. On the basis of optimal technology, the effect
of surfactant was investigated. Results showed that the optimal technological parameters included inoculum size of 200 μL,
spore suspension, fermentation broth of 80 mL, initial pH 5.3, and rotation speed of 200 r/min. Tween-80 was used as the
optimal surfactant. The glucose oxidase activity of during the fermentation reached up to 68.96 U/mL due to the addition of
3% Tween-80, which was increased by 1.4 times.

Cloning of Collagen Ⅵ COL6A2 and Its Expression in Pichia pastoris
XU Li-qun,WANG Hao,SUN Yang,WANG Gang,CHEN Huan,CHEN Guang*
2013, 34(9):  224-227.  doi:10.7506/spkx1002-6630-201309046
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The collagen Ⅵ chain α2 gene was amplified from human ollagen Ⅵ genomic DNA and subcloned into the
vector of pPIC9K, and verified by DNA sequencing. The resultant recombinant plasmid pPIC9K-COL6A2 was digested by
SalⅠ and transformed into the competent celss of Pichia pastoris strain GS115 through electroporation. Mut (methanol
utilization)+ and Muts transformants were screened with MM and MD plates. Clony PCR was used to analyze Pichia
pastoris integrants in order to determine if the gene of interest has been integrated into the Pichia pastoris genome. Cells
with stable expression of collagen were screened in a medium containing G418. Regulated by α-factor, AOX1 gene promoter
and termination signal of yeasts, the recombinant collagen was expressed and secreted from the cells. After induction with 1%
methanol, SDS-PAGE analysis showed that the molecular weight of recombinant collagen was approximately 32 kD.

Zingibain: Chemical Modification Using Dextran and Enzymatic Properties
ZHANG Jian-ping,LIU En-qi*,WU Yong-hua,CHEN Shang-long
2013, 34(9):  228-233.  doi:10.7506/spkx1002-6630-201309047
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Dextran was activated by sodium periodate for use as a modifying reagent in the chemical modification of
zingibain. In order to find an optimum method for chemcial modification, the effects of different modification conditions were
studied by orthogonal array design. Some enzymatic properties of natural and modified zingibain were investigated, and their
structures were investigated by ultraviolet absorption spectroscopy. The results showed that the modification rate of zingibain
using activated dextran was 57.8% and the relative enzyme activity was 2.0 times higher than that of the natural enzyme under
the conditions: the ratio of zingibain: activated dextran = 1:22 (m/m) and pH 6.0 at 42 ℃ for 3 h. Compared with the natural
enzyme, the optimal reaction temperature and pH and the Km of the modified enzyme using casein as the substrate were reduced,
and the thermostability of the modified enzyme was increased significantly. The spectral analysis showed that the configuration
and conformation of the modified enzyme were quite different from those of the natural enzyme.

Optimization of Enzymatic Hydrolysis of Hairtail Protein and Antioxidant Activities of Its Hydrolysates
LI Wang1,2,JIN Ting2,*,WU Yu-xue2
2013, 34(9):  234-239.  doi:10.7506/spkx1002-6630-201309048
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This study is aim to obtain enzymatic hydrolysates with high antioxidant ability from hairtail protein. Taking
scavenging activity on DPPH free radicals and hydrolysis degree as response values, four kinds of proteases were used to
hydrolyze hairtail protein and the optimal protease for preparing antioxidant hydrolysates was screened. Response surface
methodology (RSM) was used to optimize hydrolysis conditions. The molecular weight and amino acid compositions of
the hydrolysates were determined by Sephadex G-25 size-exclusion chromatography and automatic amino acid analyzer,
respectively. RSM tests showed that the optimal conditions for scavenging activity on DPPH free radicals were material-liquid
ratio of 1:3, enzyme addition amount of 1.65%, hydrolysis time of 3.80 h, and hydrolysis temperature of 48 ℃, and scavenging
activity on DPPH free radicals under the above conditions reached up to 60.13%. The resultant hydrolysate showed high
reducing power and hydroxyl radical scavenging activity. The molecular weight of the optimal hairtail protein hydrolysates
was approximately 1311 D. The hydrolysates with leucine had scavenging capability to free radicals and the content of leucine
accounted for 35.10% of total amino acids. The peptides with low molecular weights were the major components of the
hydrolysates and the content of these amino acids consisted of the peptides was approximately 89.8% of total amino acids.

Impact of Plastein Reaction on ACE Inhibitory Activity of Vinegar Egg Hydrolysates
YANG Feng1,2,CHEN Jin-ping1,MO Jin-wei2
2013, 34(9):  240-244.  doi:10.7506/spkx1002-6630-201309049
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This study was conducted to investigate the effect of plastein reaction on ACE inhibitory activity of vinegar egg
hydrolysates. A pepsin hydrolysate of vinegar egg was prepared and modified by plastein reaction with pepsin and amino
acids. The optimum plastein reaction conditions were obtained when the reaction was iniated by mixing proline with free
amino aicds in the hydrolysate in a proporption of 1 mol/mol and adjusting to pH 5.0 and procceeded at 37 ℃ for 12 h
with a substrate concentration of 50%. Under these reaction conditions, the plastein yield was 25.82%. Different plastein
products were prepared by adjusting reaction time and the ACE inhibitory activity of these products was determined. The
results showed that the ACE inhibitory activity of products after reaction for 6 h or more were improved and the highest
ACE inhibitory activity was observed when the reaction time was extended to 12 h with an IC50 of 0.17 mg/mL, compared
to 0.48 mg/mL for the origianal hydrolysate. The ACE inhibitory activity of vinegar egg hydrolysates could be improved
significantly by plastein reaction with proline for 12 h.

Over-expression of Lactate Dehydrogenase in L.casei and Fermentation Property of the Recombinant Strain
ZHU Long-bao,GE Fei,LI Wan-zhen,TAO Yu-gui*
2013, 34(9):  245-249.  doi:10.7506/spkx1002-6630-201309050
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Lactate dehydrogenase (LDH) is a key enzyme for lactic acid synthesis in L. casei. A LDH over- expressing
recombinant L.casei was constructed in this study to enhance the production of lactic acid. The lactate dehydrogenase gene ldh
of L. casei was first cloned into vector pMG36e. The recombinant vector was then transferred into L. casei. SDS-PAGE analysis
showed that LDH was successfully over expressed in the recombinant strains, which was about 37 kD. Enzyme activity analysis
showed that LDH activity of the recombinant strain increased by 3.1 times when compared to that of the wild type strain.
4 mmol/L activator FBP and 8 mg/L nicotinic acid were then added to fermentation medium to further improve lactic acid production
and analysis showed that accumulation of lactic acid was 128.2 g/L, and the lactic acid productivity was 3.04 g/(L•h), which were
increased by 35% and 53%, respectively, when compared to control. Addition of FBP and nicotinic acid significantly increased
the consumption rate of glucose and shortened fermentation time by 6 h.

Isolation, Purification and Some Characterization of Catalase from Garlic Seedlings
HU Rui-bin,SUN Fang,REN Mei-feng,TANG Yun-ming*
2013, 34(9):  250-255.  doi:10.7506/spkx1002-6630-201309051
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Electrophoresis-purity catalase from fresh garlic seedlings was obtained through homogenization, buffer solution
extraction, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration
chromatography. The specific activity of purified CAT was 25975.36 U/mg with a recovery of 40.05% and a purification
factor of 125.97. The molecular and subunit molecular weight of this enzyme was 234.93 kD and 59.16 kD, respectively.
It was relatively stable in the range of 25—45 ℃ and pH 5.0—10.0 Its optimum temperature and pH were 45 ℃ and 7.2,
respectively. Furthermore, its Km was 38.2 mmol/L under the optimum conditions. Its activity was inhibited by methanol,
ethanol, isopropanol, SDS and KSCN as well as some metal ions such as Ag+, Cu2+, Mn2+, Co2+, Cd2+, Zn2+, Ca2+, Mg2+ and
Ba2+, but was activated by Li+ and Pb2+ as well as low concentration of K+.

Optimization of Fermentation Conditions for AFB1-degrading Enzyme Production by Pseudomonas stutzeri F4
YANG Wen-hua,LIU Xiao-hua,LI Wen-ming,LI Hai-xing,CAO Yu-sheng*
2013, 34(9):  256-261.  doi:10.7506/spkx1002-6630-201309052
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Pseudomonas stutzeri F4 with high degradation activity of AFB1 was isolated and identified previously. In
this study, the fermentation conditions for AFB1-degrading enzyme (ADE) production by this strain were optimized.
The effects of inoculum age, inoculum size, initial pH, fermentation time and temperature on ADE activity were tested
by single factor tests. Based on the results of single factor tests, the fermentation conditions were then optimized
using response surface methodology. When inoculum size and initial pH were 3% and 6.5, the optimum fermentation
condition was inoculum age of 10.47 h, fermentation time of 42.52 h and fermentation temperature of 30.28 ℃. The
ADE activity in the fermented broth reached 2768.7 U under the optimized conditions, which was 28.99% higher than
before optimization.

Overexpression of Bacillus subtilis Keratinase Gene (kerC) in Pichia pastoris
HAN Xue-yi,TANG Zi-zhong,WANG Li-hua,CHEN Hui*
2013, 34(9):  262-266.  doi:10.7506/spkx1002-6630-201309053
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A stable and highly effective expression system was constructed for the expression of keratinase gene (kerC)
in Pichia pastoris. kerC gene was cloned from Bacillus subtilis B-3 by PCR, inserted into the expression vector pPICZαA and
transformed into Pichia pastoris X-33. The results showed that the kerC gene was over expressed successfully in the eukaryotic
expression system. The maximum keratinase yield of the recombinant strains reached 32.31 U/mL in the culture supernatant
grown for 108 h by 1% methanol induction, a 11.15-fold increase in comparison to that obtained with the prokaryotic expression
strain BL21-pET-kerC. The optimal temperature and pH of the recombinant keratinase were 60 ℃ and 7.5, respectively. It was
strongly stimulated by Co2+, Fe2+ and Ca2+ despite being completely inhibited by Fe3+. The recombinant keratinase with a
molecular mass of 31 kD was purified as determined by SDS-PAGE.

Changes in Molecular Subunit Composition and Allergenicity of Soybean Proteins during Enzymatic Hydrolysis
WANG Zhang-cun,LI Le-jing,YUAN Dao-qiang,CUI Sheng-wen,ZHAO Xue-wei,WANG Ji-zhong,HU Jin-qiang
2013, 34(9):  267-270.  doi:10.7506/spkx1002-6630-201309054
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This study was to investigate the effects of enzymatic hydrolysis by three enzymes (alcalase, papain and trypsin)
on molecular subunit composition and allergenicity of soybean meal proteins. The molecular subunits of soybean proteins
in soluble hydrolysate and insoluble residue were analyzed with SDS-PAGE. The contents of allergenic proteins in the two
hydrolysates were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the major α’, α,
β-subunits of β-conglycinin (7S) and the acidic polypeptide of glycinin (11S) in the two hydrolysates almost disappeared
after hydrolysis for 10 min by alcalase or papain. The content of allergenic proteins was reduced from 195.90 mg/g soybean
meal to 90.50 mg/g (alcalase) and 94.30 mg/g (papain) total hydrolysates, respectively, over half of allergenicity in soybean
meal being eliminated. After 120 min hydrolysis by alcalase and papain, the rate of eliminated allergenicity was 75.70% and
61.40% respectively. Nevertheless the effect of trypsin on the molecular subunits composition and allergenic proteins was
not evident compared to alcalase and papain. The results above indicated that enzymatic hydrolysis by alcalase or papain
could efficiently reduce the allergenicity of soybean meal.

Preparation and Purification of Anti-oxidant Peptides from Black Soybean and its Amino Acid Composition Analysis
LI Hua1,LIU En-qi1,2,TANG Shi-rong2,WU Yong-hua2
2013, 34(9):  271-276.  doi:10.7506/spkx1002-6630-201309055
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Black soybean peptides (BSP) were obtained by hydrolyzing black soybeans with three proteases alone or
in combination. Antioxidant BSP with oxygen radical absorption capacity (ORAC) were separated and purified through
ultrafiltration and macroporous resin adsorption followed by ethanol desorption. The amino acid composition of antioxidant
BSP was measured for analysis of the association of the antioxidant activity with properties of groups on amino acid side
chains. An ultrafiltaion fraction with relative molecular weight below 3000 shown to have strongly antioxidant activity
was obtaiend through the combined use of alcalase, flavourzyme and neutrase. DA201-C type of adsorption resin exhibited
improved separation efficiency than three others. The 75% ethanol eluate indicated higher average hydrophobicity and larger
amounts of basic amino acids and the strongest ORAC activity. Moreover there was an extremely significantly positive
correlation between the Trolox equivalent and GSH equivalent for ORAC (P < 0.01).

A Novel Sensitive Assay for Chitosanolytic Enzyme Activity and Its Comparison with Two Others
ZHANG Yong-qin,ZHANG Jie,CHANG Hai-yan,ZHANG Kun,LIU Zheng-dong,LUO Cai-hua,MOU Xiao-feng
2013, 34(9):  277-281.  doi:10.7506/spkx1002-6630-201309056
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A novel spectrophotometric method for the quantitative assay of chitosanolytic enzyme activity using 3-methyl-2-
benzothiazolinone (MBTH) was estalished. The proposed method was based on measuring the reducing ends released during the
enzymatic degradation of chitosan with the non-specific enzyme cellulase. The effects of several key hydrolysis parameters on
activity assay were discussed and investigated by multiple-point procedures. The results showed that the substrate concentration
was required to be no less than 2 mg/mL with an initial pH of 6.0, and the hydrolysis was allowed to proceed for no more than
60 min at 37 ℃ before end-point measurement. In comparison with the 3,5-dinitrosalicylic acid (DNS) method and the potassium
ferricyanide method, the MBTH method provided higher accuracy and sensitivity, wider detection range and simpler operation, and
showed a limit of detection of 13 mU/mL, which was 1.6 times smaller than that observed for the potassium ferricyanide method
and 116 times smaller than that observed for the DNS method. The Km for chitosan hydrolysis determined by the MBTH method
was 0.12 mg/mL, whereas accurrate Km measurement was difficult to achieve in the DNS method.

Effect of Administation of CGMP and Lactoferrin by Gavage on Colonization Resistance in the Intestine of Mice
REN Xiao-dong,CHEN Qing-sen*,LI Jun-jie,ZHOU Li-li,YAN Ya-li
2013, 34(9):  282-286.  doi:10.7506/spkx1002-6630-201309057
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This study explored the effect of casein glycomacropeptide (CGMP) and lactoferrin (Lf) on the logarithmic ratio of Bifidobacterium to Enterobacter (B/E) in the intestine of mice. BALB/c mice were randomly divided into eight groups: control (daily diet), placebo (0.2 mL of normal saline), low-, middle-, and high-dose CGMP treatments ( the same volume as normal saline at different concentrations), and low-, middle-, and high-dose Lf treatments (the same volume as normal saline at different concentrations). The administration period was 14 d. Fresh feces were collected from all mice before administration, on the 3rd, 7th, 11th and 15th days of administation and 6 d after the end of the administration period for enumeration of Bifidobacterium and Enterobacter after culture in selective media. Both CGMP and Lf at the middel dose stimulated the growth of Bifidobacterium but exerted a growth-inhibiting effect on Enterobacter, leading to an increase in B/E ratio, which was more obviously increased by CGMP. This study has demonstrated that both CGMP and Lf can increase the intestinal B/E ratio of mice and modulate the intestinal microflora.
Effect and Mechanism of Angiotensin-converting Enzyme 2 on Diabetic Renal Injury in Rats
WANG Shan-shan,MA Chang,ZHANG Wei,WANG Yan-xia,ZHANG Yuan-shu*
2013, 34(9):  287-291.  doi:10.7506/spkx1002-6630-201309058
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Objective: To compare the expression levels of ACE- AngⅡ-AT1/ACE2- Ang1-7-Mas and related regulatory
factors involved in the RAS (renin–angiotensin system) in order to explore the role and mechanism of ACE2 in the
occurrence and development of diabetes-induced renal in rats. Methods: Diabetes was induced in rats by intraperitoneal
injection of streptozotocin (STZ). The rats in the control group 1 and the model group 1 were guillotined 15 d later, and those
in the control group 2 and the model group 2 were guillotined 30 d later for measurement of the renal expression lelvels of
ACE, AT1, ACE2 and Mas mRNA and determination of the contents of AngⅠ and AngⅡ in local renal tissues. Results:
Compared with the control groups, the expression levels of ACE2 and Mas receptor mRNA were elevated in the model
group 1, ACE mRNA expression showed no significant difference, while AT1 mRNA expression and ACE/ACE2 mRNA
ratio decreased; renal ACE2 mRNA expression was lower in the model group 2 than in the control group 2, no obvisous
difference in Mas receptor mRNA expression existed, whereas the expression of ACE mRNA and AT1 receptor presented
an increasing trend and the ACE/ACE2 mRNA ratio of the modle group 2 was higher than that of the control group 2 with a
significant difference. The model group 1 showed a decrease in renin activity and AngⅠ content in local renal tissues with
a signficant difference (P < 0.05) but an increase in AngⅡ content with no significant difference (P > 0.05) as compared
to the control group 1. Compared with the control group 2, extremely significantly higher levels (P < 0.01) of renin activity
and AngⅠ content togeterh with a significant higher level (P < 0.05) of AngⅡ were observed in the model group 2.Conclusion: Abnormal expression of ACE and ACE2 is one of the reasons for the occurrence and development of diabetesinduced
renal injury in rats. The ACE2-Ang (1-7)-Mas axis activation is dominant at the initial stage of renal injury, which
is replaced by the ACE-AngⅡ-AT1 shaft activation at the serious stage. ACE2 plays a protective role by degrading AngⅠ
against the incidence of early renal disease.

Effect of 1,3-Dichloro-2-propanol on Cell Growth and Progesterone Biosynthesis of R2C Mouse Leydig Cells
BAI Shun1, SUN Jian-xia2,BAI Wei-bin3,*,ZOU Fei-yan1,ZHANG Qi-hao4,HUANG Ya-dong4
2013, 34(9):  292-295.  doi:10.7506/spkx1002-6630-201309059
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Objective: To explore the effect of 1,3-dichloro-2-propanol (1,3-DCP) on cell growth and progesterone biosynthesis
in R2C mouse leydig cells. Methods: R2C cells were exposed to 0.5, 1, 2, 4 mmol/L, and 6 mmol/L 1,3-DCP for 48 h, different
concentrations (IC25, IC50 and IC75) of 1,3-DCP were elected by MTT assay. R2C cells were stimulated by three concentrations
for 4 h or 24 h, respectively. The damage of DNA in R2C cells was investigated by single cell gel electrophoresis (SCGE).
Progesterone level in supernatant of cell culture medium was measured by radioimmunoassay (RIA). Results: 1,3-DCP could
inhibit cell growth, and IC25, IC50 and IC75 values were (1.161 ± 0.046), (1.897 ± 0.018) mmol/L and (3.100 ± 0.040) mmol/L.
In comparison with the control group, the damage of DNA were obviously higher in three concentrations of 1,3-DCP for 4 h,
and the progesterone level were also obviously lower in three concentrations of 1,3-DCP for 4 h or 24 h. Conclusion: 1,3-DCP
can directly affect cell growth and the progesterone synthesis of R2C cells.

Isolation and Purification of a Protein from Foxtail Millet Bran and Its Anti-proliferation Activities against Cancer Cells
SHAN Shu-hua1,2,WU Hai-li1,LI Zong-wei1,YUAN Lin-jie1,ZHAO Wen-bin1,LIU Qing-wu1,GUO Mao-lin1,LI Zhuo-yu1,*
2013, 34(9):  296-300.  doi:10.7506/spkx1002-6630-201309060
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FMB, a novel protein with cancer cell proliferation inhibitory activity was purified from foxtail millet bran by
using acetone precipitation, ammonium sulfate precipitation, Q-anion exchange column, SP-cation exchange column and
protein thermal denaturation. Its anti-proliferation activities against cancer cells were studied by MTT assay. The results
showed the FMB with varied concentrations (0.025, 0.05, 0.075 μg/μL and 0.1 μg/μL) could significantly inhibit the
proliferation of human colonic-cancer cell lines (DLD1) and cervical carcinoma cell line (HeLa) in dose-dependent manner.
However, under similar conditions, no significant anti-proliferation effect was observed on normal liver cell line (HL-7702).
Inverted microscope observations revealed that the DLD1 and Hela cells displayed classic morphological features of
apoptosis, e.g., obviously shrinking, floating and partial cell death, after being treated with FMB for 48 h. By contrast, the
cell line HL-7702 had no obvious changes under similar conditions.

Influence of Heat Treatment on the Osteoblast-promoting Activity of Bovine Lactoferrin
LIU Meng, DU Ming*, KONG Ying-ying,XU Wei-li,SONG Wei, ZHANG Lan-wei
2013, 34(9):  301-304.  doi:10.7506/spkx1002-6630-201309061
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Lactoferrin is a bioactive protein with multiple functions, which, however, are negatively impacted by heat
treatment. In the present work, osteoblasts of newborn SD rats were subjected to proliferation tests by MTT method to
compare the influence of different heat treatment conditions on the oteoblast-promoting activity of bovine lctoferrin. The
results of this study showed that when not subjected to heat treatment, lactoferrin had an evident proliferation-promoting
effect on rat osteoblasts in a dose-dependent manner in the range of 50 to 500 μg/mL. Moreover, lactoferrin had different
effects on the proliferation of osteoblast at different incubation time of 24, 48 h and 72 h. Compared with the native
lactoferrin, the treatments of 65 ℃/30 min and 72 ℃/10 s had only litte effects on the osteoblast-promoting activity of
lactoferrin. However, 85 ℃/10 min and 95 ℃/10 min inhibited its activity. The heat treament effect on the the osteoblastpromoting
activity of lactoferrin at higher concentrations was greater than at lower concentrations.

Antioxidant, Anti-fatigue and Immuoregulatory Functions of Red Deer (Cervus elaphus) Antler Protein Hydrolysate
HOU Xiao,GAO Jian,LI Chun-yu,WANG Zheng,JIN Li-li,WANG Qiu-yu*
2013, 34(9):  305-309.  doi:10.7506/spkx1002-6630-201309062
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The antioxidant, anti-fatigue and immuoregulatory effects of red deer (Cervus elaphus) antler protein and
hydrolysate prepared with both protamex and flavourzyme were investigated. For the determination of antioxidant effects,
pyrogallol auto-oxidation method, salicylate colorimetry and total reducing power assay were used. The in vivo antioxidant
potential of antler protein hydrolysate was analyzed by measuring SOD activity and MDA content in the serum of mice
receiving oral administration. Weight-loaded swimming test and blood lactic acid and liver glycogen assays were performed
to assess its anti-fatigue effect. Besides, the immunoregulatory effect of the hydrolysate in mice was examined by ConAinduced
mice spleen lymphocyte transformation and carbon clearance tests. Our results indicated that red deer antler protein
hydrolysate possessed scavenging activity against hydroxyl and superoxide anion radicals and improved total reducing
power compared to red deer antler protein. In addition, oral administration of this hydrolysate increased serum SOD activity,
reduced serum MDA content, obviously prolonged swimming time, decreased blood lactic acid and liver glycogen levels,
and remarkably enhanced the immune function of mice. From these observations, we concluded that red deer antler protein
hydrolysate has significantly stronger antioxidant, anti-fatigue and immuoregulatory effects than red deer antler protein.

Progress in Research on Peanut Allergens and Effect of Processing on Their Structure
HAN Yuan-long1,2,WU Zhi-hua1,*,LI Pu1,LI Xi-ying3,CHEN Hong-bing1
2013, 34(9):  310-315.  doi:10.7506/spkx1002-6630-201309063
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Peanut allergy has become a major public health concern worldwide and has been intensively researched recently.
Understanding the structure of peanut allergens can provide the basis for attenuating its allergy during processing. A variety
of approaches have been proposed to reduce or even eliminate the allergenicity of allergenic proteins in peanuts, especially
for Ara h 1, Ara h 2 and Ara h 6, and Ara h 3/4 as abundant, highly allergenic proteins that have attracted increasing concern.
This paper focuses on the recent progress in research on the structure of peanut allergens and the structural changes brought
about during processing. All of these efforts will provide more knowledge for producing peanuts products with low or no
allergenicity, and adding more choices for consumers with food allergy while effectively reducing food safety risks.

Recent Progress in Biochemical Properties and Functions of UDP-glycosyl Transferase during Plant Secondary Metabolism
JI Xiang-nan,HE Fei,DUAN Chang-qing,WANG Jun*
2013, 34(9):  316-323.  doi:10.7506/spkx1002-6630-201309064
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Uridine diphosphate glycosyltransferase (UGT) regulates the bioactivity, solubility and transportation of receptors
within the cells through modulating glycosyl transfer reaction. Glycosylation is a ubiquitous modification of plant secondary
metabolism that is involved in various functions, such as hormone homeostasis, the detoxification of exogenous toxins, and
the biosynthesis and storage of secondary compounds. Herein, we have summarized the recent advances in biochemical
properties and functions of these glycosyltransferases related to the glycosylation of secondary metabolites, phytohormones and
xenobiotics, in order to provide some references for related studies in this field.

Research Progress of Enzyme Preparation for Wheat Flour Gluten Improvement
ZHANG Chong,LU Zhao-xin*
2013, 34(9):  324-329.  doi:10.7506/spkx1002-6630-201309065
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Flour quality can be improved by improvers. Potassium bromate as a flour gluten strength improver has been
banned, and development of safe alternatives has become one of the hottest research topics in the field of food additives.
Since enzyme as one kind of protein, is harmless to human bodies after food processing, and it is a standard green food
additive that can substitute potassium bromate. This article has reviewed current research progress of enzyme preparation for
gluten improvement, including species, gluten strength mechanisms and the major problems during application.

Progress in Research on Intestinal Absorption Mechanism of Milk-derived ACE Inhibitory Peptides Using Caco-2 Cell Model
ZHU Qian1,GUO Yu-xing1,*,PAN Dao-dong1,2
2013, 34(9):  330-335.  doi:10.7506/spkx1002-6630-201309066
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The specific active peptides in milk can inhibit the activity of angiotensinⅠ-converting enzyme effectively
to execute antihypertensive effect. Due to different structures of milk-derived ACE inhibitory peptides, the peptides
are not able to pass through the intestinal mucosa into blood circulation when administered orally. Therefore, not
all ACE inhibitory peptides have the function of lowering blood pressure and a low oral bioavailability. This article
has introduced current research status and activity evaluation as well as the intestinal absorption mechanism of milkderived
ACE inhibitory peptides, which includes intestinal ingestion, metabolic activity and intestinal excretion using
Caco-2 cell model. In addition, a prospect of improving oral bioavailability of milk-derived ACE inhibitory peptides
has also been proposed.

Progress in Research on Myofibril-bound Serine Proteinase from Fish
DU Cui-hong,CAO Min-jie*
2013, 34(9):  336-339.  doi:10.7506/spkx1002-6630-201309067
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This paper reviews the recent progress in the purification, characterization, molecular cloning and recombinant
expression of myofibril-bound serine proteinases (MBSP) from different species of fish. As a weakly alkaline proteinase,
MBSP is extensively found in fish skeletal muscles. The degradation of myofibrillar proteins by MBSP is mainly resposible
for the modori phenomenon during fish surimi production. From this reviwe, we conclude that fish-derived MBSP hold great
promise as a novel tool in mass spectrometric identification of proteins due to their excellent stablity to heat and substrate
specificity to decompose the carboxyl chains of arginine or lysine residues.

Research Progress of Deamidation-induced Modification of Proteins and Peptides
LIAO Lan1,ZHAO Mou-ming2,*,WANG Shao-yun1,LI Qing-jin1
2013, 34(9):  340-345.  doi:10.7506/spkx1002-6630-201309068
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Many proteins have high levels of amide-containing amino acids such as glutamine and asparagine. These side
chains are susceptible to deamidation reaction, thus resulting in the release of ammonia, transformation to acidic groups
and partial unfolding of proteins or peptides. This conversion of the amides in acidic groups has important implication
on food and human proteins and peptides. Deamidation of food proteins offers an effective way for producing functional
food proteins, whereas deamidated human proteins or peptides undergo turnover, degradation and aging. In this review,
extensive information regarding deamidation of proteins and peptides from foods and human as well as the underlying
mechanisms has been described, which will supply useful information for the application of deamidation in food
processing and biomedical science.

Research Advance in Aqueous Enzymatic Extraction of Soybean Oil
JIANG Lian-zhou,LI Yang,WANG Yan,WANG Huan
2013, 34(9):  346-350.  doi:10.7506/spkx1002-6630-201309069
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The technological principle, basic process and characteristics of aqueous enzymatic extraction (AEE) are
introduced in this review. This paper focuses on the main factors affecting AEE extraction of soybean oil and comparison
of quality characteristics of soybean olis extracted by AEE and other approaches. Morover, we also discuss its future
application prospects.

Recent advances in high-level heterologous expression of microbial enzymes
YANG Hai-quan,LIU Long,LI Jiang-hua,DU Guo-cheng,CHEN Jian
2013, 34(9):  351-357.  doi:10.7506/spkx1002-6630-201309070
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Enzyme is a kind of biological catalyst with high specificity and has been widely used in chemical industry, agricultural industry, biopharmaceutical industry, food industry, textile industry, feed industry and other industries. The commonly used industrial enzyme expression hosts include Escherichia coli, Bacillus, filamentous fungi, yeast, and lactic acid bacteria. Different expression hosts have different characteristics. For example, the bacterium hosts can quickly and easily over-express recombinant enzymes, but the bacterium systems cannot express very large eukaryotic proteins because of proteins requiring post-translational modifications. In recent years, the great progresses have been made in the high-level expression of industrial enzymes in different hosts. In this work, we systematically summarize the recent advances in the heterologous expression of industrial enzymes, and the development trends of heterologous expression of industrial enzymes are also prospected.
Recent Advance in Research and Application of Several Glycosyltransferases in the Synthesis of Functional Oligosaccharides
LI Yu,LU Fu-ping,WANG Zheng-xiang
2013, 34(9):  358-363.  doi:10.7506/spkx1002-6630-201309071
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Isomaltooligosacharide, fructooligosaccharides, fucosylated oligosaccharide and sialyloligosaccharide have
been widely applied in milk powder for infants and young children because of its special physiological effect to promote the
growth of bifidobacterium. These functional oligosaccharides are produced by α-glycosyltransferase, fructosyltransferase,
fucosyltransferase and sialyltransferase, using glucose, sucrose and lactose as substrates, respectively. This paper
reviews recent research advances in the microbial souces, properties, activity and expression of these enzymes and their
applications in the synthesis of functional oligosaccharides in order to provide refereces for the development of highly active
glycosyltransferases and the improvement of their applicability for the production of functional oligosaccharides.

Recent Advance in Research on the Structure and Function of Trypsin Inhibitor
WANG Rong-chun1,SUN Jian-hua2,HE Shu-dong1,MA Ying1,*
2013, 34(9):  364-368.  doi:10.7506/spkx1002-6630-201309072
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The physiological function of trypsin inhibitor, a member of the family of serine protease inhibitor, is associated
with preventing or combating diseases and insect pests. In recent years, trypsin inhibitor has attracted wide attention from
researchers because of its unique physiological properties such as anti-cancer and anti-virus activity. In this paper, we review
the classification, typical structure and functions of trypsin inhibitor. Besides, the application potential of some current
methods to extract and purify trypsin inhibitor and problems exisitng in their application are introduced, aiming at providing
references for further expolration of trypsin inhibitor.

A Review on the Glycosylation of Food Proteins by Maillard Reaction or Transglutaminase-catalyzed Reaction
SONG Chun-li1,2,ZHAO Xin-huai2,*
2013, 34(9):  369-374.  doi:10.7506/spkx1002-6630-201309073
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The present paper reviews the two different approaches for the glycosylation of food proteins, i.e., Maillard
reaction as the most widely used one and transglutaminase-catalyzed reaction as a recently developed one. The chemical
mechanisms involved in the glycosylation of food proteins based on Maillard reaction as well as the structure and
important function properties of reaction products are described in this review. Meanwhile, we introduce the advantage of
transglutaminase in catalyzing protein glycosylation as well as its influence on some funtional properties of proteins and
future development potential.

Bioactive peptides: their absorption, utilization and functionality
PANG Guang-chang,CHEN Qing-sen,HU Zhi-he,XIE Jun-bo
2013, 34(9):  375-391.  doi:10.7506/spkx1002-6630-201309074
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Dietary proteins and their enzymolytic products exert a wide range of nutritional, functional and biological
activities for human health. A large number of data showed that protein hydrolytic products are absorbed and transited more
effectively than intact protein or free amino acids alone, supporting the notion that digestion is important for its uptake
and circulation even if the amino acid compositions are exactly balanced. Furthermore, when the protein was administered
orally as a hydrolysate instead of in an intact form or free amino acid, the healthy functions can be observed, suggesting
that bioactive peptides can be liberated by proteolytic enzymes. However, the body is a self-regulatory, self-renew, and selfstabilization
system, so the immune defense system never allows any dissident biomacromolecules, especially diet proteins
and peptides to be taken in, for that maybe some invaders. Even if a small amount of intact proteins are occasionally taken
up, which is due to the molecule structure recognition of substances around by sampling from intestinal tract. The major
functions are to establish as the adaptive immunity defense system for specific antigens. After absorbed, these peptides will
be rapidly hydrolyzed and processed by antigen presenting cells (APC) to present the antigenic determinants to Th cells.
The membranous microfold (M) cells can bring intact protein to B cells as antigen epitopes for antibody class switching and
secreting. Base on these investigations, diet proteins and corresponding enzymolytic peptides mainly offer their functions by
the interaction with gastrointestinal mucosal systems and intestinal microorganisms. On the other hand, the accumulated data
have demonstrated that transport of di- and tripeptides is a faster and effective route of uptake per unit of time than their free
AA or intact protein by intestinal peptide transporter, PepT1. Furthermore, the di- and tripeptides are more efficiently than
free AA or intact protein with the same respective AA compositions, and these peptides may be transported by circulation to
different tissues or organs and used for different purpose more efficiently. This review will also provide a theoretical basis
for the research and exploitation of bioactive peptides for special foods.

Application of Enzymes in Wine Production
Yanlin Liu
2013, 34(9):  392-398.  doi:10.7506/spkx1002-6630-201309075
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As efficient biocatalysts, enzymes are widely used in the winemaking process, including juice clarification,
extraction, filtration, urea removal and so on. The application of enzymes can not only increase the stability of wine
physicochemical properties, but also improve the aroma and color of wine. International Vine and Wine Organization (OIV)
provides four enzymes which can be used in the winemaking process from four kinds of sources: pectinase from Aspergillus
niger; β-glucanase from Trichoderma; urease from Lactobacillus fermentum and lysozyme from egg white. This article has
briefly described basic characteristics of pectinase, glucosidase, glucanase, lysozyme and urea enzymes, as well as their
application wine production and impact on wine quality.

Research Progress on the Antimicrobial Peptides Derived from Egg Protein
WANG Jun-jie1,2,ZHAO Yan1,2,*,TU Yong-gang3,LUO Xu-ying1,2,LI Jian-ke1,2,YANG You-xian1,2,DENG Wen-hui1,2
2013, 34(9):  399-403.  doi:10.7506/spkx1002-6630-201309076
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Antibacterial peptides are one of the research focuses in biological field, which has several features, such as small
molecular weight, high thermal stability and wide antimicrobial spectrum. Naturally, eggs with high content of proteins have
become an important source for preparation of antimicrobial peptides. Here we reviewed the antimicrobial peptides derived
from egg proteins, its antimicrobial activity and the relationship between peptide structure and antimicrobial activity. At the
end, we summarized the problems existing in the study of antibacterial peptides from egg proteins and discussed directions
for further study and their potential application in practice.

Research Progress of Metagenomic Technology and Its Application in Enzyme Preparation
YU Lei,YU Li,ZHANG Wei,LU Li-li
2013, 34(9):  404-407.  doi:10.7506/spkx1002-6630-201309077
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More than 99% of microbes in natural environment are uncultivable. These uncultivable microorganisms have been
exploited for the first time by using metagenomic technology, especially as a more powerful technique for the exploration and
mining of novel enzymes and active small molecules. In this paper, the current research advances of metagenomic technology
and successful applications in the development of new enzymes have been summarized, which will accelerate the development
of new enzymes through metagenomic technology and provide an effective strategy for its application in the food industry.