Inhibition of Thermal Degradation of Mackerel Surimi by Pig Plasma Protein and L-kininogen

ABSTRACT: Use of purified L-kininogen to prevent actomyosin (AM) degradation by cathepsins, and pig plasma proteins to reduce mackerel modori were investigated. Myosin heavy chain (MHC) degradation in AM containing various cathepsins was significantly inhibited by purified L-kininogen. The texture of mackerel gel increased to 2-fold and 1.7-fold in breaking force (g) and deformation (cm) separately after adding 1% pig plasma protein. SDSPAGE showed that degradation of MHC in mackerel surimi was significantly reduced after 1% pig plasma protein addition at several temperatures. It suggested that the pig plasma protein containing L-kininogen could effectively reduce the modori phenomenon of mackerel surimi during processing.     

Pig plasma protein: potential use as proteinase inhibitor for surimi manufacture; inhibitory activity and the active components

The inhibitory activities of pig plasma protein (PPP) against proteinases and autolysis were studied. Using casein as a substrate, it was shown that PPP exerted inhibitory activity against Pacific whiting (PW) proteinase, papain and trypsin. At levels of 10 and 20 mg ml⁻¹, PPP showed higher inhibitory activity than beef plasma protein (BPP) or egg white against PW proteinase (P < 0.05). The inhibitory activity was proportional to the concentration of PPP used up to 10 mg ml⁻¹. PPP and BPP showed marked inhibition of autolysis of PW surimi at concentrations of 10–30 mg g⁻¹. Egg white showed lower inhibition than PPP or BPP against autolysis of surimi (P < 0.05). Myosin heavy chain (MHC) in Pacific whiting surimi was better retained when higher concentrations of PPP were used, while no changes in actin were observed. Inhibitory activity staining on sodium dodecyl sulphate (SDS) substrate gels incubated with papain or trypsin indicated that residual protein bands with apparent molecular weights of 60 000–63 000 may be the active inhibitory components in PPP. These bands were found in PPP and bovine plasma fraction IV‐1, although only under non‐reducing conditions. © 2000 Society of Chemical Industry     

猪肝超氧化物歧化酶的分离纯化与初步表征

研究猪肝SOD的分离提取、纯化以及初步表征。以猪肝为原料,通过抽提液加热、丙酮沉淀和Sephadex G-200层析柱色谱提取、纯化Cu,Zn-SOD;在单因素试验的基础上,采用L(934)设计对提取工艺进行系统优化。结果表明:经过热变性,可以除去大部分杂蛋白,提高了SOD的比活力;最佳的提取条件是:在1.4%的盐浓度下,于70℃水浴中热变性10 min,然后加1.6倍体积的丙酮获得SOD粗沉淀;溶解后的粗酶液经Sephadex G-200层析柱纯化后,经SDS-PAGE电泳检测为单一条带,HPLC检测显示为单一对称峰,均表明最终得到了高纯度的SOD;紫外-可见吸收光谱和ICP-MS分析结果均表明得到的是含铜、锌的SOD,即Cu,Zn-SOD;以邻苯三酚为底物,该酶的Km值为1.574,Vmax为0.268。     

鹅血中转铁蛋白质的分离纯化及其性质

以鹅血为原料,利用醋酸纤维膜电泳,分析鹅血中各球蛋白的质量浓度.采用DEAE 52离子交换和SephdexG 100凝胶层析等方法,对转铁蛋白(Transferrin,Tf)进行了分离纯化及其性质的研究.结果表明,鹅血浆中含有较多的β球蛋白,质量浓度为34.89mg/dL,每毫升血浆可纯化得到3.1mg转铁蛋白.鹅血含铁转铁蛋白(holo Tf)的特征吸收峰为470nm,相对分子质量为77000.鹅转铁蛋白对选用的革兰氏阴性菌 大肠杆菌有抑制作用,Fe3+能够改变转铁蛋白的结构,并影响转铁蛋白的抑菌功能.     

从猪血浆蛋白质中制备无色蛋白胶

使用凝聚剂从猪血浆蛋白质中获得无色蛋白胶。通过用随机质心最优化方法找到分离最优条件为:pH3.7～3.9,№Cl浓度0.24～0.26mol/l,凝聚剂浓度1.1～1.3％,在最优化条件下制备的无色蛋白胶中蛋白质浓度几乎是猪血浆蛋白质浓度的三倍。测定了无色蛋白胶的功能特性并应用于仿真鱼圆和酸乳。     

猪肝乙醇脱氢酶的分离纯化及部分性质

新鲜猪肝经匀浆、缓冲液抽提、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析及Superdex-200凝胶过滤层析,获得电泳纯的乙醇脱氢酶（alcohol dehydrogenase,ADH）。纯化结果显示：该酶比活力为1 622.33 U/mg,回收率为29.05%,纯化倍数为34.58;该酶分子质量约为171.79 kD,亚基分子质量约为43.68 kD。ADH酶学性质研究显示：最适反应温度和pH值分别为45 ℃和10.0;在25～45 ℃及pH 7.5～9.0范围内稳定性较好;最适条件下测得该酶对乙醇的Km值为19 mmol/L;正丁醇、氯仿、异丙醇、十二烷基硫酸钠、草酸、Zn2＋、Cu2＋、Ag＋对该酶的抑制作用最强,Mg2＋对该酶有激活作用,EDTA对该酶有双重作用。     

猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质分析

研究脂肪氧合酶（lipoxygenase,LOX）在猪肉贮藏、加工过程中对脂肪氧化及风味形成的作用机制。通过序列分析和聚合酶链式反应扩增获得了猪肉12-脂肪氧合酶催化结构域（12-lipoxygenase catalytic domain,12-LOXcd）的编码基因,采用大肠杆菌表达系统,经镍柱亲和层析和Superdex G200凝胶过滤层析纯化得到12-LOXcd蛋白,并研究其酶学性质。结果表明,构建的原核表达载体pMBP-12-LOXcd在大肠杆菌中成功可溶性表达了猪肉12-LOXcd,该重组蛋白经纯化可达电泳纯。12-LOXcd以亚油酸为底物的比活力为2 826.7 U/mg,最适pH值为6.0,最适作用温度为30 ℃。亚油酸Km为0.40 mmol/L,亚麻酸Km为0.55 mmol/L,花生四烯酸Km为4.15 mmol/L,表明最适底物为亚油酸。与大豆LOX相比,该酶在较高NaCl质量分数（9%）时仍保持活性稳定;对热较不稳定,在60 ℃条件下失活,但优于大豆LOX的热稳定性;此外,12-LOXcd的pH值稳定性也优于大豆LOX,在碱性条件下仍能保留部分活力。     

Purification and Characterization of Alkaline Proteinase from Atlantic Menhaden Muscle

Two alkaline proteinases (A and B) were isolated and found to be composed of homogeneous subunits. These proteinases, A and B, were concentrated 62.9-and 986.5-fold compared to the crude muscle extract, with molecular weights of 707,000 and 450,000, respectively. Both are probably serine type proteinases, and optimum caseinolytic activity was shown at pH 8.0 and 55 °C. Both degraded actomyosin under similar conditions. Enzyme A had higher thermal stability than B. The residual activities of A and B in 3.6% NaCl solution were 95% and 85%. These data suggest that these proteinases are involved in the softening of menhaden surimi gels which occurs during heating at 50 to 70 °C.     

Purification and Characterization of Proteinase from Atlantic Menhaden Muscle

Two proteinases (A and B) were isolated from Atlantic menhaden muscle with molecular weights of 112,000 and 90,500 daltons, respectively. Proteinase B had higher activity than A for protein substrates except casein; proteinase B had no caseinolytic activity. Both proteinases hydrolyzed synthetic substrates such as Z-Phe-Arg-NMecand TAME, but not BAEE and BAPNA. Optimum Z-Phe-Arg-NMec hydrolyzing activity was shown at pH 7.4, 40 to 50 °C for both proteinases A and B. Activities of A and B in the presence of 3.0% NaCl were reduced to 71.2% and 62.2%, respectively. Both proteinases were inhibited by 1 mM TLCK, 1 mM benzamidine, 1% egg white, and 1% bovine plasma hydrolysate. Proteinases A and B are most likely tryptic serine type proteinases.     

猪肝中提取纯化动物酯酶的研究

本实验探索了一种从猪肝中提取纯化动物酯酶的有效方法,从而为农药残留的动物酯酶检测提供了一种廉价酶源。将所制动物酯酶液用于检测毒死蜱的结果表明,其检测效果良好,所制动物酯酶具有实用价值。     

鲢鱼卵高分子质量CPI-I 的纯化与鉴定

以鲢鱼卵为材料制备半胱氨酸蛋白酶抑制因子(CPIs)的粗提浓缩液,进而经Q Sepharose Fast Flow阴离子层析和Sephacryl S-200分子筛层析,获得部分纯化了的CPI-I。以偶氮酪蛋白(azocasein)法监测粗提液及层析纯化中CPIs的抑制活性。层析过程中,以灵敏度更高的荧光合成肽底物(Z-Phe-Arg-MCA)法监测洗脱物的CPIs抑制活性的热稳定性。最终鲢鱼卵CPI-I被纯化了72倍,酶回收率为10.25%。利用Sephacryl S-200分子筛层析Marker标准曲线以及反相酶谱法,初步判断CPI-I的分子质量为89kD。高碘酸-Schiff试剂(PAS)染色法证明该蛋白为糖蛋白。CPI-I能够抑制鲢鱼组织蛋白酶L,且具有显著的热稳定性。     

Preparation and Characterization of Hydrolyzed Proteins from Defibrinated Bovine Plasma

ABSTRACT: Proteins from defibrinated bovine blood plasma were enzymatically hydrolyzed with food-grade microbial proteases Alcalase 2.4 L® and Flavourzyme L&TM;, and a substrate consisting of small peptides and free amino acids was obtained. Hydrolysis of the plasma proteins with Flavourzyme resulted in a maximum degree of hydrolysis of 43% at an enzyme concentration of 110 LAPU/g protein after 15.5 h of hydrolysis. Among the free amino acids in the hydrolysate, hydrophobic amino acids were predominant. The major plasma proteins were degraded due to hydrolysis; peptides of less than 1.04 kDa were dominant in the product when a high degree of hydrolysis was employed.     

Isolation of an Angiotensin Converting Enzyme Inhibitory Peptide from Irradiated Bovine Blood Plasma Protein Hydrolysates

ABSTRACT: An angiotensin converting enzyme (ACE) inhibitory peptide was isolated and purified from the hydrolysates of the irradiated bovine blood plasma protein. Blood plasma protein was irradiated at 10 kGy to eliminate microbial contamination and was enzymatically hydrolyzed using several commercial proteases: Alcalase, Esperase, and Flavourzyne. An ACE inhibitory peptide was isolated using membrane filtration, gel permeation chromatography, normal phase and reverse phase high-performance liquid chromatography. The purified ACE inhibitory peptide was identified to be a tripeptide, His-Pro-Tyr, having IC₅₀ value of 1.68 μM.     

嗜酸乳杆菌细胞壁蛋白酶的分离纯化及酶学性质的研究

为优化CEP的分离纯化方法,采用聚乙二醇(PEG)-20000浓缩粗酶液,用超滤离心管过滤酶液,40%~60%硫酸铵盐析分级沉淀,Sephacryl-S-300HR纯化,并通过Native-PAGE切胶回收得到纯酶。所得CEP的分子质量为28.3ku,比酶活力62.066U/mg,最适温度32℃,最适pH6.5,耐热性比较强,耐酸性明显好于耐碱性。Co2+、Mg2+、Ca2+对CEP有激活作用;Ba2+、Mn2+、K+、Na+对CEP的活力影响不大;Zn2+、Ni2+则表现出较强的抑制CEP活性作用;EDTA对CEP活性有抑制作用,表明CEP是一种金属离子激活酶;PMSF对CEP有显著抑制作用,表明CEP是一种丝氨酸蛋白酶。用嗜酸乳杆菌CEP水解乳清蛋白,其酶解液对ACE的抑制率为56.31%,表明CEP水解乳清蛋白可以产生ACE抑制肽。     

低分子肝素分析及结构表征方法的研究进展

低分子肝素是一类应用广泛的抗凝血药物,由于其强极性、微不均一性以及硫酸基团的不稳定性使得分析及表征其结构的工作有困难。本文综述了低分子肝素分析及结构表征方法,重点阐述了近年低分子肝素在电泳、色谱、质谱和核磁分析领域的研究进展,并展望了低分子肝素分析的发展趋势。     

Porcine Plasma Proteins as a Surimi Protease Inhibitor: Effects on Actomyosin Gelation

Effect of porcine plasma proteins (PPP) on thermal gelation of actomyosin in the presence and absence of fish proteinase was studied using a dynamic rheological test. Substantial decreases in development rate and magnitude of gel modulus were observed by the addition of proteinase to actomyosin gels. PPP was effective in protecting a myosin-heavy chain from proteolytic degradation, however, PPP itself interfered with the formation of actomyosin gel. Lower gel modulus was observed with actomyosin gels developed with higher concentrations of PPP added. Overall, PPP reversed the loss of gel modulus by the proteinase, however, the recovered gel modulus was only as high as those containing PPP only. These results implicated that, although PPP may revert autolytic activity in surimi, it interfaces with actomyosin gelation.     

低温射频等离子体降解乙腈中黄曲霉毒素B1的效果与途径分析

采用低温射频等离子体处理溶解于乙腈中的黄曲霉毒素B1,考察了时间、等离子体发射功率、黄曲霉毒素B1初始浓度对黄曲霉毒素B1降解率的影响,通过HPLC-MS/MS分析降解产物及其可能的降解途径。结果表明,随着等离子体功率的增大、黄曲霉毒素B1初始浓度的降低,其降解率有增大的趋势。200~500μg/L黄曲霉毒素B1经120 W等离子体处理150 s以上,黄曲霉毒素B1的降解率可达95%以上。经过对比等离子体处理不同时间后的黄曲霉毒素B1的一级质谱,推测准分子离子峰157、299、339为可能的降解产物峰。结合产物峰的二级质谱碎片信息和黄曲霉毒素B1分子结构中的活性位点,推断出降解产物的结构和降解路径。     

鲢鱼卵中两种高分子半胱氨酸蛋白酶抑制因子的纯化与鉴定

以鲢鱼卵为材料,通过匀浆、酸处理和超滤制备半胱氨酸蛋白酶抑制因子（cystine proteinase inhibitors,CPIs）粗提液,进而经Sephacryl S-100分子筛层析、Blue Sepharose 6 Fast Flow染料亲和层析、SP-SepharoseFast Flow阳离子交换层析、ConA Sepharose 4B亲和层析,获得两种纯化的高分子CPIs,即ConA不吸附部分a-1和吸附部分的糖蛋白a-2。二者分别被纯化了102.62 倍和274.28 倍,酶活回收率分别为2.02%和1.42%。通过TSK G2000 SWXL凝胶过滤高效液相色谱结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及其反相酶谱法分析,表明a-2及再次经高效液相色谱法回收的a-1在电泳图上均呈单一带,a-2为单一峰,且a-1的分子质量为139 ku,a-2的分子质量为92 ku。二者均能抑制半胱氨酸蛋白酶（木瓜蛋白酶和鲢鱼组织蛋白酶L）但不抑制丝氨酸蛋白酶（胰蛋白酶和胰凝乳蛋白酶）。根据a-1和a-2的分子质量及抑制活性特征和糖蛋白特性,推测二者可能为鲢鱼卵Kininogens的不同形式。     

干酪乳杆菌6033蛋白酶降解肌肉蛋白质的作用研究

本试验将干酪乳杆菌(Lactobacillus casei，Lc)6033蛋白酶液按5％的比例分别加入肌浆蛋白和肌原纤维蛋白提取液，然后设定不同的pH值，于15℃培养7d，定时取样，测定反应后的pH值、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白质的变化。结果发现：不同设定的肌浆蛋白和肌原纤维蛋白试验组在反应初期pH值均稍稍降低，随着反应进程，各试验组的pH值呈现出逐步升高的趋势；其中，肌浆蛋白以pH5．0、4．5组的变化较为明显，肌原纤维蛋白则以pH6．0、5．0、4．5组的变化较为明显。经7d振荡培养后，电泳检测发现，各试验组肌浆蛋白均表现出分解现象，尤其以pH5．0组和pH4．5组更为明显；而肌原纤维蛋白也都表现出了明显的分解现象，尤以pH5．0组肌原纤维蛋白分解最明显。     

香蕉皮多糖提取分离纯化及分子质量测定

以废弃香蕉皮为原料,采用L9(34)正交试验的方法对提取香蕉皮多糖的条件进行优化筛选,进一步用DEAE-52色谱柱纯化,得多糖组分bpp1、bpp2和bpp3,并利用HPLC分析bpp1分子质量,结果表明：香蕉皮多糖提取最佳工艺条件为温度85℃、浸提时间5h、pH8,提取率为2.75%;bpp1分子质量为442068u。