驼乳与牛乳乳清蛋白酶解工艺优化及其产物抗氧化能力分析

为了研究不同蛋白水解酶对驼乳和牛乳抗氧化能力的影响,向驼乳和牛乳乳清蛋白中添加不同蛋白水解酶,探究乳清蛋白抗氧化活性肽的最佳制备条件,并对其抗氧化能力进行比较分析。首先从3种蛋白酶中筛选出最佳用酶,在此基础上以1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-Trinitrophenylhydrazine,DPPH)自由基的清除率为响应值,进行单因素和响应面试验,同时研究了驼乳和牛乳乳清蛋白抗氧化肽对DPPH自由基、羟自由基、超氧阴离子的清除效果。结果表明,木瓜蛋白酶水解物的能力最强,水解度可达15%。驼乳乳清蛋白最佳酶解工艺为酶解pH6.4,酶解温度55 ℃,底物浓度2.73%,DPPH自由基清除率可达71.9%。牛乳乳清蛋白最佳酶解工艺为酶解pH6,酶解温度54 ℃,底物浓度4%,DPPH自由基清除率达69.9%。在最佳酶解条件下,驼乳乳清蛋白酶解液的·OH清除率为58.2%,O₂-·清除率为67.2%;牛乳乳清蛋白酶解液·OH清除率为52.2%,·O₂-清除率为60.7%。驼乳乳清蛋白酶解液的抗氧化性在不同程度上均高于牛乳乳清蛋白酶解液,驼乳和牛乳乳清酶解液的DPPH自由基清除能力较强,其次是O₂-·清除能力,·OH清除能力最弱。     

鲫鱼鱼鳞抗菌多肽的制备纯化及其抑菌活性

以鲫鱼鱼鳞为原料,对其预处理后采用柠檬酸浸提酶解等工艺得到鱼鳞抗菌多肽粗酶解液,经透析后,再依次经Sephadex G-15、Sephadex G-50凝胶过滤层析和纤维素DEAE-52阴离子交换层析对其进行分离纯化,最后得到具有较强抑菌活性的鲫鱼鱼鳞抗菌多肽,并对其进行分子质量分析以及对多种细菌、霉菌的抑菌活性和最小抑菌浓度（minimal inhibitory concentration,MIC）测定。结果表明：经分离纯化后得到的具有抑菌活性的蛋白组分AcIII,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示为单一条带,表明其已达到电泳级纯,其分子质量约为20.1 kDa,抑菌活性测定结果表明鲫鱼鱼鳞抗菌多肽具有很好的广谱抗菌活性,对金黄色葡萄球菌、枯草芽孢杆菌、白色葡萄球菌、副溶血性弧菌、假单胞菌和希瓦氏菌的MIC为16 μg/mL,对大肠杆菌和沙门氏菌的MIC为32 μg/mL。     

牛骨胶原蛋白源抑菌肽的分离纯化及成分分析

牛骨胶原蛋白经中性蛋白酶水解,采用超滤技术和凝胶层析技术对酶解液进行分离纯化并测定对大肠杆菌的抑菌活性;借助反向高效液相色谱和基质辅助激光解析/离子化串联飞行时间质谱仪对抑菌肽进行分析.结果表明:经超滤分离后,分子质量小于10kD的组分对大肠杆菌的抑菌活性强;Sephadex G-25柱分离得到的峰Ⅰ和峰Ⅳ组分有较强的抑菌活性.经反向高效液相色谱和基质辅助激光解析/离子化串联飞行时间质谱仪分析,峰Ⅰ组分中含有的多肽种类多,分子质量集中在850～1550D之间.峰Ⅳ组分含有的多肽种类较少,分子质量集中在700～900D之间.     

芫荽抑菌成分的提取及其抑菌性能的研究

分别用石油醚、95%乙醇、水浸提芫荽,获得浸提液。抑菌实验表明,水提液对不同细菌均有明显的抑制作用,其最低抑菌浓度都为10%原液。采用沉淀蛋白、分离多糖、超滤、纳滤、离子交换等方法分离水提液组分,证明芫荽中主要抑菌成分是相对分子质量在500～3000的水溶性非肽类物质。     

驼乳生物活性肽的研究进展

驼乳被誉为"沙漠白金",具有独特的营养价值和功能特性。通过分析驼乳蛋白质组分发现驼乳中保护性蛋白质的含量明显高于牛乳。驼乳蛋白质不仅含有氨基酸等营养成分,还含有许多具有生理功能的活性肽。驼乳蛋白质中加密的氨基酸序列通过特异性蛋白酶的水解作用或者菌株发酵,释放具有抗微生物,抗氧化,抗高血压,抗炎和免疫调节等功能的生物活性肽,从而调节机体的生理功能。本文综述了驼乳蛋白质组分及其通过特异性蛋白酶水解或菌株发酵释放的抑菌肽、抗氧化肽和ACE抑制肽,为进一步研究与开发驼乳提供新的参考依据。     

核桃谷蛋白抗菌肽的分离纯化及抑菌稳定性分析

目的：以核桃粕谷蛋白为原料生产抗菌肽,研发一种新型抗生素的替代品。方法：通过菌酶协同固态发酵法（枯草芽孢杆菌和碱性蛋白酶协同）从核桃粕谷蛋白中制备抗菌肽,通过超滤、凝胶层析过滤、液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry,LC-MS/MS）和分子对接对核桃粕谷蛋白抗菌肽（walnut glutelin antimicrobialpeptide,WGP）进行分离、纯化和筛选。以对大肠杆菌和金黄色葡萄球菌的抑菌性保持率为指标,研究抗菌肽的抑菌稳定性。结果：经超滤、凝胶层析分离后,WGP-ⅢA和WGP-ⅢC组分均具有较强抗菌活性;LC-MS/MS结合虚拟筛选,从2种组分中共鉴定得到6条多肽,分子对接筛选得到3个肽段,分别为WGP-ⅢA组分的LAEAYNIPDTIARRL和WGP-ⅢC组分的SHSVIYVIR、APQLLYIVK;分子对接结果显示,WGP-ⅢC组分的抑菌活性更强;抑菌稳定性分析表明,核桃谷蛋白抗菌肽在高温热处理、紫外线、不同pH值下和不同蛋白酶处理后均表现出较好的稳定性。结论：核桃粕谷蛋白抗菌肽在食品抑菌防腐...     

猪骨免疫活性肽的分离纯化

为了从猪骨中分离纯化出免疫活性肽,使用超滤、sephadex G-25凝胶层析、SP-sephadex C-25离子交换层析等手段对鲜猪骨的Alcalase碱性蛋白酶的酶解液进行分离纯化,采用MTT法测定各分离产物对小鼠脾淋巴细胞的增殖活性。结果显示:猪骨酶解液经超滤分离获得的分子质量小于2 ku的组分对小鼠脾淋巴细胞增殖率最高;对分子质量小于2 ku的组分采用凝胶过滤层析,对层析后活性最高的组分再进行离子交换层析,最终得到的组分质量浓度为100μg/m L时,对小鼠脾淋巴细胞的增殖率为114.30%。     

青稞蛋白降血糖肽的纯化、 结构鉴定及体外降血糖和抗氧化活性

原料取青稞蛋白，通过单因素和响应面试验，以α-葡萄糖苷酶抑制活性为主要测定指标，筛选最适蛋白酶及最佳工艺条件。采用超滤和Sephadex G-15凝胶层析技术对水解肽进行分离纯化。通过液质联用获得具有较高降血糖活性的肽序列并对其氨基酸序列进行分析。结果表明：水解最适蛋白酶为胰蛋白酶，最佳水解条件参数为加酶量为12 000 U/g、底物质量浓度为3 g/100mL、酶解时间为5 h。该条件下水解液α-葡萄糖苷酶抑制活性达70.96%。水解液经过超滤获得最佳组分E-4，Sephadex G-15凝胶层析得到最优组分E-43，其对α-葡萄糖苷酶和α-淀粉酶的半抑制浓度值(IC50)分别为0.26、6.74 mg/mL；其对ABTS+、DPPH自由基清除能力的半抑制浓度值分别为2.62、4.23 mg/mL。经液相色谱－串联质谱(LC-MS/MS)测定，其氨基酸序列为Gly-Phe-Ser-Gly-Ser-Gly-Gly-Lys、Gly-Val-Gly-Ala-Gly-Ala-Ala-Arg，荷质比m/z为348.67、329.68，分子质量为695.32 u、657.36 u，具有降血糖和抗氧化活性的两条八肽中N端均为亲水氨基酸，C端均为碱性氨基酸，且均含亲水、疏水和碱性氨基酸。     

花椒籽蛋白抗菌肽的分离纯化研究

分别用胃蛋白酶、酸性蛋白酶酶解花椒籽蛋白制得两种粗酶液(A肽和B肽),以抑菌率为指标,依次采用超滤、Sephadex G-50凝胶层析进行分子截留和分离纯化,用Tricine-SDS-PAGE电泳测定其主要抗菌肽的相对分子质量。结果显示:两种酶解产物的粗酶液经超滤后,获得的相对分子质量为5~10 k D的组分(A-b肽和B-b肽)对大肠杆菌的抑菌率最高,分别为62.79%和66.94%;将A-b肽和B-b肽进行凝胶层析,分别分离得4个组分,其中A-b肽活性最大的是组分G4(A-b-Ⅳ肽),对大肠杆菌的抑菌率为100%,B-b肽活性最大的是组分F3(B-b-Ⅲ肽),对大肠杆菌的抑菌率为69.99%;A-b-Ⅳ肽和B-b-Ⅲ肽的相对分子质量分别为8.11 k D和10.80 k D。     

基于计算机虚拟技术研究牦牛乳硬质干酪苦味肽的抑菌活性差异

为研究牦牛乳硬质干酪中苦味肽的抑菌活性差异及其与不同微生物菌体蛋白相互作用的氨基酸和位置信息。运用生物信息学方法计算牦牛乳硬质干酪中4种苦味肽RPKHPIK（RK7）、TPVVVPPFL（TL9）、VYPFPGPIPN（VN10）和SLVYPFPGPIPN（SN12）的分子特性,利用分子对接工具从分子层面研究肽的抑菌活性、研究肽段和不同菌体蛋白（大肠杆菌5BNS、金黄色葡萄球菌4ALM、沙门氏菌6CH3）之间相互作用的分子机制,通过BIOPEP数据库比对表征肽段抑菌活性。研究结果表明：RK7所带净电荷为3.1,疏水性氨基酸比例为42.86%;TL9所带净电荷为0,疏水性氨基酸比例为88.89%;VN10和SN12的疏水力矩分别为0.189和0.372,疏水性氨基酸比例为70.00%和66.67%。RK7和TL9均能和5BNS、4ALM、6CH3形成配体-受体复合物构象,VN10和SN12仅能和4ALM、6CH3形成配体-受体复合物构象;将RK7、TL9、VN10和SN12与抗菌肽数据库比对后发现RK7为抑菌活性已知的肽段,最高相似度为100%,TL9、VN10和SN12为潜在的具有抑菌活性的新型抗菌肽。结合肽段分子特性分析、分子对接和数据库比对预测4种苦味肽的抑菌活性,大肠杆菌抑制活性：RK7 > TL9,金黄色葡萄球菌抑制活性：RK7 > VN10 > TL9 > SN12,沙门氏菌抑制活性：RK7 > TL9 > VN10 > SN12。该研究为分子层面研究牦牛乳硬质干酪苦味肽结构特征及其抑菌活性提供了理论参考。     

榛仁降脂活性肽分离纯化及结构鉴定

采用超滤、Sephadex G-25、Sephadex G-15、反相高效液相色谱及质谱对榛仁分离蛋白降脂活性肽进行分离纯化及结构鉴定,并通过测定3T3-L1前脂肪细胞诱导分化过程中脂质积累、总胆固醇及甘油三酯水平,筛选出具有较高降脂活性的肽段。结果表明,经Sephadex G-15分离得到的C3组分的胰脂肪酶抑制、胆固醇胶束吸附及细胞降脂活性均显著高于其他组分。进一步经质谱解析筛选出的肽段Phe-Leu-Leu-Pro-His（FLLPH）与模型组相比,可抑制26.31%的总脂形成,降低32.67%胆固醇和23.87%甘油三酯水平。FLLPH具有较好的降脂活性,本研究可为榛仁降脂活性肽的开发提供理论参考。     

Alternative splicing of lactophorin mRNA from lactating mammary gland of the camel (Camelus dromedarius).

The objective of this study was to determine the corrected structure of lactophorin, a major whey protein in camel milk. The protein had 60.4% amino acid sequence identity to a proteose peptone component 3 protein from bovine whey and 30.3% identity to the glycosylation-dependent cell adhesion molecule 1 in mice. The N-terminal heterogeneity of the protein was a result of alternative mRNA splicing. About 75% of the protein was expressed as a long variant A with 137 amino acid residues and a molecular mass of 15.7 kDa; about 25% was as a short variant B with 122 amino acid residues and a molecular mass of 13.8 kDa. Both proteins are probably threefold phosphorylated. In contrast to the related proteins, no glycosylation was found in camel lactophorin. Because of this difference, specific interaction with carbohydrate binding proteins, as reported for the murine protein, can be excluded, and a function of the protein other than cell recognition or rotaviral inhibition is proposed. The concentration of lactophorin in camel milk was found to be about three times higher than the concentration of the bovine homologue in bovine milk. Pronounced similarities existed between the primary and secondary structures of bovine and camel proteins. We speculated that camel lactophorin has a similar function to that of bovine protein in milk, which is supposed to be the prevention of fat globule aggregation and the inhibition of spontaneous lipolysis by lipoprotein lipase.     

Purification,characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.     

Antidiabetic,angiotensin-converting enzyme inhibitory and anti-inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic-fermented camel milk with molecular interaction study

This study was to separate and purify antidiabetic and angiotensin-converting enzyme (ACE) inhibitory peptides from Lactiplantibacillus plantarum KGL3A fermented camel milk. After 48 h of fermentation at 37°C, ɑ-amylase inhibition, ɑ-glucosidase inhibition, lipase inhibition and ACE inhibitory activities were 80.94%, 64.45%, 63.93%, and 77.53%, respectively in fermented camel milk. Optimisation of growth condition for the evaluation of maximum peptide production was evaluated by measuring proteolytic activity (O-phthalaldehyde, OPA method) with different inoculation rates and incubation times and highest proteolytic activity (9.21 mg/mL) was observed after 48 h of fermentation at 2.5% rate of inoculation. The antidiabetic and ACE inhibitory activity of 3 kDa permeate fraction were higher as compared with other fractions. Purification of antidiabetic and ACE inhibitory peptides from fermented camel milks was performed through sodium dodecyl-sulphate polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis and maximum number of protein bands were present in between 25 and 10 kDa. The generated peptide sequences were matched with antihypertensive peptide database (AHTPDB) and BIOPEP databases for confirming the antidiabetic and ACE inhibitory activity. Peptides, that is. TDVMPQWW and MMSLVSLLLVGILFPTIQAK were having highest peptide ranker score among the all sequences. Furthermore, anti-inflammatory activity (TNF-α, IL-6 and IL-1β) of fermented camel milk was evaluated in macrophage cell line RAW 264.7 (Ralph and William's cell line). Furthermore, peptides were predicted to have improved binding affinity against Human Angiotensin converting enzyme (hACE) through molecular docking.     

Cow and camel milk-derived whey and casein protein hydrolysates demonstrated effective antifungal properties against selected Candida species

Bioactive peptides derived from milk proteins are widely known to possess antibacterial activities. Even though the antibacterial effects of milk-derived peptides are widely characterized, not much focus is given to their antifungal characterization. Therefore, in this study, we investigated the antifungal properties of camel and cow whey and casein hydrolysates against various species of pathogenic Candida. The hydrolysates were produced using 2 enzymes (alcalase and protease) at differing hydrolysis durations (2, 4, and 6 h) and tested for their antifungal properties. The results showed that intact cow whey and casein proteins did not display any anti-Candida albicans properties, whereas the alcalase-derived 2 h camel casein hydrolysate (CA-C-A2) displayed a higher percentage of inhibition against Candida albicans (93.69 ± 0.26%) followed by the cow casein hydrolysate generated by protease-6 h (Co-C-P6; 81.66 ± 0.99%), which were significantly higher than that of fluconazole, a conventional antifungal agent (76.92 ± 4.72%). Interestingly, when tested again Candida krusei, camel casein alcalase 2 and 4 h (CA-C-A2 and CA-C-A4), and cow whey alcalase-6 h (CO-W-A6) hydrolysates showed higher antifungal potency than fluconazole. However, for Candida parapsilosis only camel casein alcalase-4 h (Ca-C-A4) and cow casein protease-6 h (Co-C-P6) hydrolysates were able to inhibit the growth of C. parapsilosis by 19.31 ± 0.84% and 23.82 ± 4.14%, respectively, which was lower than that shown by fluconazole (29.86 ± 1.11%). Overall, hydrolysis of milk proteins from both cow and camel enhanced their antifungal properties. Camel milk protein hydrolysates were more potent in inhibiting pathogenic Candida species as compared with cow milk protein hydrolysates. This is the first study that highlights the antifungal properties of camel milk protein hydrolysates.     

Yak milk casein as a functional ingredient: preparation and identification of angiotensin-I-converting enzyme inhibitory peptides

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.     

脱脂羊脑蛋白水解多肽的分离纯化及抗氧化活性

为制备羊脑蛋白抗氧化肽,本实验对脱脂羊脑蛋白含量及氨基酸组成进行了分析;采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）对枯草芽孢杆菌中性蛋白酶不同酶解时间的酶解液分子质量进行了分析;采用交联葡聚糖凝胶Sephadex G-25和Sephadex G-15对羊脑酶解产物进行了逐级分离纯化,以羟自由基（·OH）和亚硝酸根离子清除能力为指标对分离组分进行抗氧活性评价,并对纯化后的组分抗氧化活性进行了测定。结果表明,脱脂羊脑粉中蛋白含量为60.55%,在测定的17 种氨基酸中,谷氨酸和天冬氨酸这两种酸性氨基酸含量最高,且含有7 种必需氨基酸;羊脑蛋白经酶解后,分子质量集中在10 kD以下;经Sephadex G-25纯化后,得到了6 个组分,其中组分F4的抗氧化活性最强,组分F4经SephadexG-15纯化后,得到3 个组分,其中组分F4-2的抗氧化活性最强,组分F4-2对1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、·OH、超氧阴离子自由基（O2－·）、亚硝酸根离子的半数抑制率IC50分别为1.64、2.47、7.98、5.14 mg/mL。     

基于乳清蛋白的骆驼乳中掺假牛乳的检测及热处理对方法的影响

利用超高效液相色谱（ultra-high performance liquid chromatography,UPLC）技术,建立一种以牛β-乳球蛋白为掺假标识物的检测方法,用于定性定量检测骆驼乳中掺假的牛乳,并且探讨不同热处理方式对掺假标识物的影响,以期满足不同商品化驼乳制品的检测需求。结果表明：该方法能有效地检测鲜驼乳、巴氏杀菌驼乳以及驼乳粉中掺假的牛乳,3 种类型掺假乳样本的定量检测回归方程线性良好,线性相关系数（R2）分别为0.997 9、0.996 9和0.997 8;鲜驼乳、巴氏杀菌驼乳和驼乳粉中掺假牛乳的检出限分别为2%、3%和5%,可满足检测需求;利用该方法在10 种不同品牌市售纯驼乳粉中检测出4 种掺假驼乳粉产品。UPLC法可以有效地检测骆驼乳及其制品中掺假的牛乳,为骆驼乳行业的掺假检测提供一定的技术方法支持。     

海地瓜多肽分离及抗氧化活性研究

本文检测评价海地瓜体壁干粉及多肽氨基酸组成,通过超滤膜分级过滤技术及层析色谱分离技术分级分离海地瓜酶解多肽,采用生化水平及细胞水平抗氧化活性分析方法研究海地瓜多肽抗氧化活性特征。研究显示,海地瓜体壁干粉及酶解多肽氨基酸含量丰富,检测的19种氨基酸总量分别占样品干重的63.06%及82.90%;其中,必需氨基酸含量分别为10.87%及15.60%。海地瓜酶解多肽小分子肽(3 kDa)含量最高,达到43.9%;其次是分子量为3 k~10 kDa的多肽,含量为36.8%;而10 kDa的多肽含量最低,仅为19.3%。三种海地瓜多肽中,小分子多肽(3 kDa)抗氧化活性最高,ABTS法及FRAP法检测的TEAC值分别为0.61±0.03 mmol Trolox/g及0.32±0.02 mmol Trolox/g。海地瓜多肽明显增强HepG-2及HEK293细胞抗H2O2氧化损伤能力,且小分子多肽(3 kDa)活性高于其他组分;G-25层析分离海地瓜小分子多肽(3 kDa),形成6个主要吸收峰,对其样品抗氧化分析结果显示第5峰多肽样品抗氧化活性最高。