山羊乳酪蛋白抗菌肽分离纯化及结构鉴定

为制备山羊乳酪蛋白抗菌肽,阐明抗菌肽的构效关系,采用LS-106大孔吸附树脂、凝胶色谱和高效液相色谱(HPLC)对山羊乳酪蛋白酶解物进行逐级分离纯化,以对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌圈直径、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)为指标,对分离组分进行抑菌试验,采用预测软件、质谱与圆二色谱对分离抗菌肽的一、二级结构进行预测和鉴定。试验结果表明:(1)大孔吸附树脂分离酶解液,其中体积分数为60%的乙醇洗脱组分抑菌作用较强。(2)凝胶色谱将60%乙醇洗脱组分分离成3个色谱峰,峰3抑菌作用较强。(3)HPLC将峰3分离为2个组分F3-1和F3-2,其中F3-1对大肠杆菌、沙门氏菌和金黄色葡萄球菌的MIC均为297.9μg/mL,MBC为1191.6μg/mL;F3-2对大肠杆菌的MIC和MBC分别为309.2,1236.8μg/mL,对沙门氏菌和金黄色葡萄球菌的MIC均为154.6μg/mL,MBC为618.4μg/mL。(4)F3-1和F3-2的一级结构分别为Leu-Gly-Gly-Val-Gln-Glu-Pro-Asp-Pro-Asn-Thr-Asp-Val-Arg(1497.76)和Val-Val-Asn-Leu-Glu-GluLys-Leu-Pro-Gly(1242.66)。F3-1和F3-2均属阴离子抗菌肽,虽然其抑菌作用稍弱于阳离子抗菌肽,但是其二级结构为无毒性的无规则卷曲结构,与α-螺旋结构抗菌肽相比,在食品、药品和化妆品等安全应用方面潜力巨大。     

黄粉虫抗菌肽的分离纯化及生物活性研究

本研究以黄粉虫为试材,采用碱性蛋白酶酶解黄粉虫蛋白制备获得了抗菌肽,并对其进行分离纯化,比较了不同组分及纯化后抗菌肽的抑菌活性、热稳定性及分子量。结果表明:大孔吸附树脂对蛋白酶酶解液动态吸附并梯度洗脱后得到5个组分,抑菌试验表明无水乙醇洗脱组分的抑菌活性最强。采用制备型HPLC对无水乙醇洗脱组分进一步纯化,得到两个组分(H-1,H-2),其中组分H-2对大肠杆菌、沙门氏菌和金黄色葡萄球菌均表现出较强的抑菌活性,且其最小抑菌浓度(MIC)为0.512 mg/m L。热稳定性试验表明,H-2具有较好的热稳定性,121℃加热20 min仍能保持较高活性。液质联用(LC-MS)结果表明,H-2的分子量为756.82u。本研究为黄粉虫抗菌肽的高效分离和纯化提供了科学依据,为黄粉虫抗菌肽在食品防腐中的应用提供了基础数据。     

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...     

青刺果抗菌肽组分的分离及其稳定性分析

旨在高值化利用青刺果榨油后副产物青刺果粕中的蛋白质，以青刺果粕为原料，提取青刺果蛋白并制备蛋白酶解物，采用活菌吸附法联合反相高效液相色谱（RP-HPLC）从蛋白酶解物中筛选出抗菌肽组分，评价其抑菌活性，并对抑菌活性最好的组分进行稳定性分析。结果表明：筛选出的3个抗菌肽组分（F1、F2和F3）中，F2组分抑菌活性最好，当其质量浓度为10 mg/mL时对金黄色葡萄球菌和大肠杆菌的抑菌活性分别达到45.5%和39.1%；抗菌肽组分F2抑菌活性随温度升高和盐溶液浓度增加均降低，最适pH为6.0，在酸性环境中极不稳定，对酸性蛋白酶、碱性蛋白酶、胃蛋白酶和胰蛋白酶的耐受性均较差，对中性蛋白酶耐受性较好。综上，青刺果粕中的蛋白酶解物具有抑菌活性，具有开发青刺果抗菌肽的潜力。     

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.     

蛹拟青霉发酵液中抑菌活性成分的分离纯化

以大肠杆菌、白色假丝酵母、黑曲霉为指示菌株,对蛹拟青霉xzcs005菌株液体发酵液乙酸乙酯萃取相中的抑菌活性成分进行分离纯化。乙酸乙酯萃取相经硅胶柱色谱分离后得到组分Ⅴ和组分Ⅶ两个抑菌活性组分。组分Ⅴ利用反相柱分离得到抑菌活性纯品PMBA-1,其在质量浓度为1.0mg/mL时对大肠杆菌、白色假丝酵母和黑曲霉的抑菌圈直径分别为(16.21±0.27)、(23.54±0.35)、(27.33±0.54)mm;组分Ⅶ再次经过硅胶柱色谱分离后得到抑菌活性纯品PMBA-2,其在质量浓度为1.0mg/mL时对大肠杆菌、白色假丝酵母和黑曲霉的抑菌圈直径分别为(21.84±0.28)、(16.33±0.54)、(11.54±0.51)mm。     

棉籽抗菌活性肽的分离纯化及鉴定

通过体外模拟消化系统对棉籽分离蛋白(cottonseed protein isolate,CPI)进行酶解,得到具有抗菌活性的酶解产物,并采用超滤(ultrafiltration,UF)、阴离子交换色谱(anion exchange chromatography,AEC)、半制备高效液相色谱(semi-preparation high performance liquid chromatography,semi-P-HPLC)分离技术对棉籽抗菌活性肽进行分离纯化,用电喷雾串联质谱(electrospray ionization-tandem mass spectrometry,ESI-MS/MS)鉴定棉籽抗菌肽的氨基酸序列。在抗菌活性肽分离纯化过程中,用UF对具有抗菌活性的CPI酶解产物进行分离,得到3个组分U-Ⅰ~U-Ⅲ。抗菌活性检测表明U-Ⅲ的抗菌能力最强;用AEC分离U-Ⅲ得到3个组分QF-Ⅰ~QF-Ⅲ,其中QF-Ⅱ抗菌能力最强;进一步采用semi-P-HPLC分离QF-Ⅱ得到4个组分PF-Ⅰ~PF-Ⅳ,其中PF-Ⅲ的抗菌能力最强,经HPLC检测为单一峰,ESI-MS/MS检测分析得到该肽的氨基酸序列为ISGLIYEETR(Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg)。     

三斑海马蛋白ACE抑制肽的制备及其二级结构的研究

以三斑海马为原料,经碱性蛋白酶酶解,获得富含血管紧张素转化酶(angiotensin I-converting enzyme,ACE)抑制肽的酶解液。酶解液经透析、Sephadex G-25凝胶过滤色谱和反相高效液相色谱得到进一步分离纯化。结果表明,透析产物的IC50值为(0.44±0.26)mg/mL,相比酶解液(0.81±0.12)mg/mL,其ACE抑制活性更强。透析产物经Sephadex G-25凝胶柱分离纯化后,得到3种组分,其中组分F2的ACE抑制活性最强,IC50值为(0.11±0.08)mg/mL。F2经反相高效液相色谱进一步分离后,得到6个具有ACE抑制活性的组分峰,其中组分F2-4的ACE抑制活性最强,IC50值为(0.005 7±0.000 9)mg/mL。经过3步分离纯化后,成功从三斑海马蛋白中分离得到一种活性较强ACE抑制肽:ProAla-Gly-Pro-Arg-Gly-Pro-Ala(PAGPRGPA),多肽分子量为721.39 Da。圆二色谱分析多肽二级结构表明其主要含无规则卷曲。因此,从三斑海马蛋白中分离得到的多肽可能成为营养保健品和抗高血压药品及相关疾病的一种有益成分,且对海马蛋白资源的开发利用具有重要意义。     

贝莱斯芽孢杆菌抑尖孢镰刀菌脂肽类物质的鉴定

为探究贝莱斯芽孢杆菌（Bacillus velezensis）P9对于尖孢镰刀菌（Fusarium oxysporum）具有拮抗作用的主要活性成分,采用酸沉淀法和有机溶剂沉淀法提取菌贝莱斯芽孢杆菌P9发酵液中的脂肽类抗生素粗提物,研究粗提物对尖孢镰刀菌的抑菌活性。利用多功能制备液相仪对其组分进行分离,确定具有抑菌活性的组分,并利用高效液相色谱-四级杆飞行时间质谱联用（HPLC-Q-TOF-MS）仪对具有抑菌活性的组分进行鉴定。结果表明,脂肽类抗生素粗提物对尖孢镰刀菌有良好的抑菌作用,且酸沉淀法得到的粗提物抑菌活性较高,从中共分离到7种组分（编号为1#～7#）,其中4种组分具有抑菌作用,经鉴定,组分2#为C15SurfactinA或C16SurfactinB或C15SurfactinC,组分4#、6#、7#分别为C15BacillomycinD、C14BacillomycinD、C16BacillomycinD。     

草鱼蛋白源抗氧化肽的分离及鉴定

利用超滤、离子交换色谱、高效逆流色谱以及凝胶过滤色谱等系列分离纯化技术从草鱼蛋白酶解产物中分离纯化抗氧化活性肽,分离过程中发现相对分子质量1～3kD 的组分抗氧化活性最强,且碱性肽组分的抗氧化活性强于酸性或中性肽组分、疏水性肽组分的抗氧化活性强于亲水性肽组分。最终借助反相高效液相色谱在线连接的电喷雾质谱结合氨基酸分析鉴定出一种抗氧化肽,一级结构序列为Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val,相对分子质量为966.3D。     

抗菌肽的功能及生产研究进展

为进一步了解抗菌肽的研究现状,以期为抗菌肽的纯化鉴定、生产和应用提供理论依据,对抗菌肽的功能,纯化鉴定方法,人工合成和基因工程表达进行了综述。抗菌肽不仅具有广谱的抗菌活性且有抗癌、抗病毒、促进伤口愈合等作用,抗菌肽经乙酸提取,反相液相色谱纯化是较常见的方法,抗菌肽的人工合成多采用固相合成,基因重组一般包括三个内容：抗菌肽的基因结构分析,抗菌肽的基因表达调控及互补脱氧核糖核酸（cDNA）编码,抗菌肽的基因表达系统。     

优选南极磷虾蛋白肽抗氧化活性组分

目的制备南极磷虾抗氧化肽,优选出抗氧化活性最好的南极磷虾肽成分。方法采用脱脂、酶解、超滤等手段制备南极磷虾抗氧化肽;以DPPH自由基清除率、ABTS自由基清除率、抗氧化能力指数3个抗氧化指标和超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)活力2个酶活力指标为评价指标,优选出抗氧化活性最好的南极磷虾肽组分;基于G-25凝胶层析技术对抗氧化活性最好的南极磷虾肽组分进行分离纯化,进一步基于电子顺磁共振(electron paramagnetic resonance,EPR)方法测定洗脱后各峰的DPPH自由基清除率,得到抗氧化活性最好的南极磷虾抗氧化肽组分。结果通过抗氧化指标测定,截留分子量3～10 KDa的肽的DPPH自由基清除率最高,为(30.10±1.10)%,截留分子量3 KDa的南极磷虾肽的ABTS清除能力和抗氧化指数较好,则IC50值为(0.74±0.08) mg/mL、氧化自由基吸收能力(oxygen radical absorbance capacity, ORAC)值为6.39±0.21;通过酶活力指标测定,截留分子量3 KDa的肽的SOD活力和CAT活力最好,分别为(45.7±0.13)U/mg和(17.1±0.19)U/mg蛋白质。对截留分子量3KDa和3～10KDa的南极磷虾肽进行G-25分离纯化后,测定各组分的DPPH自由基清除率,可知截留分子量3～10KDa的F2-2峰清除率最好,为(51.55±1.54)%。结论基于EPR方法优选出分子量为3～10 KDa的南极磷虾肽的F2-2组分的DPPH自由基清除率最高。     

Antimicrobial peptide isolated from ovalbumin hydrolysate by immobilized liposome-binding extraction

Antimicrobial peptides are of greatest potential as a new group of antibiotics. Enzymatic hydrolysis was performed using flavourzyme, pepsin, trypsin, neutrase, papain and alcalase to obtain antimicrobial peptides from ovalbumin. Pepsin hydrolysate exhibited the highest antimicrobial activity compared with other fractions. To purify and characterize antimicrobial peptide, an immobilized liposome-binding extraction method was developed, in which the liposome was regarded as a mimic biomembrane system. The immobilized liposome stationary phase was prepared, and the maximum adsorption capacity and Langmuir constant were 81.30 μM/g and 8.19 mL/mg, respectively. Four fragments were simultaneously predicted by the comparison between reverse-phase high-performance liquid chromatography chromatograms of pepsin hydrolysate before and after interaction with immobilized liposome membrane. A novel cationic antimicrobial peptide named Opep2 was sequenced as RVASMASEKMKI. In addition, antimicrobial mode experiments showed that Opep2 interacted with cell wall membrane and caused potassium efflux, nucleotide leakage and ultimately cell death. Because of the high efficiency and procedural simplicity, the immobilized liposome-binding extraction method could be more efficient for the screening of antimicrobial peptides than conventional methods, which provides scientific references for antimicrobial peptide production.     

辣木叶蛋白质酶解产物的制备及其抗菌活性研究

为了研究辣木叶蛋白及其酶解产物的抗菌活性,利用碱提酸沉法提取辣木叶中的蛋白质,并分析其氨基酸组成;分别采用6种单酶和3种复合酶对辣木叶蛋白进行酶解,考察辣木叶蛋白及其酶解产物的抑菌活性,确定最佳蛋白酶。通过二倍稀释法,研究有效抑菌组分对不同菌的最小抑菌浓度(minimum inhibitory concentration,MIC)。结果显示:辣木叶蛋白提取率为32.67%±0.45%,必需氨基酸占氨基酸总量的39.27%。抑菌活性研究表明辣木叶蛋白仅对金黄色葡萄球菌显示抑菌活性,抑菌圈大小为(7.67±0.24) mm;胃蛋白酶结合胰蛋白酶分步酶解蛋白所得酶解产物抑菌能力均高于其他几种蛋白酶,酶解产物对化脓性链球菌的抑菌活性最强,抑菌圈大小为(10.83±0.62) mm,且其对金黄色葡萄球菌、单增李斯特菌及化脓性链球菌的MIC分别为5、10、2.5 mg/mL。说明辣木叶蛋白及其酶解产物均有抗菌活性,双酶酶解法制备的酶解产物具有较强的抑菌性及广抑菌谱。     

抗菌肽结构优化与改造策略研究进展

抗菌肽（AMPs）是一类具有抗菌活性的生物活性肽,由于热稳定性好、细胞选择性强、对哺乳动物细胞毒副作用少和不易产生耐药性等优势,成为近年来生物活性肽领域重要的研究热点。该文简要介绍了抗菌肽的分类,综述了抗菌肽的一级结构与空间结构特点,并从改变净正电荷数量与分布、调整抗菌肽α-螺旋度、提高抗菌肽蛋白酶耐受性、改变疏水性及平均疏水矩大小、改变两亲性、改变氨基酸残基位置及肽链长度、构建杂合抗菌肽、降低哺乳动物细胞毒性和制备金属抗菌肽提升抗菌活性与拓展抗菌谱等方面阐述抗菌肽的结构改造策略,并对抗菌肽结构优化策略研究进行了展望,旨在为抗菌肽结构优化策略的选择提供理论参考。     

Antilisterial activity of dromedary lactoferrin peptic hydrolysates

The aim of this study was to explore the antibacterial peptides derived from dromedary lactoferrin (LFc). The LFc was purified from colostrum using a batch procedure with a cation exchange chromatography support and was hydrolyzed with pepsin to generate peptic digest. This peptic digest was fractionated by cation exchange chromatography, and the antilisterial activity of LFc, peptic digest, and obtained fractions was investigated using the bioscreen method. The growth of Listeria innocua ATCC 33090 and LRGIA 01 strains was not inhibited by LFc and its hydrolysates. Two fractions of dromedary lactoferrin peptic hydrolysate were active against both strains. A tandem mass spectroscopy analysis revealed that the 2 active fractions comprised at least 227 different peptides. Among these peptides, 9 found in the first fraction had at least 50% similarity with 10 known antimicrobial peptides (following sequence alignments with the antimicrobial peptide database from the University of Nebraska Medical Center, Omaha). Whereas 9 of these peptides presented homology with honeybee, frog, or amphibian peptides, the 10th peptide, F₁₅₂SASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY₁₉₂ (specifically found in 1 separated fraction), exibited 54% homology with a synthetic antibacterial peptide (AP00481) derived from human lactoferrin named kaliocin-1. Similarly, the second fraction contained 1 peptide similar to lactoferrampin B, an antibacterial peptide derived from bovine milk. This result suggests that peptic hydrolysis of LFc releases more active antimicrobial peptides than their protein source and thus provides an opportunity for their potential use to improve food safety by inhibiting undesirable and spoilage bacteria.     

Bioactive properties of Kilka (<Emphasis Type="Italic">Clupeonella cultriventris caspi</Emphasis>) fish protein hydrolysates

Whole common Kilka fish was hydrolyzed separately using four commercial enzymes, Alcalase, Neutrase, Protamex at 50 °C and Pepsin at 37 °C for 30, 60 and 90 min. Degree of hydrolysis, angiotensin-I-converting enzyme (ACE) inhibitory activity and antimicrobial activity of each hydrolysate against Gram-negative (Escherichia coli, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus, Listeria innocua) bacteria were studied. Results showed that the degree of hydrolysis for all enzymes was in the range of 2.63–3.36%. Electrophoresis profiles of the Kilka protein hydrolysates showed that most of produced peptides were in the range of 30 D but Alcalase and Neutrase had a better performance in the production of low molecular weight peptides in the range of 10 D. This led to increase the antimicrobial activity against the examined bacteria at the concentration of 200 µg/mL peptide solution. The Neutrase enzyme produced hydrolysate with the highest ACE inhibitory activity (53%?±?1.8 at 500 µg/mL). Antimicrobial activity of Kilka protein hydrolysates using Protamex and Pepsin was lower than the others due to lack of considerable amount of small peptides. The current research has demonstrated that the peptides derived from the enzymatic hydrolysis of Kilka fish protein in optimum conditions are capable of being converted to antimicrobial and antihypertensive agents to be used in functional foods.     

乳酸对卵清蛋白抑菌活性的增效作用

通过添加低浓度的生物防腐剂乳酸,探讨其对食品中天然抑菌成分卵清蛋白抑菌活性的增效作用。通过单因素实验,探究乳酸浓度、处理温度和处理时间对卵清蛋白抑制致病菌(大肠杆菌O157:H7、金黄色葡萄球菌)和腐败菌(灿烂类芽胞杆菌、土变形杆菌)能力的影响,获得最佳乳酸处理条件。在此条件下确定乳酸对卵清蛋白抑菌活性的增加率以及对测试菌株的抑菌动力学曲线。结果表明:在4 ℃下,使用0.05%的乳酸处理40、60 min,可将卵清蛋白对灿烂类芽胞杆菌和金黄色葡萄球菌的抑菌活性分别增加7.802%和26.904%;在25 ℃下,使用0.15%和0.2%的乳酸分别处理土变形杆菌和大肠杆菌O157:H7 40 min,可分别将卵清蛋白的抑菌活性增加53.770%和131.321%。抑菌动力学曲线表明,添加乳酸极显著(p<0.01)增强了卵清蛋白的抑菌活性,降低了卵清蛋白的使用浓度,并延长了抑菌时间。     

Identification and molecular mechanism of action of antibacterial peptides from Flavourzyme-hydrolyzed yak casein against Staphylococcus aureus

Antibacterial peptides can be released from yak milk casein. To date, the amino acid sequences and mechanism of action of yak casein–derived antibacterial peptides remain unknown. The current study identified antibacterial peptides from yak casein and their molecular mechanism of action. Our results showed that yak α-casein, β-casein, and κ-casein could be effectively hydrolyzed by Flavourzyme (Solarbio Science and Technology Co. Ltd.), and the 2-h hydrolysate showed the highest antibacterial rate of 43.07 ± 2.59% against Staphylococcus aureus. The 1,000 to 3,000 Da fraction accounted for 23.61% of the 2-h hydrolysate and had an antibacterial rate of 62.64 ± 4.40%. Three novel peptides with antibacterial activity were identified from this fraction, and the β-casein–derived peptide APKHKEMPFPKYP showed the strongest antibacterial effect (half-maximal inhibitory concentration = 0.397 mg/mL). Molecular docking predicted that APKHKEMPFPKYP interacted with 2 important enzymes of Staph. aureus, dihydrofolate reductase and DNA gyrase, through hydrophobic, hydrogen bonding, salt bridge, and π-π stacking interactions. Our findings suggest that the yak casein–derived peptides may serve as a potential source of natural preservatives to inhibit Staph. aureus.