酪蛋白酶解物中抗菌肽的分离及其分子量的测定

使用离子交换层析分离纯化酪蛋白酶解物中的抗菌肽并测定其分子量。选用Q-Sepharose Fast Flow为分离介质,对分离条件进行了研究,并对各洗脱组分进行抑菌活性测定,确定了适宜分离条件下酪蛋白抗菌肽的洗脱体积,同时测定了酪蛋白抗菌肽的分子量。结果表明,离子交换层析对酪蛋白酶解物中的抗菌肽的最佳梯度洗脱条件为:流动相A:pH10.5的0.05mol/L乙醇胺盐酸缓冲液;流动相Β:含1mol/L氯化钠的A相溶液,pH10.5;洗脱程序:7%B,1.55CV;13%B,1.40CV;25%B,1.25CV;100%B,1.55CV,洗脱流速:0.5mL/min,检测波长:280nm,酪蛋白抗菌肽的洗脱体积为76.57mL和89.48mL。酪蛋白抗菌肽的平均分子量为3137u。     

酪蛋白酶解产物中抗菌肽的初步研究

以干酪素酪蛋白为试验原料，采用胰蛋白酶在合适的条件下水解，经过不同分子质量的中空纤维超滤膜超滤，获得具有较强抑菌活性的分子量小于3ku的活性组分．结果表明该肽可抑制多种细菌的生长，尤其是对金黄色葡萄球菌和大肠杆菌具有很强的抗菌作用。     

酪蛋白酶解产物中抗菌肽的初步研究

以干酪素酪蛋白为试验原料,采用胰蛋白酶在合适的条件下水解,经过不同分子质量的中空纤维超滤膜超滤,获得具有较强抑菌活性,分子质量<3000u的活性组分。结果表明,该肽可抑制多种细菌的生长,尤其是对金黄色葡萄球菌和大肠杆菌具有很强的抗菌作用。     

不同条件下水解酪蛋白所得到的抗菌肽抑菌效果比较

本文采用胰蛋白酶水解酪蛋白制备抗菌肽，针对不同底物浓度下，改变酶与底物浓度比，水解产物的抑菌活性随着水解度的变化情况进行对比。确定了在45℃时该反应的最适底物浓度是9％（g/ml)；最佳酶与底物浓度比为1：120。同时，通过在不同水解时间取点，发现水解所得到产物的抑菌强度随水解度的增大而增大。     

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...     

带鱼蛋白抗菌肽抑菌效果的影响因素

以舟山带鱼为原料,采用复合酶解法制备带鱼蛋白抗菌肽,探究温度、pH值、胰蛋白酶、β-内酰胺酶、反复冻融和金属离子对带鱼蛋白抗菌肽抑菌效果的影响。结果表明：带鱼蛋白抗菌肽具有较强的热稳定性;在pH值为4.8时,带鱼蛋白抗菌肽的抑菌活性最高,达到45.12%,在pH值为8.8时出现最低值40.08%;此抗菌肽在胰蛋白酶和β-内酰胺酶的作用下,依然保持了原有的抑菌活性;随着冻融次数的增多,抗菌肽的抗菌效力呈下降的趋势,但在冻融6次之后,也只下降到90.28%,;带鱼蛋白抗菌肽对Na+、K+、Ca2+、Mg2+具有较强的稳定性,抑菌活性基本不受影响;但是对Zn2+、Fe2+和Fe3+表现出较低的耐受性,其相对抗菌效力分别降低至86.25%、76.38%和74.97%,应当避免带鱼蛋白抗菌肽与这些离子的接触。该抗菌肽在各因素条件下稳定性较好,在作为天然抗菌剂上有一定的应用前景。     

响应面法优化酪蛋白抗菌肽的制备工艺

以酪蛋白为实验原料,利用胃蛋白酶水解制备抗菌肽。在单因素实验的基础上采用Box-Behnken模型响应面设计,建立了胃蛋白酶酶解酪蛋白制备抗菌肽的三元二次回归模型,确定底物浓度2.6%、水解温度37.7℃、pH2.6、酶浓度3%、酶解时间4h为最佳的酶解条件,在此条件下,酶解液对金黄色葡萄球菌的抑菌圈直径达到25.8mm。     

紫甘蓝色素的稳定性及抑菌性

从紫甘蓝叶片中提取分离紫甘蓝色素,研究紫甘蓝色素在不同条件下的稳定性及抑菌性。结果表明：紫甘蓝色素在pH 2 的酸性水溶液中呈现原植物具有的紫红色,稳定性较好;随着pH 值的增高,紫甘蓝色素发生向蓝色再到绿色的转变,且最大吸收波长发生红移。紫甘蓝色素自然光照射2d 后,色素的保存率在91% 以上,自然光照射6d 后,色素的保存率仍在79% 以上;紫甘蓝色素70℃以下加热2h,色素的保存率在97% 以上,90℃加热2h,色素的保存率仍在90% 以上,这说明紫甘蓝色素对光照和加热均有一定耐受性。紫甘蓝色素对钠、钾、钙、镁等金属离子和氧气均稳定,但铁离子会使其变质。在紫甘蓝色素溶液的质量浓度为10mg/100mL 时,对金黄色葡萄球菌和枯草芽孢杆菌的生长就具有极显著的抑制作用,随着色素质量浓度的提高,抑制作用愈来愈大,但未见紫甘蓝色素对大肠杆菌生长有抑制效果。     

红花酢浆草花色素的稳定性及抑菌性研究

实验对红花酢浆草花瓣中所含色素的稳定性和抑菌性进行了研究。结果表明:pH对红花酢浆草色素的稳定性影响较大,在pH2～3时呈稳定的红色,pH增大时颜色变紫变黑。红花酢浆草色素在暗处稳定性最高,自然光下次之,日光灯下低。红花酢浆草色素在50℃以下热稳定性较好,50℃以上热稳定性降低。蔗糖、氯化钠、苹果酸、柠檬酸和乳酸对红花酢浆草色素均有明显的增色、护色作用,可明显改善或提高其稳定性。红花酢浆草色素对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌均有抑制效果,并且随着色素溶液浓度的增加,抑菌效果也越明显。因此红花酢浆草色素在一定条件下可作食品色素兼天然防腐剂使用。     

花椒籽蛋白抗菌肽的抑菌作用及其稳定性研究

本研究采用菌落计数法对花椒籽蛋白抗菌肽的抑菌效果及稳定性进行了研究。结果表明:抗菌肽对大肠杆菌、沙门氏菌、枯草芽孢杆菌和金黄色葡萄球菌均具有抑制作用;抗菌肽的抑菌活性随浓度的升高而增强;经不同温度、加热时间处理后,抗菌肽的抑菌活性与对照相比无显著差异(p0.05);抗菌肽在经p H 2.0~12.0处理后仍有抑菌活性,p H 2.0时的抑菌率最低(58.13%),p H 12.0时的抑菌率最高(79.17%);随着金属离子浓度的增加,经K+、Ca2+和Fe3+处理后的抗菌肽的抑菌活性分别呈降低、增加、变化平缓的趋势,经0.1 mol/L K+处理后的抗菌肽的抑菌活性明显增强;经有机溶剂处理后,抗菌肽的抑菌活性有所降低;经吐温20、吐温80处理后,抗菌肽的抑菌活性极显著增强(p0.01),SDS对抑菌活性的增加不显著。因此,抗菌肽具有很好的热、酸碱、金属离子和有机溶剂稳定性,而表面活性剂(吐温20、吐温80)能使抗菌肽的抑菌活性显著增强。     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

酪蛋白水解物替代乳清蛋白的加工性能评价研究

利用复合蛋白酶水解酪蛋白制备适度及深度水解酪蛋白产品,测定酪蛋白水解物的加工性能。结果表明,经过酶解后,适度水解酪蛋白溶解度接近90%,深度水解酪蛋白溶解度接近100%,显著高于酪蛋白和乳清蛋白。此外,适度水解酪蛋白吸油性、起泡性分别约为乳清蛋白的3倍和1.5倍。深度水解酪蛋白在起泡性和乳化性上也显著高于乳清蛋白。可见,两款酪蛋白水解物在起泡性、乳化性、吸油性、溶解性等方面均在一定程度上优于乳清蛋白,可广泛替代乳清蛋白在食品工业中大规模应用。      

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.     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

Production and properties of casein hydrolysate microencapsulated by spray drying with soybean protein isolate

The aim of this work was to encapsulate casein hydrolysate by spray drying with soybean protein isolate (SPI) as wall material to attenuate the bitter taste of that product. Two treatments were prepared: both with 12 g/100 g solids and containing either two proportions of SPI: hydrolysate (70:30 and 80:20), called M1 and M2, respectively. The samples were evaluated for morphological characteristics (SEM), particle size, hygroscopicity, solubility, hydrophobicity, thermal behavior and bitter taste with a trained sensory panel using a paired-comparison test (non-encapsulated samples vs. encapsulated samples). Microcapsules had a continuous wall, many concavities, and no porosity. Treatments M1 and M2 presented average particle sizes of 11.32 and 9.18 μm, respectively. The wall material and/or the microencapsulation raised the hygroscopicity of the hydrolysate since the free hydrolysate had hygroscopicity of 53 g of water/100 g of solids and M1 and M2 had 106.99 and 102.19 g of water/100 g of solids, respectively. However, the hydrophobicity decreases, the absence of a peak in encapsulated hydrolysates, and the results of the panel sensory test considering the encapsulated samples less bitter (p < 0.05) than the non-encapsulated, showed that spray drying with SPI was an efficient method for microencapsulation and attenuation of the bitter taste of the casein hydrolysate.     

Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: Implications for the stability of casein micelles of UHT milks during their storage

Pseudomonas fluorescens grows at low temperature and produces thermo-resistant protease(s) that can destabilize UHT (Ultra High Temperature) milk during its storage. The consequences of contamination of microfiltered milk with 9 strains of P. fluorescens on the stability of the corresponding UHT milk during storage had been investigated in this study.     

A biological perspective on the structure and function of caseins and casein micelles

Caseins belong to a larger group of secreted calcium phosphate-binding phosphoproteins that have a natively unfolded conformation. Nearly all members of the group are involved in aspects of calcium phosphate biology and nearly all have recognition sites for phosphorylation by the Golgi protein kinase. In the caseins these are often close together in the primary structure, forming the so-called phosphate centres. Certain highly phosphorylated phosphopeptides derived from the calcium-sensitive caseins will combine with amorphous calcium phosphate to form defined chemical complexes called calcium phosphate nanoclusters. Both the substructure of casein micelles and the partition of salts in milk can be explained quantitatively by the ability of the calcium-sensitive caseins to sequester calcium phosphate and form nanocluster-like structures. A simple stability rule for milk can be derived by applying equilibrium thermodynamics to the process of calcium phosphate sequestration. In principle, the stability rule can be extended to problems of instability encountered in milk-processing operations and to the formulation of other types of high calcium foods.     

酪蛋白抗菌肽的酶法制备

对胃蛋白酶水解酪蛋白制备酪蛋白抗菌肽进行研究.结果表明:酪蛋白水解物中,分子量3386 Da的组分具有最强的抑菌活性和良好的热稳定性,胃蛋白酶水解酪蛋白释放抗菌肽的适宜条件为:干酪素浓度10 mg/mL、酶浓度2.0%、温度40 ℃、pH 1.5、水解时间3.5 h.     

酪蛋白抗氧化肽制备工艺及酶解产物特性研究

采用不同蛋白酶酶解酪蛋白,确定AS1,398中性蛋白酶是酶解酪蛋白制备抗氧化肽的优良酶源.通过单因素和响应面回归分析.得到AS1,398中性蛋白酶酶解酪蛋白的优化工艺条件为:底物浓度30mg/mL,水解时间90min,pH7.2.酶底比为2%(w/w),温度为50℃.优化酶解条件下,10mg/mL酶解产物的DPPH自由基清除率为80.43%,IC50为3.96mg/mL,显现出良好的抗氧化性能.高效液相排阻色谱(HPSEC)分析表明,优化条件下产物主要是3500Da以下的一些短肽.