CN114395601B - 一种具有两亲性和抗氧化性的酪蛋白酶解物及其制法和用途 - Google Patents
一种具有两亲性和抗氧化性的酪蛋白酶解物及其制法和用途 Download PDFInfo
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Abstract
本发明公开了一种具有两亲性和抗氧化性的酪蛋白酶解物及其制法和用途。该酪蛋白酶解物的制备方法包括以下步骤:取酪蛋白溶于水,剪切和搅拌,得到酪蛋白分散液;(2)调节酪蛋白分散液的pH值至7~8,加入蛋白酶酶解0.5~6h,控制水解度2‑6.5%,然后灭酶,离心取上清液;上清液适度浓缩后进行冷冻干燥或喷雾干燥,获得酪蛋白酶解物。本发明方法基于蛋白酶的酶切位点特异性,通过加酶量控制水解度,所得酶解物的水解度为2~6.5%,所带负电荷值<‑25mV,分子量分布均匀,<1000Da以上的肽段质量占比<40%,1000~5000Da的肽段质量占比>35%,保留两亲性的同时还表现出较好的抗氧化能力。
Description
技术领域
本发明属于生物活性肽领域,具体涉及一种具有两亲性和抗氧化性的酪蛋白酶解物及其制法和用途。
背景技术
水包油乳液是一种典型的食品体系,表现为油滴分散在水相中,有很多基于水包油乳液制备的食品,例如牛奶、豆奶等其他乳饮料。水包油乳液是一个高度不稳定的系统,易受众多环境因素的影响,如pH值、O2和金属离子等,容易造成乳液的物理不稳定性和和引发脂质氧化,进而导致食品感官和营养品质变差,造成安全隐患和经济损失。因此,研究开发具有提高乳液不稳定性和减缓脂质氧化的途径至关重要。
人工合成的乳化剂和抗氧化剂虽然表现出相应的稳定作用,但却具有毒副作用,且限制使用量。因此,研究开发天然来源的活性物质作为天然的乳化剂和抗氧化剂符合当今的发展趋势。
酪蛋白是食品工业生产中使用最广泛的动物蛋白,来源广泛,营养丰富。酪蛋白分子中同时具有亲水基团和疏水基团,在结构上呈现柔性结构,使其具有较好的两亲性,因此常作为乳化剂广泛应用于食品体系中。此外酪蛋白富含酪氨酸和色氨酸等抗氧化氨基酸,是制备抗氧化肽的优质蛋白来源。
生物酶法制备蛋白多肽因其来源广泛、生物相容性好、功能多样、安全性高,已逐渐成为现代功能性食品开发的重要途径。
发明内容
本发明的目的在于克服现有技术不足,提供一种具有两亲性和抗氧化性的酪蛋白酶解物及其制法和用途,通过可控酶解技术制备一种同时具有两亲性和抗氧化性的酪蛋白酶解物,其水解度在低中范围,富含中分子量的肽段,且带有较高的电荷值,同时具备一定的抗氧化能力。其在应用上不仅可以单独制备细小,均一的乳液液滴,而且还能够显著抑制乳液的脂质氧化,提供了一种运用天然活性物质改善乳液稳定性的方法。
本发明的目的通过下述技术方案实现:
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)取酪蛋白溶于水,剪切和搅拌,得到酪蛋白分散液;
(2)调节酪蛋白分散液的pH值至7.0~8.0,加入蛋白酶酶解0.5~6h,然后灭酶,离心取上清液;上清液适度浓缩后进行冷冻干燥或喷雾干燥,获得酪蛋白酶解物;
步骤(1)所述的酪蛋白优选蛋白含量90%以上的酪蛋白酸钠,水溶性较好;
步骤(1)所述的剪切,优选6000~10000rpm剪切2~5min,破坏酪蛋白溶液的胶束结构,暴露更多的酶切位点;
步骤(2)所述的蛋白酶是能够降解酪蛋白释放较多中分子量肽段,且酶解物的电荷值在<-25mV,如胰蛋白酶、中性蛋白酶或碱性蛋白酶等;
步骤(2)所述酶解的温度为30~60℃,加酶量为酪蛋白质量的0.01-0.5%,控制水解度2-6.5%;
步骤(2)所述的灭酶,是沸水浴15min;
步骤(2)所述的离心,优选在8000~10000g、4~10℃下离心15-20min;
步骤(2)所述浓缩是浓缩至固形物含量为25~35%;
步骤(2)所述冷冻干燥的温度为-50~-40℃,所述喷雾干燥的进风温度为160-210℃,排风温度为70-120℃。
上述方法制得的酶解物,其水解度为2~6.5%,所带负电荷值<-25mV,分子量分布均匀,其中分子量<1000Da的肽段质量占比<40%,1000~5000Da的肽段质量占比>35%,在保留两亲性同时还表现出较好的抗氧化能力。
一种水包油乳液,采用磷酸盐缓冲液为连续相溶剂,加入上述的酪蛋白酶解物和植物油,经过剪切和均质,得到水包油乳液;
所述的磷酸盐缓冲液优选10mM、pH值为7.0的磷酸盐缓冲液;
所述酪蛋白酶解物在连续相中的浓度优选1.0wt%;
所述水包油乳液的连续相:植物油质量份数1:10;
所述的剪切优选12000rpm剪切2min;
所述的均质优选30~35Mpa下均质若干次。
所述的植物油可以是大豆油、玉米油等含不饱和脂肪酸的植物油。
另一种水包油乳液,采用磷酸盐缓冲液为连续相溶剂,加入上述的酪蛋白酶解物和乳化剂,经过剪切和均质,得到水包油乳液;
所述的磷酸盐缓冲液优选10mM、pH值为7.0的磷酸盐缓冲液;
所述乳化剂优选吐温,尤其是吐温20或者吐温80;
所述乳化剂在连续相中的浓度优选1.0wt%;
所述酪蛋白酶解物在连续相中的浓度优选1.0wt%;
所述水包油乳液的连续相:植物油按质量份数1:10;
所述的剪切优选12000rpm剪切2min;
所述的均质优选30~35Mpa下均质若干次。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明方法基于蛋白酶的酶切位点特异性,通过控制水解度,所得酶解物的水解度为2~6.5%,所带负电荷值<-25mV,分子量分布均匀,<1000Da的肽段质量占比<40%,1000~5000Da的肽段质量占比>35%,保留两亲性的同时还表现出较好的抗氧化能力。
(2)本发明方法制得具备两亲性和抗氧化性的酪蛋白酶解物,在应用方面既可以单独制备粒径在0.4~0.48μm范围内、液滴分布均匀的乳状液,又可以抑制吐温20制备的乳状液中10~32%的POV和13~45%的TBARS值。
(3)本发明提供的方法采用一步酶解法,酶解过程反应条件温和,工艺条件简单可控,容易实现批量生产,具有较好的工业化前景。
(4)本发明提供的方法和应用为酪蛋白酶解物应用于食品中提高物理稳定性和氧化稳定性奠定了基础,对延缓食品乳液的油脂氧化,提高食品稳定性具有重要的意义。
附图说明
图1为酪蛋白酶解物的分子量分布图。
图2为酪蛋白酶解物的粒径分布和微观结构。
图3为本酪蛋白酶解物-吐温20共制备乳液储藏28天时的过氧化值(注:*p<0.05,**p<0.05)。
图4为酪蛋白酶解物-吐温20共制备乳液储藏28天时的TBARS值(注:*p<0.05,**p<0.05)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
本发明所使用的胰蛋白酶购买自Sigma公司,木瓜蛋白酶购买自庞博公司,碱性蛋白酶和中性蛋白酶购自诺维信公司。
本发明中,测定酶解液的水解度,ABTS·+清除能力,螯合金属能力,分子量分布的方法如下:
(1)酶解液水解度的测定采用邻苯二甲醛(o-Phthalaldehyde,OPA)法。
取3mL OPA试剂(含0.8mg/mL OPA,38.1mg/mL四硼酸钠,1mg/mL SDS,0.88mg/mLDTT,溶剂为去离子水)与400μL样品溶液(实施例和对比例步骤3所得的上清液)混合,避光反应2min,在340nm处测定吸光度。
计算公式如下:
式中,A空白和A丝氨酸分别为去离子水和丝氨酸标准溶液代替样品的吸光度;0.9516为丝氨酸标准溶液的浓度,单位为mmol/L。
α、β和htot均为常数:乳清蛋白的α为1.0,β为0.4,htot为8.8;酪蛋白的α为1.039,β为0.383,htot为8.2。
(2)ABTS·+清除能力的测定方法如下:
ABTS·+储备液由14mmol/L ABTS溶液和4.9mmol/L K2S2O8等体积混合,室温避光静置12-16h获得,实验反应前将其稀释一定倍数,使其在734nm时吸光度达0.70±0.02。
在96孔酶标板中依次加入50μL样品溶液(实施例和对比例步骤3所得的上清液)和150μL ABTS·+测定液,30℃下反应30min,测定734nm处的吸光度。以不同浓度的Trolox溶液代替样品进行测试绘制标准曲线。根据标准曲线计算Trolox当量值。
(3)螯合金属离子能力的测定方法如下:
取0.5mL样品溶液(实施例和对比例步骤3所得的上清液),加入50μL 2mM FeCl2溶液,涡旋混合,反应5min;先后加入100μL 5mM Ferrozine试剂和2mL超纯水,再次涡旋混合均匀,室温下反应10min,测定其562nm处的吸光度值。同等条件下,以去离子水好不同浓度的EDTA溶液代替样品,分别作为空白组及标准螯合剂组,最终结果以EDTA当量值表示。
(4)分子量的测定方法如下:
采用凝胶色谱法测定样品的分子量分布;标准肽样品为:三肽GGG(相对分子质量189Da)、四肽GGYR(相对分子质量451Da)、抑肽酶(相对分子质量6512Da)、细胞色素C(相对分子质量12384Da)、牛血清白蛋白(相对分子质量66463Da),以各标准品的分子量与高效液相色谱法检测的各标准品的保留时间拟合直线方程,通过保留时间计算样品的分子量分布。高效液相色谱条件:色谱柱TSK GEL G2000 SWXl 7.8mm×300mm,检测波长220nm,流速0.5mL/min,流动相为20%乙腈,79.92%超纯水和0.08%三氟乙酸。
本发明中使用激光粒度仪(Mastersizer 2000)对步骤(5)制备的乳状液的粒径进行测定。
本发明中对步骤(6)中蛋白肽和吐温20共制备乳液过氧化值(Peroxide value,POV值)和TBARS值进行测定。
(1)乳液POV值的分析方法如下:
吸取0.3mL样品乳液,加入1.5mL异辛烷+异丙醇(3:1,v/v)混合溶剂,剧烈涡旋三次混合均匀后,3000r/min的转速下离心2min;吸取0.2mL(取样量视过氧化物含量高低而定)上清液,加入甲醇+正丁醇(2:1,v/v)混合溶剂2.8mL;然后加入20μL硫氰酸钾(3.94M)和20μL亚铁溶液(0.132M氯化钡和0.144M硫酸亚铁);混合均匀后在室温下避光静置20min;在510nm波长下测定其吸光度。以不同浓度的过氧化氢异丙苯代替样品测定绘制标准曲线。(2)乳液TABRS值的分析方法如下:
取1mL乳液与2mL TBA溶液混合,在沸水浴中加热15min;取出冷却到室温,4200rpm离心25min。取上清液在532nm处测定;以1,1,3,3-四乙氧基丙烷做标准曲线,计算样品TBARS值。
实施例1
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)将酪蛋白和蒸馏水分别称量按照质量份数1:10比例,在搅拌中将蛋白质缓慢加入水中,加入少量可食用碱使得pH至6.5-7.0,增加溶解性;
(2)预处理的剪切参数为10000rpm,2min,得到酪蛋白溶液;
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至7.5,继续搅拌至分散液无聚集颗粒;在37℃摇床中预热10min,按照酪蛋白质量的0.01%加入胰蛋白酶,37℃酶解6h后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物,水解度为4.61%;
(5)将酪蛋白酶解物按照1wt%溶解在10mM,pH为7.0的磷酸盐缓冲液中,搅拌1~2h至充分溶解,作为连续相,然后与大豆油按照质量分数10:1混合,然后在12000rpm条件下剪切2min,在30~35mPa条件下均质2次,得到酪蛋白酶解物单独制备的水包油乳状液,测定其粒径分布和微观结构;
(6)将酪蛋白酶解物按照1wt%溶解在10mM,pH为7.0的磷酸盐缓冲液中,搅拌1~2h至充分溶解,作为连续相,加入1.0wt%的吐温20,制备条件与实施例1步骤(5)中一致,得到酪蛋白酶解物-吐温20共制备的水包油乳液,在37℃下放置28天后测定抑制脂质初级氧化产物(POV)和次级氧化产物(TBARS)的生成量。
实施例2
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)同实施例1步骤(1);
(2)同实施例1步骤(2);
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至7.5,继续搅拌至分散液无聚集颗粒;在37℃摇床中预热10min,按照酪蛋白质量的0.1%加入胰蛋白酶,37℃酶解30min后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物,水解度为6.35%;
(5)同实施例1步骤(5);
(6)同实施例1步骤(6)。
实施例3
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)同实施例1步骤(1);
(2)同实施例1步骤(2);
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至8.0,继续搅拌至分散液无聚集颗粒;在55℃摇床中预热10min,按照酪蛋白质量的0.01%加入碱性蛋白酶,55℃酶解4h后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物,水解度为3.47%;
(5)同实施例1步骤(5);
(6)同实施例1步骤(6)。
实施例4
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)同实施例1步骤(1);
(2)同实施例1步骤(2);
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至7.0,继续搅拌至分散液无聚集颗粒;在50℃摇床中预热10min,按照酪蛋白质量的0.01%加入中性蛋白酶,50℃酶解4h后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物,水解度为2.52%;
(5)同实施例1步骤(5);
(6)同实施例1步骤(6)。
对比例1
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)同实施例1步骤(1);
(2)同实施例1步骤(2);
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至7.0,继续搅拌至分散液无聚集颗粒;在55℃摇床中预热10min,按照酪蛋白质量的0.5%加入木瓜蛋白酶,55℃酶解4h后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物,水解度为4.91%,电荷值大于-25mV,分子量分布不均匀,<1000Da的肽段含量占比>50%;
(5)同实施例1步骤(5);
(6)同实施例1步骤(6)。
对比例2
一种酪蛋白酶解物的制备方法,包括以下步骤:
(1)同实施例1步骤(1);
(2)同实施例1步骤(2);
(3)使用可食用的碱调节步骤(2)中的酪蛋白溶液pH至7.5,继续搅拌至分散液无聚集颗粒;在37℃摇床中预热10min,按照酪蛋白质量的0.5%加入胰蛋白酶,37℃酶解4h后,沸水浴15min灭酶。在酶解溶液冷却至室温后,在8000g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到高水解度的酪蛋白酶解物,水解度为8.79%,电荷值小于-25mV,分子量分布不均匀,<1000Da的肽段含量占比>70%,1000~5000Da的肽段含量占比<30%;
(5)同实施例1步骤(5);
(6)同实施例1步骤(6)。
由图1所示,实施例1-4具有不同酶切位点的蛋白酶制备酪蛋白肽的水解度在2.52~6.35%之间,电荷值在-27.35~-31.10mV,小于-25mV,各分子量范围内的肽段占比分布均匀,其中<1000Da的肽段质量占比<40%,3000~5000Da的肽段质量占比>35%,带电荷值较多和中高分子量肽段含量高有利于制备粒径颗粒更小,粒径分布集中均匀的乳状液。
对比例1中酶解物的水解度在上述范围内,但电荷值大于-25mV,分子量分布不均匀,<1000Da的肽段含量占比>50%,1000~5000Da的肽段含量占比<35%;而对比例2中酶解物的电荷值小于-25mV,但是其水解度高至8.79%,且分子量分布分布不均匀,<1000Da的肽段含量占比>70%,1000~5000Da的肽段含量占比<30%,较少的电荷量以及高分子量分布较少都不利于形成稳定的乳状液。
由图2所示,实施例1-4单独制备的乳状液粒径分布呈现紧邻的双峰分布,平均体积粒径(d4,3)范围为0.39~0.48μm,乳状液液滴细小均匀。而具有接近水解度的对比例1单独制备的乳状液呈现不规则的多峰分布,其平均体积粒径为36.25μm,乳状液液滴较大;对比例2中的乳状液呈现距离较远的双峰分布,粒径为13.82μm,表明有液滴絮凝。
由图3-4所示,实施例1-4所得乳液可以显著地抑制脂质氧化产生的POV和TBARS值,特别是实施例1,抑制率分别为30.48%和45.72%,而对比例2没有表现出显著地抑制效果。
由表1可知,实施例1-4所获得的酪蛋白酶解物的水解度在低中范围内,且电荷值为-27.35~-31.10mV,具有形成乳状液的潜力。对比例1中的酪蛋白酶解物的水解度与实施例1-4相近,然而由于木瓜蛋白酶特异性水解半胱氨酸等极性不带电氨基酸,及亮氨酸和苯丙氨酸等疏水非极性氨基酸,产生的肽带的电荷较少,且其<1000Da的的肽段质量占比>50%,因此不利于形成液滴微小均匀的乳状液。而对比例2中所获得的酪蛋白酶解物虽然电荷值接近,但是其水解度过高,中等分子量1000-5000Da的肽段质量占比显著低于于实施例1,因此不利于形成液滴微小均匀的乳状液。
由表1可知,实施例1-4中酪蛋白酶解物的ABTS·+清除能力为170.81~432.62μmolTE/g样品,螯合金属离子能力171.47~206.25μmol EDTA/g样品。此外,对比例2的水解度大于实施例1,具有最强的自由基清除能力。
结合图3-4,说明酪蛋白酶解物的体外抗氧化能力不是决定乳状液氧化稳定性的唯一因素,还要考虑物理性质对其在液滴界面吸附情况的影响。
表1
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种酪蛋白酶解物,其特征在于制备方法包括以下步骤:
(1)将酪蛋白和蒸馏水按照质量份数1:10比例,在搅拌中将蛋白质缓慢加入水中,调节pH至6.5-7.0;
(2)10000 rpm,2 min剪切,得到酪蛋白溶液;
(3)调节酪蛋白溶液pH至7.5,继续搅拌至分散液无聚集颗粒;在37℃摇床中预热10min,按照酪蛋白质量的0.01%加入胰蛋白酶,37℃酶解6h后,沸水浴15 min灭酶;
在酶解溶液冷却至室温后,在8000 g,4℃条件下离心,获得上清液;
或,调节酪蛋白溶液pH至7.5,继续搅拌至分散液无聚集颗粒;在37℃摇床中预热10min,按照酪蛋白质量的0.1%加入胰蛋白酶,37℃酶解30min后,沸水浴15 min灭酶;
在酶解溶液冷却至室温后,在8000 g,4℃条件下离心,获得上清液;
或,调节酪蛋白溶液pH至8.0,继续搅拌至分散液无聚集颗粒;在55℃摇床中预热10min,按照酪蛋白质量的0.01%加入碱性蛋白酶,55℃酶解4h后,沸水浴15 min灭酶;
在酶解溶液冷却至室温后,在8000 g,4℃条件下离心,获得上清液;
或,调节酪蛋白溶液pH至7.0,继续搅拌至分散液无聚集颗粒;在50℃摇床中预热10min,按照酪蛋白质量的0.01%加入中性蛋白酶,50℃酶解4h后,沸水浴15 min灭酶;
在酶解溶液冷却至室温后,在8000 g,4℃条件下离心,获得上清液;
(4)随即取上清液浓缩至固形物含量达到30%,进行喷雾干燥,得到酪蛋白酶解物 。
2.根据权利要求1所述的酪蛋白酶解物,其特征在于:
步骤(1)所述的酪蛋白为蛋白含量90%以上的酪蛋白酸钠。
3.根据权利要求1所述的酪蛋白酶解物,其特征在于:
步骤(4)所述喷雾干燥的进风温度为160-210℃,排风温度为70-120 ℃。
4.根据权利要求1所述的酪蛋白酶解物,其特征在于:所带负电荷值<-25 mV,分子量<1000的肽段质量占比<40%, 1000~5000 Da的肽段质量占比>35%。
5.权利要求1~4任一所述的酪蛋白酶解物在制备水包油乳液中的应用。
6.一种水包油乳液,其特征在于:采用磷酸盐缓冲液为连续相溶剂,加入权利要求1~4任一所述的酪蛋白酶解物和植物油,经过剪切和均质,得到水包油乳液;
所述酪蛋白酶解物在连续相中的浓度为1.0wt%;
所述的磷酸盐缓冲液为10 mM、pH值为7.0的磷酸盐缓冲液;所述的剪切为12000 rpm剪切2 min;
所述的均质为30~35 Mpa下均质若干次。
7.一种水包油乳液,其特征在于:采用磷酸盐缓冲液为连续相溶剂,加入权利要求1~4任一所述的酪蛋白酶解物和乳化剂,经过剪切和均质,得到水包油乳液;
所述酪蛋白酶解物在连续相中的浓度为1.0wt%;
所述的磷酸盐缓冲液为10 mM、pH值为7.0的磷酸盐缓冲液;所述的剪切为12000 rpm剪切2 min;
所述的均质为30~35 Mpa下均质若干次。
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