CN112080152B - 一种热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法 - Google Patents
一种热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法 Download PDFInfo
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Abstract
本发明公开了一种热诱导花生球蛋白‑罗勒籽胶复合凝胶的制备方法,是以脱脂花生粉为原料,采用硫酸铵沉淀法得到花生球蛋白,先制备花生球蛋白溶液与罗勒籽胶溶液,充分混合后加热,后经过冷却获得花生球蛋白‑罗勒籽胶复合凝胶。本方法通过热诱导制备复合凝胶,其凝胶强度>38.72g、持水力>90.18%,凝胶性能良好,生产过程简单,对环境无污染,降低环保压力。本发明安全性高,工艺简单,无需复杂加工设备,能耗低,易于工业化生产。
Description
技术领域
本发明涉及一种热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法,属于食品蛋白质加工的技术领域。
背景技术
花生作为最主要的油料作物之一,在我国具有悠久的种植历史和广泛的种植面积,花生约含有50%的脂肪、25%-36%的蛋白质,以及部分其他活性物质。脱脂花生粉是花生榨油后的副产品,含有丰富的蛋白质,是我国具有优势的大宗蛋白质资源。但是现主要用作饲料,这不仅降低了商品的价值,而且还造成了花生资源的浪费。脱脂花生粉中蛋白质含量约为50%,氨基酸种类丰富,营养价值高,而且具有独特的香味。根据溶解性分为水溶性蛋白(10%)和盐溶性蛋白(90%),后者包括花生球蛋白(73%)、伴花生球蛋白I(6%)和伴花生球蛋白II(21%)。花生蛋白利用不充分是因为其功能特性较差,占主要部分的球蛋白结构紧密,热变性温度高,不利于加工。蛋白质的功能特性包括凝胶化、溶解性、乳化性、发泡性等等。其中凝胶性能在食品加工过程中起着关键作用,因为它会影响食品的质地和口感,从而影响消费者对产品的可接受度。
蛋白凝胶化包括蛋白质肽链的伸展、裂解、结合及聚集等四个复杂的过程,通过伸展肽链间的相互作用而形成凝胶。首先,蛋白在适当的条件下,蛋白质分子逐渐展开,使得埋藏在蛋白分子链内部的巯基、二硫键、疏水性基团等功能基团暴露出来,然后相邻的分子通过氢键、疏水相互作用、二硫键、范德华力以及静电相互作用等作用力形成网状的空间结构,从而形成凝胶。
蛋白凝胶的制备方法主要有:热诱导、酶促交联、酸诱导、盐诱导和高分子复配法等。
1、热诱导:加热是诱导凝胶形成最直接的一种方法。当蛋白质达到一定浓度时,蛋白质分子在加热过程中展开,暴露出疏水基团、游离巯基等功能性基团,然后展开的蛋白质通过分子间的氢键、静电、疏水相互作用以及游离巯基形成二硫键等使其聚集,聚集体初步形成一个凝胶网络结构。随后,将凝胶放在冰水中迅速冷却,凝胶网络进一步得到加固。
2、酶促交联:将蛋白质进行预热处理,使其分子展开,以便后续的酶促交联。一方面,蛋白质经过酶作用后,通过非共价键(疏水相互作用)促使其分子内或分子间交联,促进凝胶的形成。另一方面,酶可以催化蛋白质和蛋白质分子间或者蛋白质和其他分子间形成共价键,促进凝胶网络的形成。转谷氨酰胺酶属于酰基转移酶,可以催化蛋白质分子中的谷氨酰胺和赖氨酸之间形成异肽键,促进蛋白质分子间的交联。漆酶作为一种多酚氧化酶,能够催化氧化阿魏酸形成醌类,与蛋白质分子中的巯基、氨基、亚氨基等发生加成反应,使得阿魏酸和蛋白质分子之间形成更加稳定的C-N或C-S共价键。酶促交联所需蛋白质的浓度较低。
3、酸诱导:通常选择在蛋白质分子之间可产生强静电排斥力的pH和离子强度条件下,将较低浓度蛋白溶液加热到热变性温度以上,然后将溶液冷却至环境温度。加入酸诱导试剂如葡萄糖酸内酯(GDL)改变溶液条件pH值,以减少蛋白质分子之间的静电排斥,使蛋白质分子间发生交联,形成酸诱导凝胶。
4、盐诱导:通常选择在蛋白质分子之间可产生强静电排斥力的pH和离子强度条件下,将较低浓度蛋白溶液加热到热变性温度以上,然后将溶液冷却至环境温度。加入盐溶液改变溶液的离子强度,以减少蛋白质分子之间的静电排斥,使蛋白质分子间发生交联,形成盐诱导凝胶。
5、高分子复配:在适当浓度的蛋白质溶液中,加入高分子物质如多糖。与单纯的热诱导蛋白凝胶相似,首先将蛋白质溶液加热,使蛋白质分子展开,多糖通常会与展开的蛋白质分子通过疏水、静电、氢键等相互作用结合,促进蛋白质聚集体的形成,使得蛋白质进一步交联,形成比单蛋白质凝胶更稳定的网络结构。由于多糖和蛋白的热力学不相容性,所形成的复合凝胶分为三种网络结构:耦合型、互穿型、相分离型。
发明内容
本发明旨在提供一种热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法,本方法制得的复合凝胶具有凝胶强度高和持水性好的特点。本发明原料新颖,安全性高,加工工艺简单,过程易控,无需复杂加工设备,有利于生产性能良好的蛋白质凝胶,提高原料的附加值。
本发明通过将花生球蛋白与罗勒籽胶进行复配,加热制成复合凝胶。罗勒籽胶加入蛋白质溶液后,可吸收溶胀的蛋白质分子周围多余的水分子;由于空间位阻效应,蛋白质局部浓度增大;在加热展开的过程中蛋白质分子间距缩小,分子间的相互作用更容易发生;而且罗勒籽胶可以通过疏水、氢键相互作用与蛋白质分子结合,形成具有连续网络结构的相分离型复合凝胶。罗勒籽胶能够有效地促进花生球蛋白的凝胶功能特性和流变学特性。
本发明热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法,是以脱脂花生粉为原料,采用硫酸铵沉淀法得到花生球蛋白,先制备花生球蛋白溶液与罗勒籽胶溶液,充分混合后加热,后经过冷却获得花生球蛋白-罗勒籽胶复合凝胶。具体包括以下步骤:
步骤1:将脱脂花生粉分散在0.05M、pH为7.9的磷酸缓冲溶液中,室温下搅拌分散1小时,随后以8000g的离心力离心30分钟,收集上清液,加入硫酸铵进行盐析,将溶液置于4℃冰箱冷沉3小时;将冷沉后的溶液混匀,于4℃条件下离心30分钟,弃去上清液留取蛋白质沉淀;将沉淀溶于最小体积的磷酸缓冲溶液中,然后倒入截留分子量为10-14kDa透析袋中,在4℃的层析柜中搅拌透析48小时以上;蛋白透析液经冷冻干燥得到花生球蛋白;将花生球蛋白与0-10mM的NaCl溶液以22-26g:100mL的比例混合,置于磁力搅拌器上搅拌2小时,然后放置于4℃冰箱8-12h,使其充分水合。
步骤2:将罗勒籽在短时间内用蒸馏水冲洗,调节蒸馏水pH为8,将罗勒籽与pH为8的蒸馏水混合,在68℃水浴加热并搅拌20分钟至种籽溶胀,使用带有金属刀头的桨式搅拌器在高速旋转的条件下刮去溶胀的种籽表面胶层,得到的胶质用干酪布过滤;将胶质溶液与95%的乙醇溶液以1:3的体积比混合,搅拌后在4℃下醇沉12小时;将醇沉后的混合物在离心力为3000g的条件下离心10分钟,得到沉淀的罗勒籽胶,将其复溶于蒸馏水中,放置于50℃的真空干燥箱中干燥,得到的干燥物用研钵研磨得到罗勒籽胶粉末;将罗勒籽胶粉末与0-10mM的NaCl溶液以0.5-2g:100mL的比例混合,在室温下搅拌2小时,然后置于4℃冰箱过夜,使其充分水合。
步骤3:将步骤2获得的罗勒籽胶溶液与步骤1获得的花生球蛋白溶液混合,并在室温下充分搅拌2小时。
步骤4:调节步骤3混合溶液的pH值为7-11,然后于95℃条件下加热20-40分钟,随后立即置于冰水浴冷却至室温,然后将复合凝胶置于4℃储存8-12h。
步骤3中,所述罗勒籽胶溶液与花生球蛋白溶液以1:1的体积比混合。
与已有技术相比,本发明的有益效果体现在:
1、本发明使用多糖与蛋白复配形成复合凝胶的方法。与其他方法相比,具有简便、高效、绿色、效果好的特点。
2、本发明使用的多糖是罗勒籽胶,它是一种从溶胀罗勒籽表层收集的种子粘液,是一种新型的高分子量的阴离子杂多糖。罗勒籽胶具有原纤维及球状的结构,可作为增稠剂、稳定剂、脂肪替代剂,应用于低脂黄油、减脂蛋黄酱等产品中。由于其具有良好的凝胶特性,罗勒籽胶可应用于奶酪、面包等食品中以改善产品的质构。罗勒籽也可作为一种药用材料,对头痛、咳嗽、腹泻、便秘等具有一定的治疗功效。此外,罗勒籽胶还可被用作药物赋形剂、崩解剂、抗糖尿病剂,应用于医药领域。随着人们饮食模式的转变及对植物型健康食品的需求增大,相比其他多糖,具有优良性能的罗勒籽胶更符合人们的需求。
3、本发明制备的蛋白-多糖复合凝胶相比于单一的蛋白凝胶体系,具有较好的稳定性和理化性质。
4、本发明使用罗勒籽胶与花生球蛋白形成复合凝胶,复合凝胶的凝胶强度和持水力随胶的添加量增加而增大。例如,在花生球蛋白浓度为12%,体系pH为9的条件下,当罗勒籽胶添加量达0.75%时,复合凝胶的凝胶强度增加到38.72g,持水力增加到90.18%。由此可知,加入一定浓度的罗勒籽胶使得复合凝胶的凝胶强度和持水力显著增大,提高了花生球蛋白和罗勒籽胶在食品领域中的应用价值。
5、本发明安全性高,无环境污染,工艺简单,无需复杂加工设备,易于工业化生产。
附图说明
图1是实施例1中花生球蛋白-罗勒籽胶复合凝胶的硬度和持水力。制备11%花生球蛋白溶液,添加不同浓度的罗勒籽胶溶液,调节体系pH值为7,将混合溶液加热20分钟并冷却后,得到复合凝胶。从图1中可以看出,当添加0.25%的罗勒籽胶时,复合凝胶的凝胶强度和持水力分别为8.76g和46.32%。复合凝胶的凝胶强度和持水力随罗勒籽胶添加量增加而增大。当罗勒籽胶添加量为0.75%时,凝胶强度和持水力显著增大至19.53g和78.15%。
图2是实施例2中花生球蛋白-罗勒籽胶复合凝胶的硬度和持水力。制备12%花生球蛋白溶液,添加不同浓度的罗勒籽胶溶液,调节体系pH值为9,将混合溶液加热30分钟并冷却后,得到复合凝胶。从图2中可以看出,当添加0.25%的罗勒籽胶时,复合凝胶的凝胶强度和持水力分别为19.76g和60.99%。复合凝胶的凝胶强度和持水力随罗勒籽胶添加量增加而增大。当罗勒籽胶添加量为0.75%时,凝胶强度和持水力显著增大至38.72g和90.18%。与实施例1相比,实施案例2中相对应的复合凝胶具有较高的凝胶强度和持水力。因为在实施案例2中,蛋白质浓度增大;同时体系的pH值增大,利于提高可溶性蛋白质的含量;加热时间增加,可以让蛋白质充分变性,利于凝胶的形成。在该体系条件下,NaCl的加入有助于增强花生球蛋白分子和罗勒籽胶相互作用,从而提高凝胶强度与持水力。
图3是实施例3中花生球蛋白-罗勒籽胶复合凝胶的硬度和持水性。制备13%花生球蛋白溶液,添加不同浓度的罗勒籽胶溶液,调节体系pH值为11,将混合溶液加热40分钟并冷却后,得到复合凝胶。从图3中可以看出,添加0.25%的罗勒籽胶的复合凝胶的凝胶强度和持水力分别为13.88g和56.99%。复合凝胶的凝胶强度和持水力随罗勒籽胶添加量增加而增大。当罗勒籽胶添加量为0.75%时,凝胶强度和持水力显著增大至34.09g和85.41%。与实施例1相比,实施例3中相对应的复合凝胶具有较高的凝胶强度和持水力。与实施例2相比,实施例3中相对应的复合凝胶的凝胶强度和持水力略低,可能是由于该体系条件下,蛋白质分子和罗勒籽胶相互作用减弱。由于体系中蛋白质含量较高,复合凝胶的强度和持水力仍高于施例1中所得的复合凝胶。
通常,添加多糖能够促使蛋白质在低于其凝胶形成浓度条件下形成复合凝胶。因此,在花生球蛋白-罗勒籽胶复合凝胶中,罗勒籽胶溶液的浓度起重要作用。同时,体系的pH、蛋白质含量、盐离子(Na+)的添加、加热温度对最终的凝胶性质会有影响。
具体实施方式
非限定实施例叙述如下:
实施例1:
1、将脱脂花生粉以1g:20mL的比例分散于0.05M、pH为7.9的磷酸缓冲溶液中,室温下搅拌1小时后,以8000g的离心力离心30分钟,收集上清液以24g:100mL的比例加入硫酸铵进行盐析。将溶液置于4℃冰箱冷沉3小时;将冷沉后的溶液混匀,于4℃条件下离心30分钟,弃去上清液取蛋白质沉淀;将沉淀溶于最小体积的磷酸缓冲液中,然后倒入截留分子量为10-14kDa透析袋中,在4℃的层析柜中搅拌透析48小时以上;蛋白透析液经冷冻干燥得到花生球蛋白;将花生球蛋白与蒸馏水(即0mM NaCl溶液)以22g:100mL的比例混合,置于磁力搅拌器上搅拌2小时后,置于4℃冰箱过夜,使其充分水合;
2、将罗勒籽在短时间内用蒸馏水冲洗,调节蒸馏水pH为8,将罗勒籽与pH为8的蒸馏水以1g:68mL的比例混合,在68℃水浴加热并搅拌20分钟至种籽溶胀,使用带有金属刀头的桨式搅拌器在高速旋转的条件下刮去溶胀的种籽表面胶层,得到的胶质用干酪布过滤;将胶质溶液与95%的乙醇溶液以1:3的体积比混合,搅拌后在4℃下醇沉12小时;将醇沉后的混合物在离心力为3000g的条件下离心10分钟,得到沉淀的罗勒籽胶,将其复溶于蒸馏水,放置在50℃的真空干燥箱中干燥4小时,得到的干燥物用研钵研磨得到粉末;将罗勒籽胶与蒸馏水(即0mM NaCl溶液)以0.5-2g:100mL的比例混合,在室温下搅拌2小时后,置于4℃冰箱过夜,使其充分水合。
3、将罗勒籽胶溶液与上述花生球蛋白溶液混合,并在室温下充分搅拌2小时,使得花生球蛋白溶液和罗勒籽胶溶液充分混匀。
4、调节上述混合溶液pH值为7,将上述混合溶液在95℃条件下加热20分钟后,立即置于冰水浴冷却至室温,然后将复合凝胶置于4℃储存一夜。
经测定,如图1所示,随着罗勒籽胶的添加量从0.25%增加到0.75%,热诱导的花生球蛋白和罗勒籽胶复合凝胶的凝胶强度由8.76g增加到19.53g,持水力由46.32%增加到78.15%。
实施例2:
1、将脱脂花生粉以1g:20mL的比例分散于0.05M、pH为7.9的磷酸缓冲溶液中,室温下搅拌1小时后,在离心力为8000g的条件下离心30分钟,收集上清液以24g:100mL的比例加入硫酸铵进行盐析。将溶液置于4℃冰箱冷沉3小时;将冷沉后的溶液混匀,于4℃条件下离心30分钟,弃去上清液取蛋白质沉淀;将沉淀溶于最小体积的磷酸缓冲液中,然后倒入截留分子量为10-14kDa透析袋中,在4℃的层析柜中搅拌透析48小时以上;蛋白透析液经冷冻干燥得到花生球蛋白;将花生球蛋白与5mM NaCl溶液以24g:100mL的比例混合,置于磁力搅拌器上搅拌2小时后,置于4℃冰箱过夜,使其充分水合;
2、将罗勒籽在短时间内用蒸馏水冲洗,调节蒸馏水pH为8,将罗勒籽与pH为8的蒸馏水以1g:68mL的比例混合,在68℃下水浴加热并搅拌20分钟至种籽溶胀,使用带有金属刀头的桨式搅拌器在高速旋转的条件下刮去溶胀的种籽表面胶层,得到的胶质用干酪布过滤;将胶质溶液与95%的乙醇溶液以1:3的体积比混合,搅拌后在4℃下醇沉12小时;将醇沉后的混合物在离心力为3000g的条件下离心10分钟,得到沉淀的罗勒籽胶,将其复溶于蒸馏水,放置在50℃的真空干燥箱中干燥4小时,得到的干燥物用研钵研磨得到粉末;将罗勒籽胶与5mM NaCl溶液以0.5-2g:100mL的比例混合,在室温下搅拌2小时后,置于4℃冰箱过夜,使其充分水合。
3、将罗勒籽胶溶液与上述花生球蛋白溶液混合,并在室温下充分搅拌2小时,使得花生球蛋白溶液和罗勒籽胶溶液充分混匀。
4、调节上述混合溶液pH值为9,将上述混合溶液在95℃条件下加热30分钟后,立即置于冰水浴冷却至室温,然后将复合凝胶置于4℃储存一夜。
经测定,如图2所示,随着罗勒籽胶的添加量从0.25%增加到0.75%,热诱导的花生球蛋白和罗勒籽胶复合凝胶的凝胶强度由19.76g增加到38.72g,持水力由60.99%增加到90.18%。
实施例3:
1、将脱脂花生粉以1g:20mL的比例分散于0.05M、pH为7.9的磷酸缓冲溶液中,室温下搅拌1小时后,以8000g的离心力离心30分钟,收集上清液以24g:100mL的比例加入硫酸铵进行盐析。将溶液置于4℃冰箱冷沉3小时;将冷沉后的溶液混匀,于4℃条件下离心30分钟,弃去上清液取蛋白质沉淀;将沉淀溶于最小体积的磷酸缓冲液中,然后倒入截留分子量为10-14kDa透析袋中,在4℃的层析柜中搅拌透析48小时以上;蛋白透析液经冷冻干燥得到花生球蛋白;将花生球蛋白与10mM NaCl溶液以26g:100mL的比例混合,置于磁力搅拌器上搅拌2小时后,置于4℃冰箱过夜,使其充分水合;
2、将罗勒籽在短时间内用蒸馏水冲洗,调节蒸馏水pH为8,将罗勒籽与pH为8的蒸馏水以1g:68mL的比例混合,在68℃下水浴加热并搅拌20分钟至种籽溶胀,使用带有金属刀头的桨式搅拌器在高速旋转的条件下刮去溶胀的种籽表面胶层,得到的胶质用干酪布过滤;将胶质溶液与95%的乙醇溶液以1:3的体积比混合,搅拌后在4℃下醇沉12小时;将醇沉后的混合物在离心力为3000g的条件下离心10分钟,得到沉淀的罗勒籽胶,将其复溶于蒸馏水,放置在50℃的真空干燥箱中干燥4小时,得到的干燥物用研钵研磨得到粉末;将罗勒籽胶与10mM NaCl溶液以0.5-2g:100mL的比例混合,在室温下搅拌2小时后,置于4℃冰箱过夜,使其充分水合。
3、将罗勒籽胶溶液与上述花生球蛋白溶液混合,并在室温下充分搅拌2小时,使得花生球蛋白溶液和罗勒籽胶溶液充分混匀。
4、调节上述混合溶液pH值为11,将上述混合溶液在95℃条件下加热40分钟后,立即置于冰水浴冷却至室温,然后将复合凝胶置于4℃储存一夜。
经测定,如图3所示,随着罗勒籽胶的添加量从0.25%增加到0.75%,热诱导的花生球蛋白和罗勒籽胶复合凝胶的凝胶强度由13.88g增加到34.09g,持水力由56.99%增加到85.41%。
Claims (4)
1.一种热诱导花生球蛋白-罗勒籽胶复合凝胶的制备方法,其特征在于:以脱脂花生粉为原料,采用硫酸铵沉淀法得到花生球蛋白,先制备花生球蛋白溶液与罗勒籽胶溶液,充分混合后加热,后经过冷却获得花生球蛋白-罗勒籽胶复合凝胶;包括以下步骤:
步骤1:将脱脂花生粉分散在磷酸缓冲溶液中,室温下搅拌分散1小时,随后离心30分钟,收集上清液,加入硫酸铵进行盐析,将溶液置于4℃冰箱冷沉3小时;将冷沉后的溶液混匀,于4℃条件下离心30分钟,弃去上清液留取蛋白质沉淀;将沉淀溶于最小体积的磷酸缓冲溶液中,然后倒入截留分子量为10-14 kDa的透析袋中,在4℃的层析柜中搅拌透析48小时以上;蛋白透析液经冷冻干燥得到花生球蛋白;将花生球蛋白与5-10 mM的NaCl溶液以24-26g:100mL的比例混合,置于磁力搅拌器上搅拌2小时,然后放置于4℃冰箱8-12h,使其充分水合;
步骤2:将罗勒籽在短时间内用蒸馏水冲洗,调节蒸馏水pH为8,将罗勒籽与pH为8的蒸馏水混合,在68℃水浴加热并搅拌20分钟至种籽溶胀,使用带有金属刀头的桨式搅拌器在高速旋转的条件下刮去溶胀的种籽表面胶层,得到的胶质用干酪布过滤;将胶质溶液与乙醇溶液混合,搅拌后在4℃下醇沉12小时;将醇沉后的混合物离心,得到沉淀的罗勒籽胶,将其复溶于蒸馏水中,放置于50℃的真空干燥箱中干燥,得到的干燥物用研钵研磨得到罗勒籽胶粉末;将罗勒籽胶粉末与5-10 mM的NaCl溶液以0.5-2g:100mL的比例混合,在室温下搅拌2小时,然后置于4℃冰箱过夜,使其充分水合;
步骤3:将步骤2获得的罗勒籽胶溶液与步骤1获得的花生球蛋白溶液以1:1的体积比混合,并在室温下充分搅拌2小时;
步骤4:调节步骤3混合溶液的pH值为9-11,然后于95℃条件下加热30-40分钟,随后立即置于冰水浴冷却至室温,然后将复合凝胶置于4℃储存8-12h。
2.根据权利要求1所述的制备方法,其特征在于:
步骤1中,所述磷酸缓冲溶液为0.05M、pH为7.9的磷酸缓冲溶液。
3.根据权利要求1所述的制备方法,其特征在于:
步骤1中,所述离心时所用的离心力均为8000g;步骤2中,所述离心时所用的离心力均为3000g。
4.根据权利要求1所述的制备方法,其特征在于:
步骤2中,所述乙醇溶液的浓度为95%;胶质溶液与乙醇溶液以1:3的体积比混合。
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