CN111808903B - 一种安全高效蛋白抗冻剂及其制备方法和应用 - Google Patents
一种安全高效蛋白抗冻剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种安全高效新型蛋白抗冻剂及其制备方法和应用,所述蛋白抗冻剂的制备过程为:鱼鳞经干燥、预处理、蒸汽闪爆处理、脱钙、酶解、热水浸提及冻干后得到,所得蛋白抗冻剂可作为冷冻保护剂应用于水产品、酶及菌种的冷冻保藏;本发明原料来源广泛、成本低廉,工艺简单、高效,制备的新型抗冻剂具有安全营养和适用范围广等优点,能够有效控制冷冻食品及生物制品的品质劣变,增强其冻融稳定性。
Description
技术领域
本发明属于食品加工与贮藏领域,具体涉及一种安全高效新型蛋白抗冻剂及其制备方法和应用,可作为冷冻保护剂应用于水产品、酶及菌种的冷冻保藏。
背景技术
低温冷冻储藏是生鲜食品及其原料的主要贮藏方式之一,但冷冻处理不可避免引发冰晶形成,进一步导致原料组织破裂,食品组分发生系列变化如脂质氧化、蛋白质氧化和聚集,最终使食品的加工特性及营养品质显著降低。添加抗冻剂是增强生鲜食品冻藏稳定性普遍方法之一。传统的抗冻剂为蔗糖、山梨醇及复合磷酸盐构成的复合物,因其良好的抗冻效果以及较为低廉的成本而被广泛应用于食品的冷冻贮藏。随着食品科学的发展及消费者安全营养观念的增强,此类传统抗冻剂潜在的安全风险引起了消费者担忧。例如,蔗糖具有较高热量,会导致肥胖和龋齿等,亦不适用于糖尿病患者食用,此外蔗糖较高甜度也会改变食品本身口感;已有研究证实,复合磷酸盐存在胃肠病加重风险。由此可见,这种传统抗冻剂的安全性及适用性受到一定程度限制。作为新型抗冻剂的典型代表,抗冻蛋白和抗冻肽表现出优异的抗冻特性,但其高昂的生产成本,苛刻的制备条件以及潜在安全隐患也限制了它们的广泛应用,因此开发一种兼具安全及高效抗冻能力的新型抗冻剂成为冷冻食品行业亟待解决的关键技术问题。
目前,从现有抗冻剂如抗冻蛋白、抗冻肽以及多糖胶体的抗冻结构基础出发,通过由下到上的结构设计开发新型抗冻剂成为主要的研发思路之一。将小分子物质经化学修饰或组装改性可制备具有与上述抗冻剂抗冻能力相媲美的冷冻保护剂,已应用于生物组织及特殊材料的冷冻保护(Graham, B.,Fayter,A.E.R.et al(2019).Synthesis ofAnthracene Conjugates of Truncated Antifreeze Protein Sequences:Effect of theEnd Group and Photocontrolled Dimerization on Ice RecrystallizationInhibition Activity.Biomacromolecules,20,4611-4621)。然而以化学合成方法所制备的抗冻剂仍存在一定的安全风险,无法直接应用于食品的冷冻保护。来源于水产品加工主要副产物的鱼鳞胶兼具了抗冻蛋白的结构特性(Gly-X-Y序列分布和PPⅡ构象)以及多糖胶体的凝胶特性,具备了抗冻剂所需的分子结构基础,因此有望作为一种新型抗冻剂应用于食品的冷冻保护。此外,鱼鳞胶上述抗冻特性与分子量的大小密不可分。鱼鳞胶小分子肽已被证实具有类抗冻蛋白的抗冻活性(Fu,W.Q.,Wang,P.X.,et al.(2019).Preparation,primary structure and antifreeze activity of antifreeze peptides fromScomberomorus niphonius skin.Lwt-Food Science and Technology,101,670-677);鱼鳞胶大分子量组分的凝胶性与多糖凝胶抗冻机制类似(Gaukel,V.,Leiter,A.et al.(2014).Synergism of different fish antifreeze proteins and hydrocolloids onrecrystallization inhibition of ice in sucrose solutions.Journal of FoodEngineering,141,44-50)。因此控制鱼鳞胶分子分布的合理构成以最大程度发挥其抗冻特性是制备明胶基抗冻剂的关键。鱼鳞胶的主要制备方法包括“预处理+热水浸提法”,其中预处理方法包括酸、碱及酶处理。但由于鱼鳞本身胶原蛋白与羟基磷灰石密切结合的状态,采用上述方法制备鱼鳞胶的效率受到极大限制。
采用一定的预处理手段破坏鱼鳞原本致密的结构以实现后续高效的酶作用或是制备鱼鳞胶的有效手段之一。蒸汽闪爆技术是一种先进的生物质预处理技术,近年来受到学者的普遍关注。它具有工艺简单、高效、节能、无污染、处理成本低等优点。蒸汽爆破技术是将物料置于压力容器内,利用物料表层微孔,将高压饱和蒸汽渗透至物料组织内部,其中部分饱和水蒸汽形成高温液态水,由于物料表层微孔较小,在物料外界瞬间降为大气压时,物料内部的高压水蒸气只有极少部分可以通过物料表层的微孔释放到大气中,大部分饱和水蒸汽极速膨胀,同时高温液态水也迅速爆沸形成闪蒸,大量蒸汽在极短的时间内完成绝热膨胀做功把热能转化为机械能,蒸汽膨胀做功产生的冲击波对物料产生剪切力,从而实现破坏物料组织内部结构的目的。赵黎明公开了一种甲壳素的清洁生产工艺,通过高密度蒸汽闪爆装置对原料进行瞬时高压蒸汽闪爆预处理,通过酶解将原料中大部分蛋白质溶解提取制成食品级水解蛋白,整个工艺具有工艺简单、工序较短、操作方便的优点(CN201310056441.9);侯秀良公开了一种碱预处理、低压力蒸汽闪爆与生物酶联合处理综合利用棉秆皮的方法(CN201410298683.3)。由此可知,蒸汽闪爆技术可作为一种新型技术,能够破坏鱼鳞原有致密组织结构以增强鱼鳞胶的高效可控制备能力。
发明内容
针对当前用于生鲜食品冷冻保护的各种抗冻剂存在的不足,本发明提供了一种安全高效新型蛋白抗冻剂及其制备方法和应用。本发明蛋白抗冻剂的制备过程为:鱼鳞经干燥、预处理、蒸汽闪爆处理、脱钙、酶解、热水浸提及冻干后得到。
本发明的技术方案如下:
一种蛋白抗冻剂,按如下方法制备得到:
(1)将新鲜鱼鳞去除表面残肉,清洗并干燥后得到原料鱼鳞,将原料鱼鳞浸入氢氧化钠水溶液中,20~30℃(优选20℃)搅拌2~3h(优选2.5h),以脱除鱼鳞中的杂蛋白,之后清洗,干燥,完成预处理(储藏备用);
所述鱼鳞来自常见的淡水鱼种(例如:青、草、鲤、鲫、鲢、鳙、鲂、罗非鱼)或常见的海水鱼种(例如:大黄鱼、小黄鱼、乌鱼、鳕鱼、带鱼、石斑鱼、海鲈鱼、多宝鱼);
所述氢氧化钠水溶液的浓度为0.1~0.2M,优选0.1M;
所述氢氧化钠水溶液的体积用量以原料鱼鳞的质量计为15~25mL/g,优选20mL/g;
(2)将经过步骤(1)预处理的鱼鳞放入蒸汽闪爆设备的反应处理腔中,在蒸汽压力为1~3MPa (优选1.5MPa)下保压1~4min(优选1min),待爆破腔底盖自动下拉后,腔内压力迅速降至大气压,鱼鳞内水分形成闪蒸,由内而外对物料完成绝热做功,实现蒸汽闪爆处理;
(3)将经过步骤(2)蒸汽闪爆处理的鱼鳞加到脱钙液中,20~30℃(优选20℃)搅拌2~3h(优选2h),完成脱钙处理,接着添加酶溶液,在20~40℃、pH=2-3的条件下酶解2~5h,灭酶,清洗,之后于50~70℃(优选50℃)下,以水为溶剂恒温浸提3~5h(优选3h),过滤得到浸提液,所得浸提液经真空冷冻干燥,得到所述蛋白抗冻剂(置于避光干燥处储藏);
所述脱钙液为盐酸、醋酸或柠檬酸的水溶液,浓度为0.3~0.5M,优选0.3M盐酸;
所述脱钙液的体积用量以原料鱼鳞的质量计为20mL/g;
所述酶溶液为质量分数1%~5%蛋白酶的水溶液,所述蛋白酶选自胃蛋白酶、中性蛋白酶或碱性蛋白酶,优选胃蛋白酶,酶活为1:3000;
所述酶溶液的体积用量以原料鱼鳞的质量计为20~30mL/g;
优选的酶解条件为:温度35℃,pH=2.5,酶解时间4h;
所述灭酶的方法为:于90℃下恒温灭酶3~5min。
本发明制备的蛋白抗冻剂可作为冷冻保护剂应用于水产品、酶及菌种的冷冻保藏。
与现有技术相比,本发明具有如下优点:
1.本发明所制备的新型抗冻剂来源于水产品加工中所产生的副产物,相比于抗冻蛋白与抗冻肽较高的生产成本,本发明所制备的新型抗冻剂具有原料来源广泛、成本低廉的优点。此外,以水产品加工副产物为原料制备抗冻剂,为水产品加工副产物的高值利用和降低水产品加工业的环保成本提供了新途径。
2.本发明结合蒸汽闪爆预处理鱼鳞,相较于传统处理方法,具有工艺简单、高效、无污染、处理成本低等优点,可极大缩短鱼鳞胶的制备周期,有利于高抗冻性的鱼鳞胶的高效可控制备。
3.本发明所制备的新型抗冻剂主要组分为蛋白质,相比传统抗冻剂(蔗糖、山梨醇及复合磷酸盐复合物)的高热量及糖尿病患者的不可适性,具有安全营养和适用范围广等优点。本发明产品能够有效控制冷冻食品及生物制品的品质劣变,增强其冻融稳定性,可作为冷冻保护剂广泛应用于水产品、酶及菌种保藏等领域。
附图说明
图1本发明制备的鱼鳞胶蛋白抗冻剂。
图2鱼鳞胶对鱼糜的冷冻保护效果。
图3鱼鳞胶对过氧化氢酶的冷冻保护效果。
图4鱼鳞胶对保加利亚乳酸菌的冷冻保护效果。
图5对比例中明胶抗冻多肽的低温保护活性。
图6不同方法制备明胶得率对比。
具体实施方式
下面通过实施例对本发明进行具体描述,有必要在此指出的是以下实施例只用于对本发明作进一步说明,不能理解为对本发明保护范围的限制,该领域的技术熟练人员根据上述本发明的内容作出的一些非本质的改进和调整,仍属于本发明保护的范畴。
实施例1:
(1)将新鲜黑鱼鳞去除表面残肉,充分清洗后干燥得到原料鱼鳞。随后将50g鱼鳞浸入到 1000mL浓度为0.1M的氢氧化钠溶液,在20℃混合搅拌处理2.5h,以脱除鱼鳞中的杂蛋白,之后用去离子水清洗2次,干燥备用。
(2)将预处理后的黑鱼鳞放入蒸汽闪爆设备(QBS-200B型,鹤壁市正道生物能源有限公司) 反应处理腔中,在蒸汽压力为1.5MPa下保压1min,待爆破腔底盖自动下拉后,腔内压力迅速降至大气压,鱼鳞内水分形成闪蒸,由内而外对物料完成绝热做功,实现蒸汽闪爆处理。
(3)鱼鳞随后在1000mL浓度为0.3M的盐酸溶液中进行脱钙处理,在20℃混合搅拌2h。随后用1000mL质量分数为1%(w/w)的胃蛋白酶溶液在25℃、pH=2.5条件下进行酶解2h,酶解结束后置于90℃恒温灭酶3min。酶解后的鱼鳞用去离子水清洗1~2次,之后置于50℃下以600mL水为溶剂恒温浸提3h,过滤得到浸提液。进一步经真空冷冻干燥后得到新型蛋白抗冻剂,置于避光干燥处储藏。
(4)在新鲜黑鱼鱼糜中加入2%的食盐以及不同浓度的(0.05%,0.1%,0.3%,0.5%,w/w) 的鱼鳞胶溶液,擂溃10min,在-18℃条件下冻藏16h,融化8h,如此冻融循环4次。鱼鳞胶对鱼糜的冷冻保护效果如图2所示。
实施例2:
(1)将新鲜草鱼鳞去除表面残肉,充分清洗后干燥得到原料鱼鳞。随后将50g鱼鳞浸入到 1000mL浓度为0.1M的氢氧化钠溶液,在20℃混合搅拌处理2.5h,以脱除鱼鳞中的杂蛋白,之后用去离子水清洗2次,干燥备用。
(2)将预处理后的草鱼鳞放入蒸汽闪爆设备反应处理腔中,在蒸汽压力为1.5MPa下保压1min,待爆破腔底盖自动下拉后,腔内压力迅速降至大气压,鱼鳞内水分形成闪蒸,由内而外对物料完成绝热做功,实现蒸汽闪爆处理。
(3)鱼鳞随后在1000mL浓度为0.3M的盐酸溶液中进行脱钙处理,在20℃混合搅拌2h。随后用1000mL质量分数为1%(w/w)的胃蛋白酶溶液在25℃、pH=2条件下进行酶解2h,酶解结束后置于90℃恒温灭酶3min。酶解后的鱼鳞用去离子水清洗1~2次,之后置于50℃下以600mL水为溶剂恒温浸提3h,过滤得到浸提液。进一步经真空冷冻干燥后得到新型蛋白抗冻剂,置于避光干燥处储藏。
(4)用pH 7.0,0.05M KH2:PO4-NaOH缓冲液配制2mg/mL的鱼鳞胶溶液,与过氧化氢粗酶液等体积混合均匀,并用相同方法配制的牛血清蛋白-过氧化氢酶混合液、商业抗冻剂-过氧化氢酶混合液作为对照,取同体积的稀释酶液和等体积的缓冲液混合均匀做空白,用吸光度法测定初始过氧化氢酶酶活。然后放入-20℃条件下冷冻3h,之后在25℃解冻,如此反复冻融4次后,鱼鳞胶对过氧化氢酶酶的冷冻保护作用如图3所示。
实施例3:
(1)将新鲜鲢鱼鳞去除表面残肉,充分清洗后干燥得到原料鱼鳞。随后将50g鱼鳞浸入到 1000mL浓度为0.1M的氢氧化钠溶液,在20℃混合搅拌处理2.5h,以脱除鱼鳞中的杂蛋白,之后用去离子水清洗2次,干燥备用。
(2)将预处理后的鲢鱼鳞放入蒸汽闪爆设备反应处理腔中,在蒸汽压力为1.5MPa下保压1min,待爆破腔底盖自动下拉后,腔内压力迅速降至大气压,鱼鳞内水分形成闪蒸,由内而外对物料完成绝热做功,实现蒸汽闪爆处理。
(3)鱼鳞随后在1000mL浓度为0.3M的盐酸溶液中进行脱钙处理,在20℃混合搅拌2h。随后用1000mL质量分数为1%(w/w)的胃蛋白酶溶液在25℃、pH=3条件下进行酶解2h,酶解结束后置于90℃恒温灭酶3min。酶解后的鱼鳞用去离子水清洗1~2次,之后置于50℃下以600mL水为溶剂恒温浸提3h,过滤得到浸提液。进一步经真空冷冻干燥后得到新型蛋白抗冻剂,置于避光干燥处储藏。
(4)将过滤膜的不同浓度的鱼鳞胶(0.25mg/mL、0.5mg/mL)溶液0.9mL和稀释100倍的保加利亚乳酸菌菌液0.1mL混合均匀后将混合液接种到新液体培养基中,培养7h,测定OD600,将剩下的混合液放入-18℃冻藏22h,接种到新的液体培养中培养7h后测定OD600。以菌存活率的大小来评价鱼鳞胶对保加利亚乳酸菌的冷冻保护作用(图4)。
对比例:
李晓坤等利用猪皮明胶制备抗冻多肽及其低温保护作用研究,其明胶抗冻多肽的低温保护活性如图5所示。与本发明相比,在相同抗冻剂浓度下,保加利亚乳酸菌的存活率比本发明低。
不同方法制备明胶的得率对比如图6所示。由图6可知,本发明制备工艺所得鱼鳞胶蛋白抗冻剂的得率更高。
Claims (8)
1.一种蛋白抗冻剂,其特征在于,按如下方法制备得到:
(1)将新鲜鱼鳞去除表面残肉,清洗并干燥后得到原料鱼鳞,将原料鱼鳞浸入氢氧化钠水溶液中,20~30℃搅拌2~3h,以脱除鱼鳞中的杂蛋白,之后清洗,干燥,完成预处理;
(2)将经过步骤(1)预处理的鱼鳞放入蒸汽闪爆设备的反应处理腔中,在蒸汽压力为1~3MPa下保压1~4min,待爆破腔底盖自动下拉后,腔内压力迅速降至大气压,鱼鳞内水分形成闪蒸,由内而外对物料完成绝热做功,实现蒸汽闪爆处理;
(3)将经过步骤(2)蒸汽闪爆处理的鱼鳞加到脱钙液中,20~30℃搅拌2~3h,完成脱钙处理,接着添加酶溶液,在20~40℃、pH=2-3的条件下酶解2~5h,灭酶,清洗,之后于50~70℃下,以水为溶剂恒温浸提3~5h,过滤得到浸提液,所得浸提液经真空冷冻干燥,得到所述蛋白抗冻剂;
所述酶溶液为质量分数1%~5%蛋白酶的水溶液,所述蛋白酶为胃蛋白酶。
2.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(1)中,所述鱼鳞来自常见的淡水鱼种或常见的海水鱼种。
3.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(1)中,所述氢氧化钠水溶液的浓度为0.1~0.2M。
4.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(1)中,所述氢氧化钠水溶液的体积用量以原料鱼鳞的质量计为15~25mL/g。
5.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(3)中,所述脱钙液为盐酸、醋酸或柠檬酸的水溶液,浓度为0.3~0.5M。
6.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(3)中,所述脱钙液的体积用量以原料鱼鳞的质量计为20mL/g。
7.如权利要求1所述蛋白抗冻剂,其特征在于,步骤(3)中,所述酶溶液的体积用量以原料鱼鳞的质量计为20~30mL/g。
8.如权利要求1所述蛋白抗冻剂作为冷冻保护剂在水产品、酶及菌种的冷冻保藏中的应用。
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