CN106805252A - 一种鱼短肽及其在营养强化剂中的应用 - Google Patents
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Abstract
本发明提供一种鱼短肽及其在营养强化剂的应用。采用鱼为原料,是一种含2‑8个氨基酸分子为主的短肽制剂。该鱼短肽具有高安全性、高新鲜度和低组胺值的特点,分子量小于800Da的组分占80%以上,组胺值低于30mg/100g(干基计)。蛋白组成中含有10‑25%的胶原蛋白短肽,有利于大伤口的康复。该产品可有效地提供全面的蛋白组分,有效改善病人的营养指标,降低手术应激诱导的免疫抑制,有利于机体免疫功能的恢复,更好地促进患者康复。
Description
技术领域
本发明涉及食品、功能性食品或特殊医学用途食品开发领域,更具体涉及一种鱼短肽及其在营养强化剂中的应用。
背景技术
营养是生命物质的基础,也是治疗疾病的保证。近年来国内外学者报道住院患者普遍存在着蛋白质一热卡缺乏型营养不良。蛋白质-热量营养不良症(protein energymalnutrition,PEM)是因疾病因素引起的一种营养缺乏病,临床上表现为消瘦(marasmus)和恶性营养不良综合征(kwashiorkor)。轻型的慢性蛋白质-热量营养不良症常被忽视,它影响着患者的生长发育、免疫功能,病人不易康复。蛋白质-热量营养不良症是临床营养学上的一个重要问题。
营养与健康的关系非常密切,对住院患者尤为重要。众所周知,外科手术会对患者造成较大的创伤,且术后的较长时间会影响进食及营养吸收,因而患者术后营养不良的发生率较高。有研究表明,术后营养不良可影响手术患者的手术效果、延长患者的住院时间、增加并发症发生率及降低其生活质量。但在临床实际工作中,临床医师较少对手术患者的营养状况进行综合、系统的评价,对住院患者中存在营养不良状况普遍地认识不足。
2009年版的《中国国家处方集》中将肠内营养制剂按氮源分为3大类,即氨基酸型、短肽型 (前两类也称为要素型,elementaltype)、整蛋白型(也称为非要素型non—elemental Iype)。前两类制剂以蛋白水解物为氮源,经少量消化过程便可吸收;整蛋白,适用于胃肠功能较好的患者。
短肽型肠内营养制剂是随着科学技术水平和医疗技术水平的发展提高而研究出一种新型肠内营养剂,该营养剂中的营养成分是经过水解处理预消化型营养制剂,其主要构成成分包括人体所需的多种糖类、蛋白、维生素等等,其脂肪源为中链甘油三脂和植物油,碳源主要包括蔗糖和麦芽糖糊精,氮源主要是乳清蛋白水解产物(短肽),同时在组成成分融入了适量的的微量元素、矿物质以及维生素等等。常用的短肽型肠内营养制剂最大的好处就是无需消化分解即可直接被肠上皮细胞吸收利用,在重症监护室得到了广泛的应用,尤其是适用于胰液分泌不足、胃肠功能不全、吸收面积减少的患者;除此之外,预消化型短肽制剂的渗透压也比较低,因此可以降低胃肠道副反应的发生率,提高患者的耐受性;在营养剂的配方过程中将低脂肪的含量适量降低,更适合脂肪代谢障碍以及消化道功能不全的患者。
许多研究证实,在手术之后,患者肠内营养有助于维持肠黏膜细胞结构与功能的完整性,维护肠道黏膜屏障功能,刺激消化液和胃肠道激素的分泌,增加内脏血流,使代谢更符合生理需求,减少中心静脉置管、代谢、肝胆并发症的发生。
2003 年,欧洲肠内肠外营养学会(ESPEN)发表了一种新的营养评价方法——营养不良风险筛查方法(nutritional risk screening 2002,NRS2002),后被中华医学会采用并大力推广,NRS2002 是一种结合人体测量、疾病严重程度、近期体重变化和近期营养摄入的变化等四项指标,采用评分的方法,以NRS评分≥3分作为营养不良风险的标准。
临床研究表明,胃肠道肿瘤术后第9天,供给短肽营养剂的试验组病人营养不良风险的发生率明显低于对照组,表明胃肠道肿瘤术后给予短肽制剂可增加肝蛋白质的合成,减少术后应激期的负氮平衡,在促进蛋白质合成和加快正氮平衡方面优于TPN。肠道内营养物质摄入后,可明显增加肠道和门静脉的血流量,促使胃肠蠕动和胆囊收缩、促进胃肠道激素的合成及释放,调节胃液、胆汁和胰液的分泌,使营养物质吸收利用更合理,且能维护肠黏膜屏障功能。
营养与免疫是互为相关的。在临床实验研究中,试验组病人未发生术后并发症。这一结果提示,病人营养状况的改善、免疫功能的恢复、食糜刺激以及术后通过肠道摄取营养素,稳定了肠道内微生物环境和肠粘膜屏障功能,从而减少了术后引发内源性感染并发症的机会,缩短了病人的住院时间。
重症监护室患者不同于普通患者,其应激系数一般都比较高,能量和营养的消耗主要用于维持重要内脏器官的功能代谢,这样就降低了消化道的消化吸收功能顺应性,这时候给予患者实施短肽型肠内营养剂可以有效的将消化道吸收负担减轻,从而增加机体能量及蛋白的供给量;同时,短肽型肠内营养剂可以有效的保护肠道正常菌群、防止肠黏膜萎缩、维持免疫活性以及降低创伤后的应激反应。
短肽类营养剂所含的蛋白质为蛋白水解物,在小肠中也有运输低聚肽的体系,低聚肽经小肠黏膜刷状缘的肽酶水解后进入血液,容易被机体利用。除以上特点外,此类营养药不含乳糖,避免了乳糖不耐受引起的腹泻和脂代谢障碍等一系列问题。这类药物几乎完全吸收,低渣,需少量消化液吸收,排粪便量少。适用于有胃肠道功能或部分胃肠道功能的患者,如胰腺炎、肠道炎性疾病、放射性肠炎和化疗、肠瘘、短肠综合征、艾滋病病毒感染等;也可作为营养不足患者的手术前后喂养及肠道准备,能补充人体日常生理功能所需的能昔及营养成分。
国内已有多种全蛋白质类营养剂商品,多为各种植物蛋白粉。植物种子提取油之后剩下的渣子,如豆粕、玉米粕、花生饼等为原料制成的植物水解蛋白粉,由于其必需氨基酸比率的不平衡,难于体现其营养价值。
鱼水解蛋白粉与人体需求蛋白组成比率相似,是一种优质蛋白质。将鱼蛋白经酶水解到易溶于水的小分子短肽或游离氨基酸,将更有利于人体消化吸收。
本发明提供一种鱼短肽作为制备营养强化剂的应用。采用鱼为原料,是一种含2-8个氨基酸分子为主的短肽制剂。该鱼短肽具有高安全性、高新鲜度和低组胺值的特点,分子量小于800Da的组分占80%以上,组胺值低于30mg/100g(干基计)。蛋白组成中含有10-25%的胶原蛋白短肽,有利于大伤口的康复。该产品可有效地提供全面的蛋白组分,有效改善病人的营养指标,降低手术应激诱导的免疫抑制,有利于机体免疫功能的恢复,更好地促进患者康复。
发明内容
本发明的目的在于提供一种鱼短肽及其在营养强化剂中的应用。
所述鱼短肽的制备方法为:鱼绞碎后,脱脂处理1-3次,得到脱脂鱼糜浆;加入水,鱼与水的质量体积比为1:2-20,调节pH至3-8;采用蛋白酶酶解工艺,酶的添加量为0.01-3wt.%,反应温度为20-80℃,酶解时间1-12小时;酶解液经SEC-HPLC检测,蛋白质分子量80%以上低于800Da,终止酶反应;酶解产物固液分离后收集上清液,采用纳滤膜进行脱盐和浓缩处理,至产品组胺含量低于30mg/100g(干基计),终止膜处理。
具体为:取小杂鱼绞碎后,脱脂处理3次,得到脱脂鱼糜浆;加入水,小杂鱼与水的质量体积比为1kg:4L,用10wt.%的醋酸溶液调节pH,使最终pH为3.5;采用二步酶解工艺,第一步酶解采用木瓜蛋白酶,酶的添加量为0.8wt.%,调节pH值至3.0,反应温度为40℃,酶解时间8小时;第二步酶解采用胰蛋白酶,酶的添加量为0.7wt.%,调节pH值至4.5,反应温度为40℃,酶解时间12小时;酶解液经SEC-HPLC检测,蛋白质分子量80%以上低于800Da,终止酶反应;酶解产物经高速连续离心机离心分离,收集上清液,采用250Da膜膜分离装置处理上述物料,RO水水洗,鱼短肽溶液最终浓缩至200L用于喷雾干燥,收集的鱼短肽胨粉。所述鱼短肽是一种含2-8个氨基酸分子为主的短肽制剂,分子量小于800Da的组分占80%以上。该鱼短肽具有高安全性、高新鲜度和低组胺值的特点,组胺值低于30mg/100g(干基计)。
本发明的显著优点是:蛋白组成中含有10-25%的胶原蛋白短肽,有利于大伤口的康复。该产品可有效地提供全面的蛋白组分,有效改善病人的营养指标,降低手术应激诱导的免疫抑制,有利于机体免疫功能的恢复,更好地促进患者康复。
附图说明
图1 不同浓度鱼短肽对HSF细胞增殖的影响。
图2 鱼短肽对HSF迁移的影响。其中a为常规营养组0h,b为0.5 mg/ml鱼短肽强化组0h,c为常规营养组 24h,d为0.5 mg/ml鱼短肽强化组 24h。
图3小鼠实验白蛋白的含量。
图4鱼短肽分子量分布(SEC-HPLC谱图)。
具体实施方式
以下通过试验例来进一步阐述具体实施方式,但不用来限制本发明的范围。
实施例1
取小杂鱼100公斤,用绞肉机进行绞碎,加入300升60号溶剂油脱脂处理3次,得到脱脂鱼糜浆。加入400L水,用10wt.%的醋酸溶液调节pH,使最终pH达到3.5。采用二步酶解工艺。第一步酶解采用木瓜蛋白酶,酶的添加量为0.8wt.%,调节pH值至3.0,反应温度为40℃,酶解时间8小时;第二步酶解采用胰蛋白酶,酶的添加量为0.7wt.%,调节pH值至4.5,反应温度为40℃,酶解时间12小时。酶解液经SEC-HPLC检测,蛋白质分子量80%以上低于800Da,终止酶反应。酶解产物经高速连续离心机离心分离,收集上清液,采用膜分离装置(250Da膜)处理上述物料,RO水水洗800L,鱼短肽溶液最终浓缩至200L用于喷雾干燥。收集的鱼短肽粉进行分析,鱼短肽分子量分布采用SEC-HPLC法检测,蛋白质分子量低于800Da占比达 91.6%,组胺含量14mg/100g(参照GB/T 5009.45-2003)。
表1鱼短肽产品分子量分布数据
表2组胺含量检测结果
实施例2
37℃、5% CO2条件下常规培养人皮肤成纤维细胞。人皮肤成纤维细胞接种于96孔板中,细胞浓度为5000个/孔,鱼短肽用培养基溶解分别配制成浓度为0.2mg/ml、0.4mg/ml、0.6mg/ml和0.8mg/ml,培养24h后采用MTT法检测鱼短肽对细胞的增值情况,结果如图1所示,鱼短肽样品随浓度的增大增殖效果逐渐增强,浓度为0.6 mg/ml时,细胞增殖率为120%。
6孔细胞培养板中加入约5×105个HSF细胞,细胞融合率达到100%后,做一条笔直的划痕。空白对照组加入2ml无血清培养基,实验组加入浓度为0.5 mg/ml的鱼短肽无血清培养基。放入37℃、5% CO2培养箱培养。0h和24h拍照比较细胞生长迁移效果,结果如图2所示,添加0.5mg/ml鱼短肽培养24h后,与常规营养组相比,细胞迁移显著,说明在细胞培养中添加鱼短肽有利于促进细胞生长。
取昆明小鼠40只,按体重分为4组,每组10只,分别设为鱼短肽低剂量组,鱼短肽高剂量组,营养缺陷组和正常营养组。正常营养组喂养含20%蛋白的饲料,其他各组喂养8%蛋白含量的饲料,自由采食和饮水。每天对小鼠进行灌胃,其中鱼短肽低剂量组为200mg/ml鱼短肽,高剂量鱼短肽组为400mg/ml鱼短肽,正常营养组和营养缺陷组为0.9%生理盐水,灌胃体积均为0.5ml,四周后摘眼球取血液,进行血清中白蛋白含量的检测。结果如图3所示,营养缺陷组小鼠血清中白蛋白含量最低,仅为 30.42 g/L,正常营养组白蛋白含量为33.65g/L,鱼短肽低剂量组和鱼短肽高剂量组的白蛋白含量分别为35.35g/L和39.31g/L,动物实验结果表明,鱼短肽具有营养强化作用。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (4)
1.一种鱼短肽,其特征在于:采用鱼为原料,原料经绞碎、脱脂得到鱼糜浆,加入蛋白酶酶解,进一步用膜分离进行脱盐和浓缩,喷雾干燥后的粉末即蛋白胨。
2.根据权利要求1所述的鱼短肽,所述鱼短肽的制备方法为:鱼绞碎后,脱脂处理1-3次,得到脱脂鱼糜浆;加入水,鱼与水的质量体积比为1 kg:(2-20)L,调节pH值至3-8;采用蛋白酶酶解工艺,酶的添加量为0.01-3wt.%,反应温度为20-80℃,酶解时间1-12小时;酶解液经SEC-HPLC检测,蛋白质分子量80%以上低于800Da,终止酶反应;酶解产物固液分离后收集上清液,采用纳滤膜进行脱盐和浓缩处理,至产品组胺含量以干基计低于30mg/100g,终止膜处理。
3.根据权利要求1所述的鱼短肽,其特征在于:所述鱼短肽是一种含2-8个氨基酸分子为主的短肽制剂,分子量小于800Da的组分占80%以上;组胺含量以干基计低于30mg/100g。
4.如权利要求1所述的鱼短肽在营养强化剂中的应用。
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