CN106578836A - 一种多肽在食品防腐中的应用 - Google Patents
一种多肽在食品防腐中的应用 Download PDFInfo
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- CN106578836A CN106578836A CN201611243276.8A CN201611243276A CN106578836A CN 106578836 A CN106578836 A CN 106578836A CN 201611243276 A CN201611243276 A CN 201611243276A CN 106578836 A CN106578836 A CN 106578836A
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- food
- polypeptide
- nisin
- polypeptides
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
本发明属于食品添加剂领域,本发明公开了一种多肽在食品防腐中的应用,所述多肽对引起食品腐败的革兰氏阴性与革兰氏阳性微生物有较好的抑菌效果,延长食品的货架寿命,安全高效。本发明还提供了一种复配天然生物食品防腐剂,其中,所述多肽含量为0.01%‑0.2%,纳他霉素含量为0.001%‑0.03%,Nisin含量为0.004%‑0.05%。本发明所述多肽及其复配食品添加剂为天然食品添加剂,安全性高,效果好,有望代替化学食品防腐剂。
Description
技术领域
本发明涉及食品防腐领域,具体涉及一种多肽在食品防腐中的应用。
背景技术
腐败变质会导致食品营养和食用价值降低,还可引起食物中毒。食品的腐败变质原因较多,包括物理、化学和生物性因素,如动、植物食品组织内酶的作用,昆虫、寄生虫以及微生物的污染等。其中由微生物污染所引起的食品腐败变质是最为重要和普遍的。微生物广泛分布于自然界,食品中不可避免的会受到一定类型和数量的微生物的污染,当环境条件适宜时,它们就会迅速生长繁殖,造成食品的腐败与变质。
为防止食品的腐败和变质,本领域技术人员采取了各种方法,如冷冻、冷藏、干燥、腌制、加热,以及添加食品防腐剂。然而,低温保藏虽能对食物中微生物的繁殖加以控制,但却不能杀灭微生物,食品的质量变化并未完全停止。高温处理能杀灭绝大部分微生物,但却会破坏食品本身的营养成分,并显著影响食品本身的风味。干燥能提高食品的保藏性,但也会引起褐变、变色、褪色、异味、异嗅及组织上的变化。而腌制食品,虽有一定的防腐性能和独特的风味,但对健康有负面影响(高盐或高糖)。
目前食品工业常用的防腐剂以化学类防腐剂居多,如苯甲酸及其钠盐、山梨酸及其钾盐、对羟基苯甲酸酯类及其钠盐、丙酸类、亚硫酸及其盐类、硝酸盐及亚硝酸盐等。
然而,随着科技进步,化学防腐剂被曝有各种副作用,如用于肉类着色和防腐的NaNO2在消化系统中易与脯氨酸形成一种强致癌剂N-亚硝胺类化合物,用于水果防腐保鲜的三唑类化合物有潜在致癌风险,二氧化硫对气喘患者的支气管具刺激性,亚硝酸盐是潜在致癌的,苯甲酸盐与各种过敏、哮喘、皮疹以及脑损伤有关。
基于此,广谱、高效、安全、稳定的天然防腐剂已成为人心所向。
乳酸链球菌素(Nisin)是由多种氨基酸组成的多肽类化合物,可作为营养物质被人体吸收利用。1969 年,联合国粮食及农业组织/世界卫生组织(FAL/WHO)食品添加剂联合专家委员会确认乳酸链球菌素可作为食品防腐剂。90年代被批准为我国食品防腐剂,它能有效抑杀革兰氏阳性细菌,但却不能抑制革兰氏阴性菌、酵母和霉菌(缪存影,天然食品防腐剂——乳酸链球菌素,中国食物与营养,2008,4:30-32.)。
纳他霉素(Natamycin)是一种由链霉菌发酵产生的天然抗真菌化合物,属于多烯大环内酯类,1982年6月,美国FDA正式批准纳他霉素可以用作食品防腐剂。1996年,中国食品添加剂标准化学技术委员会正式批准纳他霉素可作为食品防腐剂。纳他霉素依靠其内酯环结构与真菌细胞膜上的甾醇化合物作用,形成抗生素—甾醇化合物,从而破坏真菌的细胞质膜的结构,进而引起菌内氨基酸,电解质等物质渗出,菌体死亡。当某些微生物细胞膜上不存在甾醇化合物时,纳他霉素就对其无作用,因此纳他霉素只对真菌产生抑制,对细菌和病毒不产生抗菌活性(阎永贞等,食品工业科技,2010,31(4):365-373.)。
现有技术中,专利号为ZL200910069419.1的专利报道了一种复合生物防腐剂,该生物防腐剂由乳酸链球菌素、木醋液和蒸馏水组成,每100mL 食品的复合生物防腐剂中含乳酸链球菌素350~400mg、木醋液15~25mL 和其余为蒸馏水。申请号为201310719332.0的专利公开了一种冷鲜肉的复合生物防腐剂,其由乳酸链球菌素、纳他霉素、乳酸和茶多酚的配置比例是按肉类原料重量的百分比确定,其中乳酸链球菌素为0.01%-0.05%、纳他霉素为0.003%-0.01%、乳酸为0.1%-0.2%,茶多酚为0.03%-0.05%。申请号为201610173023.1的专利公开了一种鱼类用生物复配保鲜剂及其制备方法,本发明生物复配保鲜剂,有效成分按重量份计包括:普鲁兰多糖5-30份,纳他霉素0.1-0.5份,乳酸链球菌素0.1-1份,中药提取物0-10份,黄酮类化合物0-20份;所述中药提取物为金银花提取物、黄芪提取物、地榆提取物、大黄提取物、五倍子提取物中的一种或几种。
发明内容
本发明的第一目的是提供一种多肽在食品防腐中的应用,广泛提高食品的保存性;本发明的第二目的是提供一种食品防腐保存方法,在不损害食品品质的基础上保存食物。本发明的第三目的是提供一种复配天然生物食品防腐剂。
本发明所述多肽的氨基酸序列为SEQ ID:NO.1。
本发明中的含量,无特殊说明下均为质量百分数。
本发明提供一种多肽在食品防腐中的应用,所述多肽浓度为0.01%-0.5%,优选浓度为0.02%-0.2%,更加优选浓度为0.02%-0.1%。
本发明提供一种食品制造方法,将多肽添加于食品中,所述多肽的浓度0.01%-0.5%,优选浓度为0.02%-0.2%,更加优选浓度为0.02%-0.1%。
本发明所述多肽可单独使用,也可根据其特性,与其他防腐剂及防腐技术等结合,以形成多重防腐栅栏技术而应用于食品加工和贮藏过程中。
一个含所述多肽的组合物也可以是包含本领域技术人员添加了其他化合物的配方。
当本发明所述多肽与作为食品添加剂使用的具有防腐作用的物质联合使用时,所述物质不限于现有作为防腐剂允许使用的食品添加剂,也包括未来允许使用的其他物质。
作为食品添加剂使用的具有防腐作用物质可以是:苯甲酸及其钠盐、丙酸及其钠盐、钙盐、单辛酸甘油酯、对羟基苯甲酸酯类及其钠盐、二甲基二碳酸盐、2,4-二氯苯氧乙酸、二氧化硫,焦亚硫酸钾,焦亚硫酸钠,亚硫酸钠,亚硫酸氢钠,低亚硫酸钠、二氧化碳、、联苯醚、硫磺、肉桂醛、山梨酸及其钾盐、双乙酸钠、脱氢乙酸及其钠盐、稳定态二氧化氯、硝酸钠,硝酸钾、亚硝酸钠,亚硝酸钾、液体二氧化碳、乙二胺四乙酸二钠、乙酸钠、乙氧基喹等。
本发明所述多肽也可与其他天然生物防腐剂联合使用,Nisin、ε-聚赖氨酸及其盐酸盐、纳他霉素、溶菌酶、鱼精蛋白、壳聚糖等。
与本发明所述多肽并用的物质,不限于一种,几种组合使用可能更为有效,按照食品种类、组成、预想到的致食品腐败的微生物、pH值、水分活性、所要求的保存温度、保存期限等,适宜地将几种组合使用。各物质使用的量应在国家允许的作为防腐剂的食品添加剂最大限量以内。
与本发明所述多肽联合使用的其它防腐技术包括但不限于:热处理、冷处理、高密度二氧化碳、超声波、臭氧处理、非热力杀菌技术有高压脉冲电场(PEF)、超高压(HPP)、真空包装、酶制剂、金属螯合剂,如EDTA、三磷酸钠(STPP)等。
不同的防腐剂及防腐技术与本发明所述多肽联合使用可使微生物细胞膜削弱或变得容易受到额外多肽的攻击,从而增强多肽的抑菌效果。
本发明惊讶的发现,将本发明所述多肽与纳他霉素和乳酸链球菌素结合能产生协同抗菌作用。因此,优选纳他霉素和乳酸链球菌素用于增强所述多肽的抗菌活性,该组合的使用可能减少其他防腐技术的使用,如加热法,保留食物自身风味;该组合的使用也可能减少其他化学防腐剂的使用,进而减少其副作用。
因此,本发明还提供一种使用所述多肽复配而成的天然生物食品防腐剂,其中,所述多肽含量为0.01%-0.2%,纳他霉素含量为0.001%-0.03%,Nisin含量为0.004%-0.05%。
本发明的复配天然食品防腐剂,添加量为食物重量的 0.2%,就可以对食物进行有效的防腐作用。
本发明中所述多肽可以用来预防或抑制革兰氏阳性细菌或革兰氏阴性细菌引起的食品变质腐败。相较于化学防腐剂山梨酸钾,本发明的多肽对食品防腐效果更佳;相较于生物防腐剂Nisin及纳他霉素,本发明的多肽抗菌谱更广。本发明中用于食品防腐的多肽安全性高,经口可为消化酶分解为氨基酸,进而作为营养物质吸收;急性经口毒性,对雌雄大鼠经口LD50值大于2000mg/kg b.wt.,为实际无毒级。本发明所述多肽与Nisin及纳他霉素组成的天然食品防腐剂作为食品添加剂使用具有良好的抗菌防腐作用。
附图说明
图1多肽对牛奶菌落总数的影响
图2多肽对牛奶OD595值的影响
图3平板法检测多肽对牛奶的抑菌效果
图4多肽对橙汁菌落总数的影响
图5多肽对橙汁OD595值的影响
图6平板法检测多肽对橙汁的抑菌效果。
具体实施方式
本发明所述多肽对食品的防腐可以发生于食品的储存过程、运输过程、处理操作过程、加工过程或展示过程。
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
本发明所述多肽无特殊说明均为氨基酸序列为SEQ ID:NO.1。
本发明所述多肽可按照申请人在先申请专利(申请号:201310245373.0,发明名称:一种抗菌肽的制备方法和应用)中所述多肽的制备方法。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,实施例中,加入的各原料除特别说明外,均为市售。
本发明中所述多肽对食品的防腐可表现为对微生物抑菌生长的作用,与相同条件下但无多肽时相比所述多肽降低了10%的生长率,更优选的降低了20%的生长率,更优选的降低了30%的生长率,更优选的降低了40%的生长率,更优选的降低了50%的生长率,更优选的降低了75%的生长率,更优选的降低了90%的生长率,更优选的降低了95%的生长率,更优选的降低了98%的生长率,更优选的降低了99%的生长率,最优选的,多肽能防止微生物的生长。
本发明所述多肽在食品防腐中应用时,所述多肽优选使用有效剂量,该剂量是指能够抗菌和/ 或抑菌,能达到防止或抑制腐败的可检测水平;优选的,是同与相同条件的不加该多肽时对比。
本领域技术人员能够通过常规方法确定一个合适的剂量浓度。优选的,例如,多肽的浓度至少为0.01%,浓度至少为0.0125%,浓度至少为0.02%,浓度至少为0.05%,浓度至少为0.1%,浓度至少为0.2%,直到浓度为0.5%,直到浓度为1%,直到浓度为10%。优选的,使用多肽的浓度选自0.01%和0.2%之间,更优选的0.02%和0.1%之间。所述多肽的有效剂量对人或动物无毒。
含所述多肽的组合物可以混合掺入食品中,或应用于食品表面( 如作为液态膜或喷雾)。食品也可以浸入( 并可选的保持于) 所述组合物中,如将所述多肽溶液涂在鲜肉后结合真空包装。
本发明使用所述多肽复配的天然生物食品防腐剂,其中,所述多肽含量为0.01%-0.2%,纳他霉素含量为0.001%-0.03%,Nisin含量为0.004%-0.05%。
实施例1 抑菌试验
取0.0125%所述多肽进行抑菌试验,采用GB15979中附录C4的方法进行,结果见表1,试验表明,本发明所述多肽对多种微生物有显著的抑菌效果。铜绿假单胞菌和枯草芽孢杆菌可以导致食品腐败变质;大肠杆菌能引起pH≥4.5的食品腐败,产酸并产气;金黄色葡萄球菌是引起食品腐败,引起食物中毒的常见细菌;沙门氏菌通过病畜或健康带菌动物污染肉类食品,随食物进入人体肠道;污染阪崎肠杆菌的奶粉对新生儿致死率达50%。所述多肽对其100%抑菌。
表1 多肽的抑菌性
实施例2 防腐效果试验
1.实验方法与材料
1.1试剂
山梨酸钾、Nisin、多肽冻干粉、无水乙醇、细菌培养基、灭菌超纯水。
1.2试验食品
1.2.1牛奶
巴氏消毒牛奶,从超市采购。
1.2.2橙汁
浓缩橙汁由农夫山泉有限公司提供,按照水:浓缩橙汁=6:1配制,配制好的橙汁置于空气中放置3小时,以得到初始菌落。
1.2.3果冻
制备(浸泡法):将1g卡拉胶与0.1g魔芋胶混匀后于20ml水中,糖18g于70ml水中溶解;柠檬酸0.1g于10ml水中溶解,使料液冷却至70℃左右加入,搅匀料液趁热灌装,密封后倒置,自然冷却或放入4℃保鲜柜中冷却至28℃左右,翻转使之自然凝冻即得成品。
1.3防腐效果检测
防腐效果实验设置以下六组:空白对照组、山梨酸钾、Nisin、0.02%多肽、0.1%多肽、0.2%多肽。每组设3个以上重复。
1.4总菌落数及酶标仪检测
用平板涂布法,隔12 h测定一次总菌落数;用酶标仪测定OD595值,并且拍照。每个处理最少测定3次。
1.5 数据分析
采用DPS 9.5数据处理系统进行单因素方差分析,用origin 9.0软件及Excel作图。
抑菌率%=100×(对照-处理)/对照
2.结果
2.1多肽对牛奶的防腐效果
试验以0.02%Nisin,0.02%山梨酸钾为阴性对照,以无菌水为阳性空白对照,检测不同浓度多肽(0.02%、0.1%、0.2%)添加于牛奶后对总菌落数的抑制效果,各组物质对牛奶的防腐效果见图1、图2、图3。图1中,24h、48h、72h、84h连续检测结果显示,添加多肽可显著减少牛奶的菌落总数,抑菌效果与添加量成正比。图2中,用酶标仪对不同浓度多肽(0.02%、0.1%、0.2%)添加于牛奶后0-48h抑菌效果的检测结果,其效果与菌落计数法检测结果基本一致。
总体上多肽的抑菌效果优于山梨酸钾。0.1%多肽与0.02%Nisin效果类似,0.2%多肽效果优于0.02%Nisin。
表2 为不同防腐剂对巴氏消毒奶的抑菌率(根据菌落数统计)影响显著性分析,抑菌率%计算公式见1.5数据分析。抑菌率以无菌水作为阳性空白对照,抑菌率值越大代表抑菌效果越好,由表1数据方差分析可见,0.02%山梨酸钾对牛奶无抑菌作用,0.02% Nisin不同浓度多肽对牛奶有显著的抑菌作用,且随着培养时间的延长,抑菌效果降低,其中0.02% Nisin抑菌效果下降最为明显。24 h-84h期间,0.1%多肽的抑菌效果优于0.02%Nisin,显著性水平<0.01。总体上,0.1%多肽的抑菌效果优于0.02% Nisin,可作为新型防腐剂应用于巴氏消毒奶。
表2 牛奶中添加多肽、山梨酸钾和Nisin的抑菌效果比较
本试验通过菌落总数计数法和酶标仪OD595值检测,进行了多肽对巴氏消毒牛奶的防腐效果观察。菌落总数结果显示,0.02% 山梨酸钾与空白对照的趋势相近,无显著抑菌作用。在实验期间0.02% Nisin,0.02%、0.1%、0.2%多肽对牛奶具有显著的抑菌效果;24h-84h,0.1%多肽的抑菌效果超越了0.02% Nisin抑菌效果。酶标法测定OD595数据证实了菌落总数计数法的结果。实验结果证明多肽可以作为巴氏消毒牛奶的新型防腐剂。
2.2多肽对橙汁的防腐效果
试验以0.02% Nisin,0.02% 山梨酸钾为阴性对照,以无菌水为阳性空白对照,检测不同浓度多肽(0.02%、0.1%、0.2%)添加于橙汁后对总菌落数的抑制效果,各组物质对橙汁的防腐效果见图4、图5、图6。图4中,24h、48h、72h、84h连续检测结果显示,添加多肽可显著减少橙汁的菌落总数,抑菌效果与添加量成正比。图5中,用酶标仪对不同浓度多肽(0.02%、0.1%、0.2%)添加于橙汁后0-48h抑菌效果的检测结果,其效果与菌落计数法检测结果基本一致。
0.2%多肽对橙汁的抑菌效果优于0.02%山梨酸钾的效果,而0.02%Nisin对橙汁没有抑菌效果。
表3为不同防腐剂对橙汁的抑菌率(根据菌落数统计)显著性分析,抑菌率%计算公式见1.5数据分析。抑菌率以无菌水作为阳性空白对照,抑菌率值越大代表抑菌效果越好。由表3数据方差分析可见,培养24h后0.02%Nisin对橙汁的抑菌效果降低,无显著抑菌作用,而0.02%山梨酸钾以及不同浓度多肽对橙汁有显著的抑菌作用,且抑菌效果与添加量成正比,差异显著性水平<0.01。随着培养时间的延长,0.02%Nisin与0.02%多肽抑菌效果降低,其中0.02%Nisin抑菌效果下降最为明显,与空白对照对比,无显著抑菌作用;48h-84h期间,0.2%多肽的效果同0.02%山梨酸钾抑菌效果近似。各时间段抑菌效果分析结果显示,0.2%多肽与0.02%山梨酸钾的抑菌效果无显著性差异。
表 4 多肽对果冻抑菌实验菌落总数统计结果(浸泡法)
注:∞:样品菌落总数多不可计;
实施例3 一种复配天然生物食品防腐剂
按照表5中配方1-3数据称取原料,混合均匀后,即可获得本发明所述的复配天然生物食品防腐剂。
表5 食品防腐剂配方表
现将表5中的各组进行防腐对比试验,实验材料为豆腐、荷包蛋、蛋糕,将表5各组成分分别加入上述食品,在25℃下保存,结果见表6 :
表6 防腐效果
实施例4 大鼠急性经口试验
健康SD大鼠20只,体重182-215g,雌雄各半,试验方法为一次最大限度试验,设计剂量为2000mg/kgb.wt., 称取本发明所述多肽12000mg加纯净水至60ml配制成受试物,大鼠禁食过夜后,经口一次灌胃给予,灌胃容量为10 ml/kg b.wt.。灌胃后4h给食,自由摄食。观察期14d。大鼠灌胃后未见中毒表现,观察期内无动物死亡。结果表明本发明所述多肽对雌雄大鼠经口LD50值均大于2000mg/kg b.wt.,属实际无毒级。
SEQUENCE LISTING
<110> 江苏吉锐生物技术有限公司
<120> 一种多肽在食品防腐中的应用
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> PRT
<213> 人工序列
<400> 1
Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys Lys
1 5 10 15
Leu Ser
Claims (10)
1.一种多肽用于食品防腐的用途,其特征在于,所述多肽的氨基酸序列为SEQ IDNO.1。
2.如权利要求1所述的用途,其特征在于,所述多肽的浓度为0.01%-0.5%。
3.如权利要求2所述的用途,其特征在于,所述多肽的浓度为0.02%-0.2%。
4.如权利要求3所述的用途,其特征在于,所述多肽的浓度为0.02%-0.1%。
5.一种食品制造方法,其特征在于,将如权利要求1所述的多肽添加于食品中。
6.如权利要求5所述的方法,其特征在于,所述多肽的浓度为0.01%-0.5%。
7.如权利要求6所述的方法,其特征在于,所述多肽的浓度为0.02%-0.2%。
8.如权利要求7所述的方法,其特征在于,所述多肽的浓度为0.02%-0.1%。
9.一种复配天然生物食品防腐剂,其特征在于:所述食品防腐剂由如权利要求1所述的多肽、Nisin和纳他霉素组成。
10.如权利要求9所述的食品防腐剂,其特征在于:所述多肽含量为0.01%-0.2%,纳他霉素含量为0.001%-0.03%,Nisin含量为0.004%-0.05%。
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CN108771028B (zh) * | 2018-05-28 | 2021-08-06 | 南京千济诺生物科技有限公司 | 一种动物饲料添加剂及其制备方法与应用 |
CN110156884A (zh) * | 2019-05-22 | 2019-08-23 | 天津科技大学 | 一种重组Mytichitin-CB抗菌肽的应用 |
CN112457376A (zh) * | 2020-12-02 | 2021-03-09 | 中国科学院成都生物研究所 | 一种结构改造的抗菌肽bmap-14及其应用 |
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