CN113278049B - 一种从富硒海藻中分离的富硒呈味肽、制备方法和应用 - Google Patents
一种从富硒海藻中分离的富硒呈味肽、制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种从富硒海藻中分离的富硒呈味肽、制备方法和应用。本发明的方法是(1)将海藻粉经生物酶分段酶解,以便获得酶解液;(2)将所述酶解液进行超滤分离处理,以便获得混合呈味肽;以及(3)将所述混合呈味肽进行反相高效液相色谱纯化分离,以便获得目标呈味肽。所述呈味肽具有SEQ ID NO:1所示的氨基酸序列。本发明的呈味肽呈味效果明显,可同时呈鲜味和甜味,阈值低、纯度高,可应用于食品领域的多个分支,提高食品风味的同时,也能提高食品的营养价值。同时由于其含有有机硒,可以将其制备成抗癌功能和/或防癌功能的保健品。
Description
技术领域
本发明属于生物技术领域,更具体地,涉及一种从富硒海藻中分离的富硒呈味肽、制备方法和应用。
背景技术
食盐和味精是人们生活中不可或缺的调味品,但是过量食用将大大增加高血压、肾脏病、骨质疏松等多种相关疾病的发病率。由于目前我国绝大多数地区人们的食盐摄入量为推荐量的2到3倍,因此,低钠调味品的研究和开发已经成为一种趋势。目前市面上出现的低钠盐是用氯化钾替代部分氯化钠以降低钠摄入量,但氯化钾具有苦涩味,影响低钠盐的推广。现在市场上不少产品已经采取将盐、味精、营养素等其他调料混合制成营养性复合调味料。但此类营养性复合调味料,由于都是各种原料的粗加工提取物,因此用量大,增咸、增鲜效果也不够显著。
食品中存在许多呈味物质(例如甜味物质、鲜味物质、咸味物质、酸味物质等),这些物质一般为游离氨基酸、肽、呈味核苷酸、无机盐等。在肉及肉制品中呈味物质以游离氨基酸和肽占有较重要的角色,这是由人类的味觉受体——味蕾与呈味物质的结构和性质所决定的。呈味肽,又称作美味肽或风味增强肽,它是一类能产生酸、甜、苦、咸、鲜等不同味感的小分子肽,通常是通过酶解、微生物发酵或化学合成的方式制得。其呈味特性往往与氨基酸组成、氨基酸序列有关,不同的呈味肽会产生不同的味觉感受和呈味强度;部分呈味肽还具有风味提升的作用,与鲜味剂、酸味剂协同使用时可提升原有风味,并增强食品的厚感。除呈味效果外,作为一种小分子肽,人体对它的吸收、利用效率要高于大分子蛋白和游离氨基酸,营养特性优良;同时还伴随着一定的抗氧化活性,有利于食品的加工和保藏。因此,将呈味肽作为一种呈味基料或辅料应用于食品领域中,具有较大的前景。这些年来,科研工作者们都尝试着从各式各样的美味食物中分离提取起关键作用的呈味肽(也称风味增强肽、美味肽等),例如:Yamasaki等人(A peptide with delicious taste.Agricultural andBiological Chemistry.1978,42,1761-1765.) 从奶 酪,豆浆,酪蛋白、大豆蛋白、鸡蛋白质酶解物,乳蛋白酶解物和火腿酶解 物中分离并鉴定出大量的呈味肽(例如Glu-Glu、Glu-Val、Ala-Asp-Glu、 Ala-Glu-Asp、Asp-Glu-Glu、Ser-Pro-Glu、Glu-Glu-Asn、 Leu-Ser-Glu-Arg-Tyr-Pro等)。申请号为201210102244.1的专利公开了一种从海豚鱼中分离纯化出一种具 有鲜甜味道的呈味肽,其氨基酸序列为Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val;申请号为201810007698的专利公开了一种从蛹虫草粉中分离出的呈味肽,其氨基酸序列为AYM;申请号为CN201711308936 的专利公开了一种草菇呈味肽,其氨基酸序列为Ala-Ser-Asn-Met-Ser-Asp-Leu。这些研究进展都证实了呈味肽具有良好的加工特性、营养功能和生理活性,并且对食品的滋味具有很大的影响,可以用于食品佐料,例如可以用作调味品或香精香料的重要基料。目前国内对呈味肽的研究主要停留在发掘阶段,有研究表明肽链的长短不同,其味感会不同,肽链越长,味感越不明显,但关于呈味肽与味觉感受器相互作用而呈味的机理还不是十分清晰。但是还没有一种从味精的初始发现来源-海藻中分离提取出的呈味肽,而且也都仅仅集中在调味功能上,并不具有更深入的保健功能。
“海藻”是海带、紫菜、裙带菜、石花菜等海洋藻类的总称,是生长在海中的藻类,是植物界的隐花植物,藻类包括数种不同类以光合作用产生能量的生物。硒是人体必需的微量矿物质营养素,具有无机和有机两种化学形态;植物在硒的生态链中可以更有效地将无机硒转化为有机硒,植物硒的生物效价相对于动物硒更高。实验证明,植物活性硒被人体吸收率是无机硒的20倍。海藻富含大量的有机硒,是天然的有机硒携带者和人体食物有机硒的主要来源。硒被科学家称之为人体微量元素中的“抗癌之王”。科学界研究发现,血硒水平的高低与癌的发生息息相关。大量的调查资料说明,一个地区食物和土壤中硒含量的高低与癌症的发病率有直接关系,但是,目前的常见的硒补充剂多数为无机硒,吸收率不好。为解决这种技术问题,需要出现一种使用方便,配比简单,吸收率好,明显提高食品风味的同时又可以补充有机硒的产品。
发明内容
本发明的目的在于根据现有技术中存在的上述不足,提供一种具有强烈增鲜提味功能的富硒呈味肽。
进一步的,本发明提供一种富硒呈味肽的制备方法,所述方法以海藻为原料,通过改进酶解、分离纯化技术,制得了一种具有强烈增鲜提味功能的富硒呈味肽,可同时呈鲜味和甜味,而且其中含有有机硒,该呈味肽可应用于食品领域的多个分支,如调味品、休闲食品、饮料等,更重要的是,在提高食品风味的同时,也能提高食品的营养价值和保健功能。
所述制备方法包括:(1)将海藻粉经生物酶分段酶解,以便获得酶解液;(2)将所述酶解液进行超滤分离处理,以便获得混合呈味肽;以及(3)将所述混合呈味肽进行反相高效液相色谱纯化分离,以便获得目标呈味肽。
其中,酶解步骤为取海藻(最好是经检测后具有较高硒含量的海藻)于70℃烘干,经球磨粉碎机或者大功率破壁机破壁粉碎,过100-120目筛后按照海藻粉:水(重量比)为1:5-20的比例在50-65℃条件下混合超声均质,超声功率100-200W、处理15-20分钟。处理后的均质液冷却到室温后调节pH至2.0~3.0,按均质液质量的0.5~2%加入果胶酶和胃蛋白酶在25-30℃下酶解4-6h后,调节pH至6.5-7.0,按照按均质液质量的0.5~2%加入纤维素酶、风味蛋白酶和中性蛋白酶,在45~55℃下搅拌酶解4~6h后升温至80-100℃灭酶10min。
优选的,为了提高酶解效率,可以在酶解过程中辅以微波、超声、震荡等多种方式,可以根据本领域的常规手段进行优化和调整。
其中超滤分离处理步骤为:将酶解灭酶后的均质液过滤后,8000-12000rpm离心10-30分钟,收集上清液。 用0.45μm的滤膜抽滤,滤液再依次利用分子量截留范围为1000Da的超滤膜和200Da的纳滤膜对其进行超滤分离,收集分子量在200-1000Da的超滤组分,冷冻干燥后储存于-80℃冰箱中
其中反相高效液相色谱纯化分离步骤为:将前述分子量<1000Da的超滤组分冷冻干燥后利用超纯水配置成浓度为10mg/mL,取1mL溶液上柱(1.6cm×100cm),凝胶层析柱的填料为Sephadex G-15葡聚糖凝胶树脂,用蒸馏水洗脱,收集吸收峰获得的3个洗脱组分。将收集的3个层析组分分别冷冻干燥后储存于 -80℃冰箱中。对层析后选择的浓厚感和鲜味最强的组分进一步采用反相高效液相色谱(RP-HPLC)方法进行层析,再选择其中鲜味和甜味最强的组分进行成分鉴定,确定氨基酸序列为Ala- Pro-Met*- Leu,其中Met*为硒取代甲硫氨酸。
根据本发明的实施例,所述呈味肽具有SEQ ID NO:1所示的氨基酸序列。
进一步的,本发明提出了一种分离的核酸。根据本发明的实施例,所述核酸编码前面所述的呈味肽。利用根据本发明实施例的核酸导入宿主细胞,使得宿主细胞表达前面所述的呈味肽,呈味肽呈味效果明显,可同时呈鲜味和甜味,且阈值低。
进一步的,可以将上述核酸导入宿主细胞中,在相应启动子的调控表达下,宿主细胞高效表达前面所述的呈味肽,呈味肽呈味效果明显,可同时呈鲜味和甜味,且阈值低。
进一步的,本发明提供一种构建体。根据本发明的实施例,所述构建体携带前面所述的核酸。利用根据本发明实施例的核酸导入宿主细胞,使得宿主细胞表达前面所述的呈味肽,呈味肽呈味效果明显,可同时呈鲜味和甜味,且阈值低。
进一步的,本发明提出了一种重组细胞。根据本发明的实施例,所述重组细胞是通过前面所述的核酸或前面所述的构建体导入宿主细胞获得的。根据本发明实施例的重组细胞可以表达和/或分泌前面所述的呈味肽,可用于呈味肽的制备,所得呈味肽呈味效果明显,可同时呈鲜味和甜味,且阈值低。
进一步的,本发明提供一种富硒呈味肽的应用,所述应用为食品领域或者保健品领域,所谓食品包括选自调味品、休闲食品、饮料的至少之一,所述保健品选自具有抗癌功能和/或防癌功能的保健品。
需要说明的是,制备前面所述的呈味肽不限于前面所述的基因工程的方法或酶解纯化海藻粉的方法,本领域技术人员亦可根据实验需要,选择固相合成等化学合成等其他工艺手段获得。
附图说明
图1是根据本发明实施例的柱层析分离结果图;
图2是本发明呈味肽的反相HPLC图谱。
具体实施方式
以下结合实施例对本发明作进一步详细描述。
实施例1 一种富硒呈味肽的制备方法
1)将洗净除杂的海藻(经检测,其中的硒含量为0.22μg/g)于70℃烘干,经球磨粉碎机充分粉碎30-45min,过100目筛后按照海藻粉:水(重量比)为1:20的比例在65℃条件下混合超声均质,超声功率150W、处理20分钟。
2)处理后的均质液冷却到室温后调节pH至2.0,按均质液质量的1%分别加入果胶酶和胃蛋白酶在25-30℃下酶解4-6h后,调节pH至6.5-7.0,按照按均质液质量的1%分别加入纤维素酶、风味蛋白酶和中性蛋白酶,在45~55℃下搅拌酶解6h后升温至90℃灭酶10min。
3)将酶解灭酶后的均质液用三层纱布过滤后, 12000rpm离心10分钟,收集上清液。 用0.45μm的滤膜抽滤,滤液再依次利用分子量截留范围为1000Da的超滤膜和200Da的纳滤膜对其进行超滤分离,收集分子量在200-1000Da的超滤组分,冷冻干燥后储存于-80℃冰箱中。
4)将前述分子量<1000Da的超滤组分冷冻干燥后利用超纯水配置成浓度为10mg/mL,取1mL溶液上柱(2.0cm×100cm),凝胶层析柱的填料为Sephadex G-15葡聚糖凝胶树脂,用蒸馏水洗脱,收集吸收峰获得的3个洗脱组分(如图1所示)。将收集的3个层析组分分别冷冻干燥后储存于 -80℃冰箱中(分别记为成分1-3)。根据现有技术中常用的人工感官评定方法和电子舌系统测定方法,确认第三个峰的洗脱成分浓厚感和鲜味最强。
5)对层析后选择的浓厚感和鲜味最强的组分进行反相脱盐处理。脱盐纯化在在Pharmacia公司的AKTA蛋白纯化系统上完成,采用分离柱为Phenomenex C18柱(4.6 mm ×250 mm)。一次进样200~300 mL。先用100% ddH2O(含0.1%TFA)冲洗20 min,将混在样品中的盐分洗涤干净(紫外检测吸收值为零)之后,用乙腈(含0.1%TFA)溶液进行梯度洗脱。流速为1.0 mL/min,检测波长为280nm,柱温为40℃,收集洗脱峰。
6)目的样品再在反相HPLC纯化系统(Waters公司,Alliance 2690 HPLC &Millennium32 高效液相色谱工作站),996 PDA检测器上进行纯化。分离柱为PhenomenexC18柱(4.6 mm × 250 mm);洗脱液分别为:A液(0.1% TFA/H2O)、B液(0.1% TFA/CAN),流速为1.0 mL/min,检测波长为280 nm,柱温箱温度为40℃。
7)用MALDI-TOF质谱仪鉴定样品的纯度,目的样品的冻干粉末置于-20℃冰箱储存备用。
8)在 Perkin Elmer Procise 491A型气相测序仪(美国Applied Biosystem公司产品)上进行氨基酸序列测定,确定氨基酸序列为Ala- Pro-Met*- Leu(SEQ ID NO.1),其中Met*为硒取代甲硫氨酸。
与此同时,利用胃蛋白酶、风味蛋白酶和中性蛋白酶的组合进行酶解获得的200-1000Da的超滤组分记为对照1;果胶酶、胰蛋白酶和风味蛋白酶的组合进行酶解获得的200-1000Da的超滤组分记为对照2;果胶酶、碱性蛋白酶、中性蛋白酶和风味蛋白酶的组合进行酶解获得的200-1000Da的超滤组分记为对照3(其余步骤与上同)。
实施例2 Ala- Pro-Met*- Leu序列呈味肽的感官评价
为了验证呈味肽的具体性能,委托天根生化(北京)有限公司合成本发明的Ala-Pro-Met*- Leu序列呈味肽(命名为A),同时,为了进行对照试验,同时制备了之前专利申请中的呈味肽Ala-Asp-Glu(命名为B),Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val(命名为C),Ala-Ser-Asn-Met-Ser-Asp-Leu(命名为D)。
采用滋味稀释分析法(TDA)对上述不同序列的呈味肽和层析组分1-3、对照组份1-3进行评价。
滋味稀释分析(TDA)法如下:取本发明的呈味肽配置成5g/L的评价溶液,其基础溶液采用纯水和空白鸡汤,再依次按照1:1的比例用水和空白鸡汤对评价溶液进行逐步稀释,逐步稀释后的评价溶液按照浓度递减的顺序呈给经过训练的评价人员(6名:3男,3女,年龄在25-45岁间),每个稀释水平溶液采用三角实验测定法进行评定。当某个稀释水平的溶液与两个空白(水或空白鸡汤)之间的厚味差异不能被任何一位评价人员识别出来时,记录此时样品浓度,即为样品在该溶液中的阈值。此时的稀释倍数即稀释因子系数(TDA系数)。阈值及TDA系数的最终结果值取各评价人员评定结果的平均值。每份样品在不同时间内重复三次,均为常温下评定。每位感官人员还需要评价每次呈上样品的呈味特性。记录结果如表1所示。
表1:呈味肽的呈味特性、TDA系数、阈值
样品名称 | TD值 | 阈值 | 呈味特性 |
组分1 | - | - | 无明显味感 |
组分2 | 2 | 0.25% | 咸味 |
组分3 | 64 | 0.008% | 咸味、鲜味、甜味 |
对照1 | 4 | 0.125% | 苦味、咸味 |
对照2 | 2 | 0.25% | 咸味 |
对照3 | 4 | 0.125% | 咸味、鲜味 |
A | 64 | 0.008% | 咸味、鲜味、甜味 |
B | 32 | 0.016% | 咸味、鲜味 |
C | 24 | 0.023% | 咸味、鲜味 |
D | 12 | 0.047% | 咸味、鲜味 |
食盐 | - | 0.047% | 咸味 |
鸡精 | - | 0.125% | 咸味、鲜味 |
味精 | - | 0.125% | 咸味、鲜味 |
白砂糖 | - | 0.023% | 甜 |
注:“-”表示因组分无明显味感而无法测定、计算。
实施例3 呈味肽的生物保健功能验证
将利用获得的Ala- Pro-Met*- Leu序列呈味肽利用蒸馏水解配制成20%的肽溶液,进行小鼠饲喂实验。具体为:将6周龄清洁小鼠随机分为3组,每组12只,饲以无硒饲料建立低硒模型,自由采食饮水,室温保持(25±1)℃。给药前6h对小鼠禁食,自由饮水。三组小鼠分别灌胃100ml肽溶液 (实施例1的呈味肽,5%亚硒酸钠溶液,生理盐水),并于灌胃前和灌胃后0,2,4,6,8,10,15,30min尾部取血600μL,置于EDTA抗凝管中,3000r/min下离心10min,吸取上层血浆于离心管中,做好标记。采用氢化物发生-原子荧光光谱法(HG-AFS)检测小鼠血浆中的硒含量(如表2所示)。
表2 小鼠血液中硒含量
组别 | 小鼠血液中的血硒值的峰值总量(mg/L) | 小鼠血液中的血硒值的峰值时间(min) |
呈味肽 | 2.56 | 6 |
亚硒酸钠溶液 | 0.68 | 10 |
生理盐水 | - | - |
从表中可以看出,在实施例呈味肽和阳性对照(亚硒酸钠)后,都不同程度的出现了体内硒含量提升的现象,但是从表中可以发现,呈味肽组小鼠血液中的血硒值的峰值显著高于亚硒酸钠样品,而且血硒值的峰值出峰时间明显的早于阳性对照。说明本发明的富硒活性肽样品中的硒更容易被吸收,补充硒的效果好于亚硒酸钠,这可能是因为本发明的呈味肽仅仅为4肽小分子,可以伴随着水快速经胃排空后进入小肠等重要消化系统,由于其分子量小,更易被小肠和十二指肠吸收。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
<110>国民康健(北京)自然科学研究有限公司
〈120〉一种从富硒海藻中分离的富硒呈味肽、制备方法和应用
〈160〉 1
〈170〉 PatentIn version 3.3
〈210〉 1
〈211〉 28
〈212〉 proetein
〈213〉peptide
〈400〉Ala- Pro-Met*- Leu,其中Met*为硒取代甲硫氨酸
Claims (3)
1.一种呈味肽,其特征在于,所述呈味肽的氨基酸序列如SEQ ID NO.1: Ala- Pro-Met*- Leu所示,其中Met*为硒取代甲硫氨酸。
2.如权利要求1所述的呈味肽的应用,所述应用为食品领域,所述食品选自调味品、休闲食品、饮料的至少一种。
3.含有权利要求1所述的呈味肽的产品,所述产品为食品。
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