CN109601793A - 大鲵活性肽酵素 - Google Patents
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Abstract
本发明公开一种大鲵活性肽酵素,采用大鲵活性肽与多种辅料同时发酵的方法进行制备,利用苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜、桔梗、人参、大枣、蓝莓、山葡萄、木耳、枸杞子及沙棘在烧酒曲、酵母、乳酸菌及醋酸菌发酵作用下产生的酶,将大鲵活性肽进行酶解,使其甜味氨基酸显著高于苦味氨基酸(甜味氨基酸9.75mg/g、苦味氨基酸5.24mg/g),不仅改善的大鲵活性肽的品质,同时所得到的大鲵活性肽酵素可快速解酒且可明显减轻乙醇对肝组织急性损伤。
Description
技术领域
本发明涉及一种酵素,尤其是一种具有可快速解酒且可明显减轻乙醇对肝组织急性损伤的大鲵活性肽酵素。
背景技术
酵素是近年流行的保健饮料,大多以苹果、梨、柠檬、柑橘、橙、草莓、猕猴桃及葡萄等水果为原料,采用传统发酵工艺制备而成,是一种天然的、无任何添加剂的保健饮品。研究表明,酵素饮品在营养保健、临床医学、美容护理、减肥瘦身、亚健康调理等领域有广泛的应用。
中国专利申请号为201610314751.X的发明专利申请公开了一种“大鲵活性肽及用途”,大鲵活性肽是按照如下步骤制备:
a. 将大鲵肉匀浆,向匀浆中加入1~3倍体积pH 7~8的0.01~0.1M磷酸缓冲液及与匀浆重量比为0.1~1%的复合酶,酶解12~48 h;所述复合酶为海洋碱性蛋白酶和木瓜蛋白酶按活力单位比为7:3比例混合;
b. 离心取上清,采用截留分子量为4000 Da以下用物理吸附法制备的胰蛋白酶固定超滤膜分离器分离,取透过超滤膜的液体;
c. 通过Sephadex LH-20分子筛层析柱,按每管1ml收集层析产物,取第30~36管层析产物进行冷冻干燥;
d.将c步骤所得到的冷冻干燥物制备成10~50mg/ml的去离子水溶液,8000 rpm、4℃条件下离心10 min,用0.22 µm水膜过滤,得到上样上清液;
e. 使用ddH2O 配制5%甲醇缓冲溶液,通过孔径0.45 µm水膜抽滤,通过超声波除气作为高效液相色谱柱流动相A,使用ddH2O 配制95%甲醇缓冲溶液,通过孔径0.45 µm油膜抽滤,通过超声波除气作为高效液相色谱柱流动相B;使用流动相A和流动相B进行梯度洗脱,洗脱条件:流速为1ml/min,洗脱梯度分配:0~20min,流动相A100%;20.5~40min,流动相A50%,流动相B50%;40.5~60min, 流动相B100%;将上液上清液注入C18柱高效液相色谱仪进样口,收集紫外检测波长280 nm的吸收峰;
f. 挥发收集物中的甲醇后真空冷冻干燥,得到大鲵肽制品。
该专利申请说明书公开了所制备的大鲵活性肽可清除自由基、提高机体免疫力及促进皮肤纤维原细胞增殖。
但是,实践中发现大鲵活性肽所含有的苦味氨基酸较多(甜味氨基酸为6.58mg/g,苦味氨基酸为7.71mg/g),口感较腥,影响了大鲵活性肽产品的开发,同时现有大鲵活性肽并不具备解酒及减轻乙醇对肝组织急性损伤的作用。
发明内容
本发明是为了解决现有技术所存在的上述技术问题,提供一种可快速解酒且可明显减轻乙醇对肝组织急性损伤的大鲵活性肽酵素。
本发明的技术解决方案是:一种大鲵活性肽酵素,其特征在于依次按照如下步骤进行制备:
a. 将苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜、桔梗洗净并沥干,切成1~3cm块状;将大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘洗净并沥干;各取相同质量进行混合,得混合物A;
b. 将混合物A与食醋、红糖、大鲵活性肽按质量比15:1:0.5:0.5进行混合,得混合物B,再将烧酒曲、酵母、乳酸菌及醋酸菌加入混合物B中,10~25℃于发酵罐中发酵50~60天,得一次发酵液;所述烧酒曲、酵母、乳酸菌及醋酸菌的加入量为混合物B质量的0.5%~1%、1%~2.5%、1%~1.5%、1%~1.5%,所述烧酒曲、酵母、乳酸菌及醋酸菌的菌数为15~26×106个/ml;
c. 将一次发酵液置于发酵罐中,所述一次发酵液体积为发酵罐容积的2/3,发酵罐保持36~38℃恒温发酵至少60天,发酵的同时于发酵罐底向发酵罐内通入空气,每立方米发酵罐空气通入量为0.5-1L/min,即得大鲵活性肽发酵液;
d. 将所得大鲵活性肽发酵液过滤,滤液放入罐中浸泡竹炭贮藏30~60天,即得大鲵活性肽酵素。
本发明采用大鲵活性肽与多种辅料同时发酵的方法进行制备,利用苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜、桔梗、人参、大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘在烧酒曲、酵母、乳酸菌及醋酸菌发酵作用下产生的酶,将大鲵活性肽进行酶解,使其甜味氨基酸显著高于苦味氨基酸(甜味氨基酸9.75mg/g、苦味氨基酸5.24mg/g),不仅改善的大鲵活性肽的品质,同时所得到的大鲵活性肽酵素可快速解酒且可明显减轻乙醇对肝组织急性损伤。
附图说明
图1是本发明实验各小鼠肝组织病理图(HE,100×)。
具体实施方式
实施例1:
本发明的大鲵活性肽酵素,依次按照如下步骤制备:
a. 将苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜及桔梗洗净并沥干,切成2cm块状;将大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘洗净并沥干;各取相同质量(1000g)进行混合,得混合物A;
b. 将混合物A与食醋、红糖、大鲵活性肽(按照背景技术所述方法制备)按质量比15:1:0.5:0.5进行混合,得混合物B,再将烧酒曲、酵母、乳酸菌及醋酸菌加入混合物B中,20℃于发酵罐中发酵50天,得一次发酵液;所述烧酒曲、酵母、乳酸菌及醋酸菌的加入量为混合物B质量的0.5 %、1%、1.5%、1.25%,所述乳酸菌及醋酸菌的菌数为26×106个/ml;
c. 将一次发酵液置于另一发酵罐,一次发酵液体积为发酵罐容积的2/3,将发酵罐保持37℃恒温发酵60天,发酵的同时于发酵罐底向发酵罐内通入空气,每立方米发酵罐空气通入量为0.75L/min,即得大鲵活性肽发酵液;
d. 将所得大鲵活性肽发酵液过滤,滤液放入罐中浸泡竹炭贮藏50天,即得大鲵活性肽酵素。
除大鲵活性肽,所有原料均外购所得。
将实施例1所得大鲵活性肽酵素按照食品安全国家标准《食品中氨基酸的测定》(GB5009.124-2016)规定的方法进行检测,甜味氨基酸9.75mg/g、苦味氨基酸5.24mg/g。
实施例2:
本发明的大鲵活性肽酵素,依次按照如下步骤制备:
a. 将苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜及桔梗洗净并沥干,切成1cm块状;将大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘洗净并沥干;各取相同质量(1000g)进行混合,得混合物A;
b. 将混合物A与食醋、红糖、大鲵活性肽按质量比15:1:0.5:0.5进行混合,得混合物B,再将烧酒曲、酵母、乳酸菌及醋酸菌加入混合物B中,10℃于发酵罐中发酵55天,得一次发酵液;所述烧酒曲、酵母、乳酸菌及醋酸菌的加入量为混合物B质量的0.5%、2.5%、1%、1.5%,所述所述烧酒曲、酵母、乳酸菌及醋酸菌的菌数为20×106个/ml;
c. 将一次发酵液置于另一发酵罐,一次发酵液体积为发酵罐容积的2/3,将发酵罐保持36℃恒温发酵60天,发酵的同时于发酵罐底向发酵罐内通入空气,每立方米发酵罐空气通入量为0.5L/min,即得大鲵活性肽发酵液;
d. 将所得大鲵活性肽发酵液过滤,滤液放入罐中浸泡竹炭贮藏30天,即得大鲵活性肽酵素。
原料来源同实施例1。
实施例3:
本发明的大鲵活性肽酵素,依次按照如下步骤制备:
a. 将苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜及桔梗洗净并沥干,切成3cm块状;将大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘洗净并沥干;各取相同质量(500g)进行混合,得混合物A;
b. 将混合物A与食醋、红糖、大鲵活性肽按质量比15:1:0.5:0.5进行混合,得混合物B,再将烧酒曲、酵母、乳酸菌及醋酸菌加入混合物B中,25℃于发酵罐中发酵50天,得一次发酵液;所述烧酒曲、酵母、乳酸菌及醋酸菌的加入量为混合物B质量的1%、1%、1.5%、1%,所述所述烧酒曲、酵母、乳酸菌及醋酸菌的菌数为26×106个/ml;
c. 将一次发酵液置于另一发酵罐,一次发酵液体积为发酵罐容积的2/3,将发酵罐保持38℃恒温发酵60天,发酵的同时于发酵罐底向发酵罐内通入空气,每立方米发酵罐空气通入量为1L/min,即得大鲵活性肽发酵液;
d. 将所得大鲵活性肽发酵液过滤,滤液放入罐中浸泡竹炭贮藏60天,即得大鲵活性肽酵素。
实验例1:
将本发明实施例1所得大鲵活性肽酵素冻干,得到大鲵活性肽酵素粉。取大鼠40只,禁食6h,随机分成对照组及实验组,每组20只。用生理盐水灌胃空白对照组。实验组灌胃相同体积生理盐水溶解的大鲵活性肽酵素粉按0.3g/kg·d·w灌胃30min后按12.0g/kg·d·w给予酒精,酒精事先进行稀释使用。以翻正反射消失为醉酒指标,计算醉酒耐受时间及醉酒维持时间,具体结果如表1。
表1 本发明实施例1对大鼠抗醉作用的影响
实验结果表明,服用本发明大鲵活性肽酵素的实验组大鼠的醉酒耐受时间明显长于对照组,而醉酒维持时间与对照组相比明显缩短,两组数据相比均有显著性差异(p<0.01)。因此,本发明大鲵活性肽酵素可用于快速解酒,减少醉酒时间,缓解醉酒症状。
应用例2:
将本发明实施例1的大鲵活性肽酵素冻干,得到大鲵活性肽酵素粉。选昆明种小鼠50只,随机分成空白对照组、模型对照组和硫普罗宁阳性对照组(50mg/kg·d),大鲵活性肽酵素低浓度(50mg/kg˙d)、高浓度(150mg/kg˙d)剂量组,每组10只,于12h黑暗,12 h光亮的塑料饲养箱内饲养,室温(20±2)℃,湿度40%~60%,自由采食和饮水。各给药组每天灌胃1次,连续2周。对照组和模型组给予同样量的生理盐水,于每次给药3h后,除空白对照组外,各组均灌胃56度商品二锅头16mL/(kg˙d)。各组均于末次给药后禁食不禁水。
小鼠于末次给药后16h,断头取血,在30min内,3500r/min,离心5min后得上层血清备用。快速处死小鼠取肝脏,并在肝左叶组织取约0.3g放冰冷的生理盐水中漂洗,取出后用滤纸将水吸干,制成10%肝组织匀浆液(生理盐水的体积总量是组织块体积的9倍),制备好的10%的肝组织匀浆液在低温低速离心机中以2000r/min左右离心10~15min,将离心好的匀浆保留上清液弃沉淀进行生化指标测定。血清中谷草转氨酶(AST)与谷丙转氨酶(ALT)活性,肝脏组织中丙二醛(MDA)含量,超氧化物岐化酶(SOD)活性,蛋白质含量均按试剂盒的要求进行测定。处死小鼠后,取肝脏右叶组织约0.3g,用10%的中性甲醛溶液进行固定,石蜡包埋切片。苏木精-伊红(HE)染色,在显微镜下进行病理学观察。结果如表2所示。
表2 大鲵皮胶原蛋白肽对乙醇诱导肝损伤小鼠生化指标的影响
注:与乙醇模型组相比, *: p < 0.01
结果表明:饲喂大鲵活性肽酵素组的ALT、AST明显小于乙醇模型组(p<0.01)。大鲵活性肽酵素低剂量及高剂量组与乙醇模型组肝组织中MDA含量相比显著下降(p<0.01)。大鲵活性肽酵素低剂量及高剂量组SOD含量均高于乙醇模型组肝组织中的SOD含量。
各实验组的肝脏组织病理变化如图1所示:图1中A:对照组;B:硫普罗宁阳性对照组;C:乙醇模型组;D:大鲵活性肽酵素低剂量组(50mg/kg·d);E:大鲵活性肽酵素高剂量组(150mg/kg·d)
对照正常的肝组织切片,乙醇模型组肝细胞出现肿胀,出现大小不等的脂肪滴,伴有炎症细胞浸润;硫普罗宁对照组,基本与正常肝组织一致,肝细胞结构清晰,形态较正常;大鲵活性肽酵素低剂量组肝组织轻微损伤,肝细胞轻微肿胀,脂肪滴空泡较乙醇模型组轻;大鲵活性肽酵素高剂量组肝组织细胞正常,肝索排列整齐,与正常肝组织细胞差别不大。生物化学指标及组织病理结果表明,大鲵活性肽酵素具有明显减轻乙醇对肝组织的急性损伤的作用。
Claims (1)
1.一种大鲵活性肽酵素,其特征在于依次按照如下步骤进行制备:
a. 将苹果、梨、木瓜、胡萝卜、山药、冬瓜、辣椒、大白菜、萝卜、桔梗洗净并沥干,切成1~3cm块状;将大枣、蓝莓、黑果花楸、黑加仑、山葡萄、木耳、枸杞子及沙棘洗净并沥干;各取相同质量进行混合,得混合物A;
b. 将混合物A与食醋、红糖、大鲵活性肽按质量比15:1:0.5:0.5进行混合,得混合物B,再将烧酒曲、酵母、乳酸菌及醋酸菌加入混合物B中,10~25℃于发酵罐中发酵50~60天,得一次发酵液;所述烧酒曲、酵母、乳酸菌及醋酸菌的加入量为混合物B质量的0.5%~1%、1%~2.5%、1%~1.5%、1%~1.5%,所述烧酒曲、酵母、乳酸菌及醋酸菌的菌数为15~26×106个/ml;
c. 将一次发酵液置于发酵罐中,所述一次发酵液体积为发酵罐容积的2/3,发酵罐保持36~38℃恒温发酵至少60天,发酵的同时于发酵罐底向发酵罐内通入空气,每立方米发酵罐空气通入量为0.5-1L/min,即得大鲵活性肽发酵液;
d. 将所得大鲵活性肽发酵液过滤,滤液放入罐中浸泡竹炭贮藏30~60天,即得大鲵活性肽酵素。
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