CN107119096A - 一种滑菇活性肽的制备方法及其应用 - Google Patents
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Abstract
本发明公开了一种滑菇活性肽的制备方法及其应用。本发明的滑菇活性肽以滑菇子实体为原料,通过粉碎、制浆、浸提、酶解、浓缩、喷雾干燥等工艺制备而成。本发明的滑菇活性肽制备工艺简单,产品安全,具有体外抗氧化和抗肿瘤活性,以及体内降血脂作用,可应用于食品、保健食品、药品等领域,有利于滑菇蛋白质资源的开发与利用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种滑菇活性肽的制备方法及其应用。
背景技术
滑菇(Pholiota nameko)是一种冬、春季发生的菌盖粘滑的木腐菌,又名光帽鳞伞、光帽黄伞、珍珠菇、滑子蘑,是世界上五大宗人工栽培食用菌之一。滑菇味道鲜美,营养丰富,药用价值高,是一种集美味、营养、保健于一身的优质食用菌,其天然提取物作为功能性食品或药品已被视为安全有效的途径。
近年来,国内外学者对滑菇的营养成分及其功能进行了大量研究,在其菌丝体、子实体中分离到多种活性物质,具有免疫调节、降血糖、抗肿瘤、抗氧化等功能,但是,有关研究主要集中在多糖以及生物碱、萜类、黄酮类等小分子次生代谢产物上。如李海平公开了滑菇多糖提取物的制备方法(CN101863999B)和一种滑菇不溶性膳食纤维的制备方法(CN103284167B)。
另外,CN103864944 B中提供一种滑菇多糖粗品的制备方法,涉及抗肿瘤药物领域,其具体工艺过程如下:粉碎、筛分→微波处理→浸提→酶解→膜过滤→提取→脱色→干燥八个步骤。但是该方法也没有涉及滑菇蛋白或多肽。 吴艳丽等在杏鲍菇多肽对铅致大鼠氧化损伤的干预作用中公开了杏鲍菇多肽的提取方法,但是该方法在提取效率以及多肽的提取总量上还有待提高,而且也不是特异性针对滑菇的。
目前,有关滑菇活性蛋白或肽的报道极少。为深度开发、利用滑菇的蛋白质资源,本发明公开了一种滑菇活性肽的制备方法及其应用。
发明内容
本发明的目的在于提供一种滑菇活性肽的制备方法及其应用。
本发明涉及的滑菇活性肽的制备方法包括以下步骤:
(1)预处理:将干燥的滑菇子实体粉碎,过60目筛,按1:20-1:30(W/V)的料液比与蒸馏水混合,制浆,即滑菇浆液;
(2)浸提:将滑菇浆液置于超声波提取器中,超声波频率控制在300W-500W之间,按原料质量的0.1%-0.3%(W/W)加入漆酶(104U/g),调节pH值至5.0-6.0,在40-50℃下处理1-2小时;然后在80-100℃下处理10-20min,6000-10000r/min离心20-40min,收集上清液,即滑菇浸提液;
(3)酶解I:将滑菇浸提液置于酶解反应器中,按原料质量的0.1%-0.3%(W/W)加入中性蛋白酶(105U/g),调节pH值至6.0-7.0,在40-50℃下水解2-4小时;然后在80-100℃下处理10-20min,灭活蛋白酶,6000-10000r/min离心20-40min,收集上清液,即酶解液I;
(4)酶解II:将酶解液I置于酶解反应器中,按原料质量的0.1%-0.3%(W/W)加入风味蛋白酶(105U/g),调节pH值至6.0-7.0,在50-60℃下水解2-4小时;然后在80-100℃下处理10-20min,灭活蛋白酶,6000-10000r/min离心20-40min,收集上清液,即酶解液II;
(5)浓缩:将酶解液II置于旋转蒸发器中,在50-60℃下处理2-4小时;然后采用MWCO1000和MWCO3000的超滤膜,在0.30-0.50Mpa下处理1-2小时,获得分子量在1000Da-3000Da的浓缩液,即滑菇活性肽浓缩液;
(6)干燥:将滑菇活性肽浓缩液在0.05-0.10Mpa、50-60℃下喷雾干燥,即得滑菇活性肽。
本发明涉及的滑菇活性肽具有抗氧化活性、抗肿瘤活性和降血脂作用,在体外能够清除DPPH自由基和羟基自由基,对肝癌细胞HepG2具有抗增殖活性,在体内对SD大鼠的降血脂作用与调节血清TC、TG、MDA、NO含量和SOD 活性相关。
本发明具有的优点和有益效果:
本发明以滑菇子实体为原料,将漆酶预处理与超声波辅助提取相结合,有利于增加提取液中活性成分的含量;工艺简单,成本低,适于工业化生产;生产过程无污染,产品安全性好;具有抗氧化、抗肿瘤活性和降血脂作用,可应用于食品、保健食品和药品。
具体实施例
下面结合具体实施例对本发明作进一步的说明。
实施例1 滑菇活性肽的制备
(1)预处理:将干燥的滑菇子实体粉碎,过60目筛,按1:20(W/V)的料液比与蒸馏水混合,制浆,即滑菇浆液;
(2)浸提:将滑菇浆液置于超声波提取器中,超声波频率控制在500W,按原料质量的0.1%(W/W)加入漆酶(104U/g),调节pH值至5.0,在40℃下处理2小时;然后在80℃下处理20min,6000r/min离心40min,收集上清液,即滑菇浸提液;
(3)酶解I:将滑菇浸提液置于酶解反应器中,按原料质量的0.1%(W/W)加入中性蛋白酶(105U/g),调节pH值至6.0,在40℃下水解4小时;然后在80℃下处理20min,灭活蛋白酶,6000r/min离心40min,收集上清液,即酶解液I;
(4)酶解II:将酶解液I置于酶解反应器中,按原料质量的0.1%(W/W)加入风味蛋白酶(105U/g),调节pH值至6.0,在50℃下水解4小时;然后在80℃下处理20min,灭活蛋白酶,6000r/min离心40min,收集上清液,即酶解液II;
(5)浓缩:将酶解液II置于旋转蒸发器中,在50℃下处理4小时;然后采用MWCO1000和MWCO3000的超滤膜,在0.30Mpa下处理2小时,获得分子量在1000-3000Da的浓缩液,即滑菇活性肽浓缩液;
(6)干燥:将滑菇浓缩液在0.05Mpa、50℃下喷雾干燥,即得滑菇活性肽。
实施例2 滑菇活性肽的制备
(1)预处理:将干燥的滑菇子实体粉碎,过60目筛,按1:25(W/V)的料液比与蒸馏水混合,制浆,即滑菇浆液;
(2)浸提:将滑菇浆液置于超声波提取器中,超声波频率控制在400W,按原料质量的0.2%(W/W)加入漆酶(104U/g),调节pH值至5.5,在45℃下处理1.5小时;然后在90℃下处理15min,8000r/min离心30min,收集上清液,即滑菇浸提液;
(3)酶解I:将滑菇浸提液置于酶解反应器中,按原料质量的0.2%(W/W)加入中性蛋白酶(105U/g),调节pH值至6.5,在45℃下水解3小时;然后在90℃下处理15min,灭活蛋白酶,8000r/min离心30min,收集上清液,即酶解液I;
(4)酶解II:将酶解液I置于酶解反应器中,按原料质量的0.2%(W/W)加入风味蛋白酶(105U/g),调节pH值至6.5,在55℃下水解3小时;然后在90℃下处理15min,灭活蛋白酶,8000r/min离心30min,收集上清液,即酶解液II;
(5)浓缩:将酶解液II置于旋转蒸发器中,在55℃下处理3小时;然后采用MWCO1000和MWCO3000的超滤膜,在0.40Mpa下处理1.5小时,获得分子量在1000-3000Da的浓缩液,即滑菇活性肽浓缩液;
(6)干燥:将滑菇浓缩液在0.08Mpa、55℃下喷雾干燥,即得滑菇活性肽。
实施例3 滑菇活性肽的制备
(1)预处理:将干燥的滑菇子实体粉碎,过60目筛,按1:30(W/V)的料液比与蒸馏水混合,制浆,即滑菇浆液;
(2)浸提:将滑菇浆液置于超声波提取器中,超声波频率控制在300W,按原料质量的0.3%(W/W)加入漆酶(104U/g),调节pH值至6.0,在50℃下处理1小时;然后在100℃下处理10min,10000r/min离心20min,收集上清液,即滑菇浸提液;
(3)酶解I:将滑菇浸提液置于酶解反应器中,按原料质量的0.3%(W/W)加入中性蛋白酶(105U/g),调节pH值至7.0,在50℃下水解2小时;然后在100℃下处理10min,灭活蛋白酶,10000r/min离心20min,收集上清液,即酶解液I;
(4)酶解II:将酶解液I置于酶解反应器中,按原料质量的0.3%(W/W)加入风味蛋白酶(105U/g),调节pH值至7.0,在60℃下水解2小时;然后在100℃下处理10min,灭活蛋白酶,10000r/min离心20min,收集上清液,即酶解液II;
(5)浓缩:将酶解液II置于旋转蒸发器中,在60℃下处理2小时;然后采用MWCO1000和MWCO3000的超滤膜,在0.50Mpa下处理1小时,获得分子量在1000-3000Da的浓缩液,即滑菇活性肽浓缩液;
(6)干燥:将滑菇浓缩液在0.10Mpa、60℃下喷雾干燥,即得滑菇活性肽。
实施例4 滑菇活性肽的抗氧化活性
(1)方法
DPPH自由基清除活性:1mL浓度为50μmol/L 的DPPH溶液含有不同浓度的待测样品,混匀,25℃反应90min,517nm波长处测定吸光值。以不含待测样品为空白对照,丁化羟基苯甲醚(BHA)为正对照。清除率的计算公式为:清除率(%)=(A0-A)/A0×100%,其中A0为空白对照的吸光值,A为待测样品的吸光值。
羟基自由基清除活性:3mL浓度为0.15mmol/L 的PBS缓冲液(pH 7.4)含有100mmol/L Vc,100μmol/L CuSO4,10μmol/L细胞色素C和不同浓度的待测样品,混匀,25℃反应90分钟,550nm波长处测定透光值。以不含待测样品为空白对照,丁化羟基苯甲醚(BHA)为正对照。清除率的计算公式为:清除率(%)=(T0-T)/T0×100%,其中T0为空白对照的透光值,T为待测样品的透光值。
超氧阴离子自由基清除活性:1mL浓度为16mmol/L 的Tris缓冲液(pH 8.0)含有80μmol/L NADH、50μmol/L NBT、10μmol/L PMS和不同浓度的待测样品,混匀,25℃反应90分钟,560nm波长处测定吸光值。以不加待测样品为空白对照,没食子酸为正对照。清除率的计算公式为:清除率(%)=(A0-A)/A0×100%,其中A0为空白对照的吸光值,A为待测样品的吸光值。
(2)原理
DPPH(1,1-二苯基-2-三硝基苯肼)是一种稳定的氮中心的自由基,其乙醇溶液呈紫色,在517nm波长处有最大吸收。当自由基被清除时,吸光值减小,溶液颜色变浅,借此评价DPPH自由基清除活性。
VC和CuSO4反应产生羟基自由基,可以氧化浅红色的还原型细胞色素C生成浅黄色的氧化型细胞色素C,通过测定550nm波长处的透光值,能够间接测定反应体系中羟基自由基的含量,从而检测羟基自由基清除活性。
在吩嗪硫酸甲酯(PMS)-还原型烟酰胺腺嘌呤二核苷酸(NADH)体系中,PMS氧化NADH产生超氧阴离子自由基,可以还原氮蓝四唑(NBT)为蓝色产物—甲腙,在560nm波长处有最大吸收,借此评价超氧阴离子自由基清除活性。
(3)结果
由表1可知,在100-500μmol/L的浓度范围内,滑菇活性肽清除自由基的活性随着浓度的增加而增强。与正对照相比,滑菇活性肽清除DPPH自由基和羟基自由基的活性高于BHA,但无显著差异(P>0.05),而滑菇活性肽的超氧阴离子自由基清除活性较低,显著低于没食子酸(P<0.05),表明滑菇活性肽具有抗氧化活性,与清除DPPH自由基和羟基自由基相关。
表1 滑菇活性肽的抗氧化活性
注:与正对照相比,*P<0.05
实施例5 滑菇活性肽的抗肿瘤活性
(1)将乳腺癌细胞MCF7、子宫颈癌细胞HeLa、肺癌细胞A549、肝癌细胞HepG2按1×105cfu/mL的浓度接种于96孔板,每孔100μL,37℃培养24小时;
(2)每孔加入10μL不同浓度(0.125mg/mL、0.25mg/mL、0.5mg/mL、1.0mg/mL、2.0mg/mL)的滑菇活性肽,37℃培养48小时;
(3)每孔加入25µL三氯醋酸(500mg/mL),4℃放置1小时;
(4)蒸馏水洗涤5次,每孔加入4mg/mL SRB,染色30min;
(5)醋酸洗涤5 次,每孔加入100µL Tris缓冲液(10mmol/L),测定490nm处吸光值。
半抑制浓度(IC50)是评价肿瘤细胞增殖抑制活性的常用指标,滑菇活性肽对乳腺癌细胞MCF7、子宫颈癌细胞HeLa、肺癌细胞A549、肝癌细胞HepG2的IC50分别为13.5μmol/L、8.9μmol/L、26.1μmol/L、2.7μmol/L,表明本发明的滑菇活性肽对肝癌细胞HepG2具有较强的抗增殖活性。
实施例6 滑菇活性肽的降血脂作用
(1)方法
70只SD大鼠随机分为2组,空白组10只,普通饲料喂养,高脂组60只,高脂饲料喂养。10天后,高脂组SD大鼠眼眶静脉采血,测定总胆固醇(TC)和甘油三酯(TG),然后随机分为模型组,血脂康组,滑菇活性肽灌胃高、低剂量组,腹腔注射高、低剂量组,连续给药4周。末次给药后禁食12h,股主动脉取血,测定总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、丙二醛(MDA)、超氧化物歧化酶(SOD)、一氧化氮(NO)。
(2)结果
由表2可知,与空白组相比,模型组TC、TG升高(P<0.05),表明模型组大鼠存在血脂代谢紊乱。与模型组相比,滑菇活性肽各剂量组TC、TG降低(P<0.05),HDL-C无明显差异(P>0.05),表明滑菇活性肽的降血脂作用与调节TC、TG含量相关。
表2 滑菇活性肽对大鼠TC、TG、HDL-C的影响
注:与空白组相比,△P<0.05,与模型组相比,*P<0.05
由表3可知,与空白组相比,模型组MDA、NO值升高,SOD值降低(P<0.05)。与模型组相比,滑菇活性肽各剂量组MDA、NO值降低,SOD值升高(P<0.05),表明滑菇活性肽的降血脂作用与调节SOD 活性、MDA和NO含量相关。
表3 滑菇活性肽对大鼠MDA、SOD和NO的影响
注:与空白组相比,△P<0.05,与模型组相比,*P<0.05
以上仅为本发明的优选实施例而已,并不用于限制本发明,凡在本发明的原则内所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种滑菇活性肽的制备方法,其特征在于包括以下步骤:
(1)预处理:将干燥的滑菇子实体粉碎,过60目筛,按1:20-1:30(W/V)的料液比与蒸馏水混合,制浆,即滑菇浆液;
(2)浸提:将滑菇浆液置于超声波提取器中,超声波频率控制在300W-500W之间,按原料质量的0.1%-0.3%(W/W)加入漆酶(104U/g),调节pH值至5.0-6.0,在40-50℃下处理1-2小时;然后在80-100℃下处理10-20min,6000-10000r/min离心20-40min,收集上清液,即滑菇浸提液;
(3)酶解I:将滑菇浸提液置于酶解反应器中,按原料质量的0.1%-0.3%(W/W)加入中性蛋白酶105U/g,调节pH值至6.0-7.0,在40-50℃下水解2-4小时;然后在80-100℃下处理10-20min,灭活蛋白酶,6000-10000r/min离心20-40min,收集上清液,即酶解液I;
(4)酶解II:将酶解液I置于酶解反应器中,按原料质量的0.1%-0.3%(W/W)加入风味蛋白酶105U/g,调节pH值至6.0-7.0,在50-60℃下水解2-4小时;然后在80-100℃下处理10-20min,灭活蛋白酶,6000-10000r/min离心20-40min,收集上清液,即酶解液II;
(5)浓缩:将酶解液II置于旋转蒸发器中,在50-60℃下处理2-4小时;然后采用MWCO1000和MWCO3000的超滤膜,在0.30-0.50Mpa下处理1-2小时,获得分子量在1000Da-3000Da的浓缩液,即滑菇活性肽浓缩液;
(6)干燥:将滑菇活性肽浓缩液在0.05-0.10Mpa、50-60℃下喷雾干燥,即得滑菇活性肽。
2.一种权利要求1所述的滑菇活性肽的制备方法制备得到活性肽的应用,其特征在于所述的滑菇活性肽在抗氧化、抗肿瘤和降血脂中的用途。
3.一种权利要求1所述的滑菇活性肽的制备方法制备得到活性肽的应用,其特征在于所述的滑菇活性肽在制备抗氧化、抗肿瘤和降血脂的药物中的用途。
4.根据权利要求4所述的用途,其特征在于滑菇活性肽在体外能够清除DPPH自由基和羟基自由基,对肝癌细胞HepG2具有抗增殖活性,在体内对SD大鼠的降血脂作用与调节血清TC、TG、MDA、NO含量和SOD 活性相关。
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