CN114621980A - 一种提升羊栖菜提取物降血糖活性的方法 - Google Patents
一种提升羊栖菜提取物降血糖活性的方法 Download PDFInfo
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Abstract
本发明涉及微生物领域和食品加工领域,具体涉及一种提升羊栖菜提取物降血糖活性的方法。所述羊栖菜提取物制备过程包括以下步骤:羊栖菜与保藏编号为:CGMCC No.23295的米曲霉共同发酵得到发酵基质提取物。本发明提供的羊栖菜提取物为羊栖菜与保藏编号为:CGMCC No.23295的米曲霉共同发酵后提取得到的,相比未发酵处理的羊栖菜提取物,其降血糖活性具有显著提升的效果,具有较大的市场应用前景。
Description
技术领域
本发明涉及微生物领域和食品加工领域,具体涉及一种提升羊栖菜提取物降血糖活性的方法。
背景技术
羊栖菜(Sargassum fusiforme)隶属于褐藻门墨角藻目马尾藻科,别名鹿角尖、海菜芽等。羊栖菜在我国有着悠久的食用、药用历史,据《本草纲目》记载,羊栖菜咸能润下,寒能泄热引水,有消瘿瘤、结核,除水肿,利小便等功效。现代科学研究表明,羊栖菜富含多糖、甾醇类化合物、氨基酸、脂肪酸、微量元素等成分,具有抗肿瘤、降血糖、降血脂、抗氧化、抗疲劳、增强免疫能力等多种生物学活性(王百龙,孙稚颖,周凤琴.羊栖菜成分以及药理研究进展.山东中医杂志,2015,34(9):722-725)。降血糖是羊栖菜最主要的保健功效之一,但是作为植物材料,本身降血糖活性不强,需要进行分离纯化制备其活性成分(丁浩淼,孙弢,夏彭奎,汤倩,王忠华,汪财生,陈海敏,钱国英,2019.羊栖菜组分多糖对α-葡萄糖苷酶的抑制作用.核农学报,33(2):0297-0304),过程复杂,含量低,因此有必要对此进行改进。
发酵是提升植物源材料的有效方法之一,食药用真菌在发酵过程中能对植物源材料中的纤维、糖类、蛋白等物质加以利用,产生的酶具有破壁作用而使有效成分溶出,并可能对植物源材料中的成分进行生物转化,提高药效。但是,不同食药用真菌菌种的作用千差万别,而且就是同一菌种的不同菌株的作用也完全不一样,但是目前没有专门的菌株可以以提升其降血糖活性为目的进行羊栖菜的发酵提取。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供一种提升羊栖菜提取物降血糖活性的方法。
本发明提供提升羊栖菜提取物降血糖活性的方法,包括以下步骤:羊栖菜与上述的保藏编号为:CGMCC No.23295的米曲霉共同发酵得到发酵基质提取物。所述米曲霉(Aspergillus oryzae)菌株于2021年11月10日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编:100101。该菌株的菌株名称是:WZ-212;分类命名是米曲霉(Aspergillus oryzae);保藏编号为:CGMCC No.23295。该米曲霉(Aspergillus oryzae)的核苷酸ITS序列如SEQ IDNO:1所示。
进一步的,所述方法包括以下步骤:将羊栖菜粉末置于发酵容器中,添加蒸馏水至含量水量为50-70%,加入葡萄糖,灭菌后加入包含保藏编号为:CGMCC No.23295的米曲霉的微生物体系,于26-34℃条件下静置发酵2-8天。
进一步的,所述微生物体系的制备过程包括以下步骤:将保藏编号为:CGMCCNo.23295的米曲霉接种到新鲜的种子培养基中,深层发酵得到米曲霉种子液。
进一步的,所述种子培养基组成为:黄豆芽含量200g/L、葡萄糖含量20g/L、玉米粉含量为3-5g/L、羊栖菜含量5-10g/L。
本发明还提供如上所述的羊栖菜提取物在制备降血糖的药物或保健品的应用。
本发明还提供一种降血糖的药物,其包含如上所述的羊栖菜提取物。
本发明还提供一种降血糖的保健品,其包含如上所述的羊栖菜提取物。
本发明的有益效果如下:本发明提供的羊栖菜提取物为羊栖菜与保藏编号为:CGMCC No.23295的米曲霉共同发酵后提取得到的,相比未发酵处理的羊栖菜提取物,其降血糖活性具有显著提升的效果,具有较大的市场应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1采用MEGA7.0软件,邻位连接法显示菌株CGMCC No.23295与GeneBank数据库中相关种的ITS序列系统发育树;
图2羊栖菜原料的HPLC-MS总离子流图(正离子);
图3羊栖菜经米曲霉CGMCC No.23295发酵后样品的HPLC-MS总离子流图(正离子);
图4小鼠降血糖试验过程中空腹血糖值的变化。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
以下实施例中所用的菌种培养基如下:
米曲霉筛选培养基(g/L):黄豆芽200,葡萄糖20,琼脂15,pH6.8,链霉素1万U;
斜面培养基(g/L):黄豆芽200,葡萄糖20,琼脂15,pH6.8;
种子培养基(g/L):黄豆芽200,葡萄糖20,玉米粉5,羊栖菜10;
实施例1米曲酶(Aspergillus oryzae)WZ-212的筛选分离与鉴定
一、米曲酶(Aspergillus oryzae)WZ-212是从自然发酵的植物材料(铁皮石斛叶)分离而来的。取自然发酵的样品10克,加入50毫升无菌生理盐水,将其稀释10-5,10-6,10-7倍,取100微升涂布在米曲霉筛选培养基平板,每个浓度重复三次,28℃培养72小时,产生孢子的菌株即为霉菌,挑选孢子多次纯化,将纯化后的菌株接种到斜面培养基上,28℃培养72小时后放于4℃冰箱保存待用。
将4℃冰箱保存待用的菌株接种到种子培养基中,28℃培养72小时后,分别取10毫升种子液接种于100克铁皮石斛叶进行发酵,测定发酵基质提取物抑制抑制α-葡萄糖苷酶活性,从中筛选出一株提升铁皮石斛叶抑制α-葡萄糖苷酶活性效果强的菌株,定名为WZ-212。
筛选过程中,挑选出的各种霉菌(具有提升铁皮石斛叶抑制α-葡萄糖苷酶活性能力的部分霉菌)以及市场上直接采购过来的其它菌株检测得到提升铁皮石斛叶抑制α-葡萄糖苷酶活性对比如表1所示,可见霉菌WZ-212提升铁皮石斛叶抑制α-葡萄糖苷酶活性效果的能力非常显著。
表1不同菌株发酵铁皮石斛叶提升抑制α-葡萄糖苷酶活性
菌种 | 抑制α-葡萄糖苷酶活性(阿卡波糖当量) |
未发酵对照组 | 0.435mg/g |
红曲霉 | 0.884mg/g |
蛹虫草菌 | 0.580mg/g |
灵芝菌 | 0.775mg/g |
云芝菌 | 0.803mg/g |
真姬菇菌 | 0.828mg/g |
鸡腿菇菌 | 0.663mg/g |
猴头菌 | 0.609mg/g |
霉菌WZ-201 | 6.772mg/g |
霉菌WZ-202 | 10.597mg/g |
霉菌WZ-203 | 17.629mg/g |
霉菌WZ-204 | 1.926mg/g |
霉菌WZ-205 | 28.766mg/g |
霉菌WZ-212 | 44.235mg/g |
二、米曲酶(Aspergillus oryzae)WZ-212的分子生物学鉴定
将目的菌株在斜面培养基上培养72小时,提取基因组DNA,利用PCR技术扩增该菌株的ITS,所用引物为ITS1(5'-TCCGTAGGTGAACCTGCGG-3')和ITS4(5'-TCCTCCGCTTATTGATATGC-3');
PCR反应体系组成如下表:
PCR条件如下表所示:
对PCR产物进行DNA测序,序列提交GeneBank,与GeneBank数据库中的ITS进行同源性比对,结果见图1,发现与Aspergillus oryzae F8224(GenBank accessionNo.MN429273.1)的同源性最高,高达100%,确定该菌株为米曲酶(Aspergillus oryzae),已于2021年11月10日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为:CGMCC No.23295。
实施例2羊栖菜发酵前后成分变化的HPLC-MS分析
(1)样品处理
5g样品加入100mL 80%的乙醇溶液中,50℃条件下超声提取120min,8000r/min离心10min,收集上清液。
(2)仪器及测定条件
超高效液相色谱(U3000,Thermo Scientific)与质谱仪(TripleTOF5600+,ABSCIEX)联用。
色谱条件:Waters BEH C18色谱柱(150mm×2.1mm,1.7μm)。洗脱液,0.1%甲酸(A)和乙腈(B)。梯度洗脱:0min,95%A、10min,50%A、15min,5%A、17min 5%A、20min,95%A。柱温35℃,流速为0.3mL/min,进样量为10μL。
质谱条件:电子轰击离子源(EI),离子源温度500℃(正离子)和450℃(负离子),接口温度280℃,扫描质量范围100Da-1200Da,碎片扫描范围50Da-1200Da。
(3)数据处理
通过Analysis Base File Converter软件将LC-MC获得的原始数据转换后,通过MS-DIAL 4.60软件进行数据处理。
实施例3羊栖菜体外降血糖活性能力测定
(1)样品制备
称取2g羊栖菜样品,加入50mL 95%的乙醇后,在45℃下超声提取1小时,4000rpm下离心15min,取上清液减压蒸干后,用15mL的DMSO溶液,制得待测溶液。
(2)抑制α-葡萄糖苷酶活性能力测定
依据反应体系依次加入3mL 0.1mol/L磷酸缓冲液(pH 6.8)和100μL1.625U/mL的α-葡萄糖苷酶溶液,混合均匀,于37℃水浴保温10min,取出,然后加入200μL样品溶液和100μL 5mmol/L PNPG(对硝基苯-α-D葡萄糖苷,4-Nitrophenylα-D-glucopyranoside),充分混匀,于37℃水浴反应10min,反应结束后迅速加入200μL 0.1mol/L的Na2CO3溶液终止反应,在405nm处测定吸光值(As)。同等条件下,分别用磷酸缓冲液代替样品溶液测定吸光值(Ac)和酶溶液测定吸光值(Ab)。根据下列公式计算出α-葡萄糖苷酶抑制率。
抑制率%=[Ac-(As-Ab)/Ac]×100%
其中:Ac:对照组吸光度值;As:样品组吸光度值;Ab:空白组吸光度值。
同时制备阿卡波糖浓度与α-葡萄糖苷酶抑制百分比之间的标准曲线,计算每克羊栖菜中含与阿卡波糖活性相当的当量(阿卡波糖当量/克)。
实施例4
将米曲酶(Aspergillus oryzae)CGMCC No.23295接种到斜面培养基上,在28℃条件下培养72小时;然后将斜面上长好的米曲霉孢子接种到新鲜的种子培养基中,在温度为28℃、转速为170r/min条件下回转式深层发酵72小时,得到米曲霉种子液;称取100g羊栖菜粉末,置于发酵容器中,添加蒸馏水至含量水量为50%,加入2%的葡萄糖,搅拌均匀,121℃灭菌30min;冷却后接入10%体积质量比的米曲霉种子液,搅拌均匀,28℃条件下静置发酵4天。取发酵前后的羊栖菜按照实施例2的方法进行成分检测,羊栖菜原料和经米曲霉发酵后的羊栖菜物质成分的总离子流图如图2、图3所示。由图中可知,发酵明显改变羊栖菜中成分的组成和含量。羊栖菜原料和米曲霉发酵后的羊栖菜成分见表2。采用实施例3的方法测定样品抑制α-葡萄糖苷酶活性,结果表明,样品阿卡波糖当量从4.20mg/g上升为6.85mg/g。
表2羊栖菜原料和米曲霉发酵后的羊栖菜中的成分
实施例5
米曲霉活化和种子制备同实施例4;称取100g羊栖菜粉末,置于发酵容器中,添加蒸馏水至含量水量为60%,加入2%的葡萄糖,搅拌均匀,121℃灭菌30min;冷却后接入10%体积质量比的米曲霉种子液,搅拌均匀,30℃条件下静置发酵4天。发酵结束后经HPLC-MS法分析羊栖菜原料和米曲霉发酵后的羊栖菜成分,达到了如实施例4类似的结果,样品阿卡波糖当量从4.20mg/g上升为10.08mg/g。
表3为实施例1中挑选出的各种霉菌(具有抑制α-葡萄糖苷酶活性能力的部分霉菌)以及市场上直接采购过来的其它菌株检测得到发酵羊栖菜提升抑制α-葡萄糖苷酶活性对比,可见米曲酶(Aspergillus oryzae)CGMCC No.23295的发酵羊栖菜提升羊栖菜提取物降血糖能力非常显著。
表3不同菌种发酵羊栖菜提升抑制α-葡萄糖苷酶活性
实施例6
米曲霉活化和种子制备同实施例4;称取100g羊栖菜粉末,置于发酵容器中,添加蒸馏水至含量水量为80%,加入2%的葡萄糖,搅拌均匀,121℃灭菌30min;冷却后接入10%体积质量比的米曲霉种子液,搅拌均匀,30℃条件下静置发酵4天。发酵结束后经HPLC-MS法分析羊栖菜原料和米曲霉发酵后的羊栖菜成分,达到了如实施例4类似的结果,样品阿卡波糖当量从4.20mg/g上升为9.46mg/g。
实施例7羊栖菜体内降血糖活性能力测定
1、发酵羊栖菜样品的制备同实施例6。
2、试验动物分组与给药
10只C57BL/6小鼠和40只C57BKSdb/db糖尿病模型小鼠(5周龄),在25±2℃,12h照明,12h黑暗,相对湿度50-60%的动物实验室适应性饲养2周。第七周早上8点测量所有小鼠的空腹血糖与体重,血糖值在11~25mmol/L的是高血糖小鼠,然后按照血糖值与体重的数据将40只模型小鼠均匀分为4组(模型组,阳性对照组,未发酵样组,发酵样组)。饲养过程中保证充足的鼠粮和饮水,及时更换老鼠垫料,保证小鼠生活环境的清洁。每天给药,具体给药情况如表2。
表2小鼠分组以及灌胃剂量
3、小鼠空腹血糖测定
分别于给药后的第7d、14d、21d、28d上午8:30~9:30,剪小鼠尾尖取血,用血糖仪进行采血并测量空腹血糖,测量前小鼠禁食(不禁水)12h。
4、结果
结果见图4,羊栖菜经米曲酶(Aspergillus oryzae)CGMCC No.23295发酵后,灌胃糖尿病小鼠4周,与对照组和未发酵羊栖菜组相比,小鼠空腹血糖值显著降低,与阿卡波糖的效果相当,降血糖作用明显提升。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
序列表
<110>温州大学
<120>一种提升羊栖菜提取物降血糖活性的方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 506
<212> DNA
<213>米曲霉(Aspergillus oryzae)
<400> 1
ttctagcgag cccaacctcc cacccgtgtt tactgtacct tagttgcttc ggcgggcccg 60
ccattcatgg ccgccggggg ctctcagccc cgggcccgcg cccgccggag acaccacgaa 120
ctctgtctga tctagtgaag tctgagttga ttgtatcgca atcagttaaa actttcaaca 180
atggatctct tggttccggc atcgatgaag aacgcagcga aatgcgataa ctagtgtgaa 240
ttgcagaatt ccgtgaatca tcgagtcttt gaacgcacat tgcgccccct ggtattccgg 300
ggggcatgcc tgtccgagcg tcattgctgc ccatcaagca cggcttgtgt gttgggtcgt 360
cgtcccctct ccggggggga cgggccccaa aggcagcggc ggcaccgcgt ccgatcctcg 420
agcgtatggg gctttgtcac ccgctctgta ggcccggccg gcgcttgccg aacgcaaatc 480
aatcttttcc aggtgacctc ggatca 506
Claims (4)
1.一种提升羊栖菜提取物降血糖活性的方法,其特征在于,包括以下步骤:羊栖菜与保藏编号为:CGMCC No.23295的米曲霉共同发酵得到发酵基质提取物。
2.根据权利要求1所述的提升羊栖菜提取物降血糖活性的方法,其特征在于,包括以下步骤:将羊栖菜粉末置于发酵容器中,添加蒸馏水至含量水量为50-70%,加入葡萄糖,灭菌后加入包含保藏编号为:CGMCC No.23295的米曲霉的微生物体系,于26-34℃条件下静置发酵2-8天。
3.根据权利要求2所述的提升羊栖菜提取物降血糖活性的方法,其特征在于:所述微生物体系的制备过程包括以下步骤:将保藏编号为:CGMCC No.23295的米曲霉接种到新鲜的种子培养基中,深层发酵得到米曲霉种子液。
4.根据权利要求3所述的提升羊栖菜提取物降血糖活性的方法,其特征在于:所述种子培养基组成为:黄豆芽含量200g/L、葡萄糖含量20g/L、玉米粉含量为3-5g/L、羊栖菜含量5-10g/L。
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