CN108424863B - 纳豆菌及其在发酵扇贝裙边制备ace抑制肽中的应用 - Google Patents
纳豆菌及其在发酵扇贝裙边制备ace抑制肽中的应用 Download PDFInfo
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Abstract
本发明公开了枯草芽孢杆菌纳豆亚种ACE‑IP,Bacillus subtilis subsp.natto ACE‑IP,在发酵扇贝裙边制备ACE抑制肽中的应用,其保藏编号为CCTCC NO:M 2018081。枯草芽孢杆菌纳豆亚种ACE‑IP的筛选鉴定方法包括:原料处理和氨基酸成分分析;制备扇贝裙边发酵培养基;纳豆芽孢杆菌的初筛;纳豆芽孢杆菌的复筛;菌株的鉴定。本发明筛选得到的纳豆芽孢杆菌,通过发酵扇贝裙边制备得到ACE抑制肽,充分发挥了纳豆芽孢杆菌发酵时能够产生多种蛋白酶的功效,将扇贝裙边大分子蛋白分解成小分子ACE抑制肽,并具有较高的抑制活性;不仅为产业化生产安全、无副作用且活性高的ACE抑制肽提供了优良菌株,也为扇贝裙边的开发利用和进一步加工提供了理论依据。
Description
技术领域
本发明涉及功能性食品的加工领域,具体地说,涉及一株纳豆菌,及其在发酵栉孔扇贝裙边制备ACE抑制肽中的应用。
背景技术
栉孔扇贝(Chlamysfarreri)隶属珍珠贝目(Pterioida),珍珠亚贝类(Pteriina),扇贝科(Pectinidae),栉孔扇贝属(Chlamys)动物,广泛分布于中国北部沿海,如辽宁的旅大、山东的青岛、长岛、荣成、日照等地。扇贝裙边是去除扇贝贝柱后的剩余部分,约占整个扇贝的30%,仅山东省产量每年高达30万吨之多,但在生产过程中由于季节性的的限制直接被丢弃,至今未得到合理有效的应用,造成了资源的浪费和环境的污染。研究表明扇贝裙边含有大量的蛋白质、脂肪、碳水化合物等营养成分,而且含有不饱和脂肪酸、抗肿瘤、抗氧化等有效活性物质。因此如何实现扇贝裙边的高值化利用,减少资源的浪费成为当前亟待解决的问题。
纳豆芽孢杆菌(Bacillus natto)属芽孢杆菌科、纳豆芽孢杆菌属,是纳豆的生产菌种,目前已有2000多年的食用历史。纳豆菌在发酵过程中能够分解蛋白质、碳水化合物、脂肪等大分子物质,因而纳豆菌发酵的产品中含有大量的氨基酸、寡聚糖、有机酸等容易被人体消化吸收的营养成分。
高血压是一种以动脉血管收缩压或舒张压升高为特征的临床综合征,常伴有大脑、肾脏和心血管等器官和结构的损害,是导致心血管疾病如心力衰竭、中风、冠心病和心肌梗塞的重要危险因子。据报道我国有1亿多人患有高血压,每年近100万人因高血压而死亡。化学合成的血管紧张素转移酶(angiotensin converting enzyme,ACE)抑制剂是治疗高血压疾病的临床药物;自1977年Cheung等研发了第一个化学合成的ACE抑制剂卡托普利(Captopri)以来,人工合成的ACE抑制剂已得到医学界的普遍认可。然而,药物治疗高血压往往会产生咳嗽、肾功能可逆性降低、皮疹等副作用,所以人们越来越青睐于食物疗法。因此亟需一种既具有高蛋白酶活性,又能以扇贝裙边为原料进行发酵的纳豆菌菌株。
发明内容
本发明所要解决的技术问题是提供一种发酵菲律宾蛤仔裙边的纳豆菌株的筛选和鉴定方法,以及利用该菌株制备ACE抑制肽。
为实现上述目的,本发明采用以下技术方案:
利用微生物发酵法,以多肽含量和ACE抑制率为指标,从7种纳豆(见表1)中筛选出一株蛋白酶活性较高的菌株,将其命名为Bacillus subtilis subsp.nattoACE-IP,Bacillus subtilis subsp.nattoACE-IP,保藏编号为CCTCC NO:M 2018081。
枯草芽孢杆菌纳豆亚种ACE-IP发酵扇贝裙边的发酵物用于制备降血压功能性食品。
利用该菌株发酵扇贝裙边,用于生产安全性高、无副作用的食物源ACE抑制肽,减少化学合成的抗高血压药物引发的副作用,同时提高扇贝裙边的资源利用度,减少环境污染。
表1.七种纳豆菌名称及来源
具体技术方案如下:
(1)原料处理和氨基酸成分分析:取新鲜的栉孔扇贝,去除扇贝贝柱,清洗沥干,匀浆,得扇贝裙边,然后用氨基酸自动分析仪对扇贝裙边氨基酸组成进行分析,结果见表2;
表2.扇贝裙边氨基酸的组成
(2)扇贝裙边发酵培养基的制备:称取处理好的扇贝裙边5-10g,加入蒸馏水20-40mL,放入121℃高压灭菌锅灭菌20min,制得扇贝裙边发酵培养基,备用;
(3)纳豆芽孢杆菌的分离纯化:称取0.2-0.5g纳豆于20-50mL 0.9%的生理盐水中振荡40-70min,此菌悬液作为原始菌悬液;取原始菌悬液0.5mL加入4.5mL无菌生理盐水中制成10-1稀释菌悬液,如上操作梯度稀释制备10-2~10-8稀释菌悬液;各取200-300μL上述10-5~10-8稀释菌悬液加至含盐量2%的牛肉膏蛋白胨培养基平板,每处理设3个平行,涂匀稀释菌悬液;将上述平板移至37℃恒温培养箱中倒置培养24-26h;观察菌落生长情况,选择菌落分布均匀、菌落数介于30~80的平板,挑取不同菌落形态的单菌落进行革兰氏染色,显微镜下观察;对芽孢杆菌反复纯化,直到获得纯种;
(4)纳豆芽孢杆菌的初筛:利用纳豆枯草芽孢杆菌产蛋白酶的特性,用灭菌牙签沾取少许培养24-26h的待测菌株,点种于脱脂牛奶培养基上,于37℃恒温培养箱中倒置培养18-20h,选取菌落周围产生水解圈相对直径大的菌株,进一步用福林酚法测定蛋白酶活力,选取蛋白酶活力较高的菌株进行发酵;
(5)纳豆芽孢杆菌的复筛:分别取初筛纳豆菌的菌悬液,以5%的接种量接种到5g/20mL的扇贝裙边发酵培养基中,于37℃恒温摇床中发酵,分别培养12、24、36、48、60、72h;将发酵液以8000-10000r/min离心10-15min,取上清液,分别测多肽含量和ACE抑制率,每组做三个平行,筛选出具有较高ACE抑制率的纳豆芽孢杆菌;
(6)菌株的鉴定:筛选出的菌株提取基因组后进行16S rDNA菌种鉴定,鉴定结果在NCBI网站进行比对,并根据相似性分析建立菌株***发育树。
本发明的有益效果:
1)本发明选用的发酵原料为扇贝加工副产品(扇贝去除扇贝柱后剩余部分),经氨基酸自动分析仪分析显示,非极性氨基酸和芳香族氨基酸的含量占总氨基酸的40.99%,符合具有较高ACE抑制率活性肽的结构特点;因此本发明选用扇贝裙边作为发酵底物时更容易得到具有较高抑制活性的ACE抑制肽,而且原料处理方面简单易行,节约生产成本,大大提高了扇贝裙边的资源利用度;
2)目前纳豆菌主要用于制作纳豆面膜、纳豆饲料、纳豆胶囊等产品,而作为一种发酵菌株被应用却鲜有报道,本发明选用纳豆菌作为制备ACE抑制肽的发酵菌株,不仅为产业化生产食物源ACE抑制肽提供了一种有用的菌株,也为开发新的纳豆菌产品提供了试验依据和指导;
3)本发明筛选得到的纳豆芽孢杆菌,通过发酵扇贝裙边,制备得到ACE抑制肽,充分发挥了纳豆芽孢杆菌发酵时能够产生多种蛋白酶的功效,将扇贝裙边大分子蛋白分解成容易被人体消化吸收的小分子ACE抑制肽,并具有较高的抑制活性;而且操作简单,对设备的要求低;不仅为产业化生产安全、无副作用且活性高的ACE抑制肽提供了优良菌株,也为扇贝裙边的开发利用和进一步加工提供了理论依据。
附图说明:
图1为本发明酪氨酸溶液标准曲线;
图2为本发明复筛得到的纳豆芽孢杆菌蛋白酶的活性比较;
图3为本发明Gly-Gly-Tyr-Arg四肽溶液标准曲线;
图4为本发明BN-3在不同发酵时间的多肽含量和ACE抑制率变化;
图5为本发明BN-7在不同发酵时间的多肽含量和ACE抑制率变化;
图6为本发明BN-15在不同发酵时间的多肽含量和ACE抑制率变化;
图7为本发明BN-16在不同发酵时间的多肽含量和ACE抑制率变化;
图8为本发明BN-25在不同发酵时间的多肽含量和ACE抑制率变化;
图9为本发明BN-30在不同发酵时间的多肽含量和ACE抑制率变化;
图10为本发明BN-35在不同发酵时间的多肽含量和ACE抑制率变化;
图11为本发明纳豆菌菌株BN-30的基因组;
图12为本发明纳豆菌菌株BN-30的PCR扩增产物电泳图;
图13为本发明纳豆菌菌株BN-30的***发育树。
具体实施方式
以下结合附图对本发明做进一步的详细描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例1纳豆芽孢杆菌的分离纯化
称取0.2g纳豆于20mL 0.9%的生理盐水中振荡1h,此菌悬液作为原始菌悬液;取原始菌悬液0.5mL加入4.5mL无菌生理盐水中制成10-1稀释菌悬液,如上操作梯度稀释制备10-2~10-8稀释菌悬液;各取200μl上述10-5~10-8稀释菌悬液加至含盐量2%的牛肉膏蛋白胨培养基平板,每处理设3个平行,涂匀稀释液;将上述平板移至37℃恒温培养箱中倒置培养24h;观察菌落生长情况,选择菌落分布均匀、菌落数介于30~80的平板,挑取不同菌落形态的单菌落进行革兰氏染色,显微镜下观察;对芽孢杆菌反复纯化,直到获得纯种。
按照上述方法从7种纳豆中分别分离纯化得到5个单菌落,总共分离出35个单菌落,依次命名为BN-1~BN-35。
实施例2纳豆芽孢杆菌的初筛
将上述分离得到的35株纳豆芽孢杆菌用无菌牙签点种至脱脂牛奶固体培养基上,37℃倒置培养18小时,测量菌落及其周围水解圈的直径,粗略计算蛋白酶活性。
分别取7株水解圈直径大的纳豆芽孢杆菌制备粗酶液,采用Folin-酚法测定粗酶液的蛋白酶活性。蛋白酶活性的定义:在40℃、pH7.2的条件下,每毫升发酵液生成酪氨酸的微克数(单位:IU/mL)。
配置0、20、40、60、80、100μg/mL的酪氨酸标准溶液,分别取上述标准溶液1mL,各加入0.4mol/L的碳酸钠溶液5mL,再各加入福林酚试剂l mL,漩涡震荡混匀后置于水浴锅中,40℃反应20min。最后在660nm处测定吸光度值,重复三次取平均值。以吸光度A为纵坐标,酪氨酸的浓度为横坐标,绘制酪氨酸溶液标准曲线,如图1所示,回归方程为y=0.0103x-0.0258,R2=0.9976。
根据回归方程计算出当吸光度A为1时的酪氨酸的量(μg),即为吸光度常数K。
粗酶液的的制备:将初筛分离得到的7株纳豆菌,进行菌种活化后,从斜面上挑1~2环菌种接到装有种子液体培养基的三角瓶中,于37℃,200r/min下摇床培养24-26h,制得种子菌悬液,其浓度为1x108~8x108cfu/mL。其中,种子液体培养基为:蛋白胨1%、牛肉膏0.5%、氯化钠0.5%、葡萄糖0.5%,调整pH为7.2~7.4。将活化好的菌液用无菌水稀释10倍制成粗酶液进行蛋白酶活力的测定。
样品测定:分别取粗酶液1mL,再加入酪蛋白1mL,精确保温10min后加入0.4mol/L三氯乙酸2mL,以终止反应,继续加入水浴锅中保温20min。过滤后取上清液1mL,再加入0.4mol/L的碳酸钠溶液5mL,再各加入福林酚试剂l mL,漩涡震荡混匀后置于水浴锅中,40℃反应20min,最后在660nm处测定吸光度值,测三次取平均值。空白对照测定方法同上,只需在加入酪蛋白之前先加0.4mol/L三氯乙酸2mL,使酶失活,再加入酪蛋白。
计算公式:蛋白酶活力(IU/mL)=(AxBxKx4)/10
式中:A-菌液平均吸光度;B-菌液稀释倍数;K-吸光常数;4-反应试剂的总体积(mL);10-反应时间为10min。
测定结果如图2所示,初筛得到的BN-3、BN-7、BN-15、BN-16、BN-25、BN-30、BN-357株纳豆芽孢杆菌,其产蛋白酶活性强弱依次为BN-30﹥BN-7﹥BN-16﹥BN-15﹥BN-3﹥BN-35﹥BN-25,表明纳豆菌在生长的过程中可分泌出大量的蛋白酶,可用于酶解扇贝裙边蛋白产生大量的多肽。
实施例3纳豆芽孢杆菌的复筛
将上述7株蛋白酶活性较高的纳豆芽孢杆菌活化后分别发酵扇贝裙边,取发酵上清液,以多肽含量和ACE抑制率为指标进行复筛。
利用双缩脲法进行扇贝裙边发酵液多肽含量的测定。用5%的三氯乙酸(TAC)依次配置0.0、0.2、0.4、0.6、0.8、1.0和1.2mg/mL的Gly-Gly-Tyr-Arg四肽标准溶液,然后分别取3.0mL上述标准溶液,加入2.0mL双缩脲试剂,于涡旋混合仪上混合均匀,静置5min,取上清液于540nm下测定吸光度值(以第一管做空白对照),以肽浓度为横坐标,OD540值为纵坐标,制作Gly-Gly-Tyr-Arg四肽溶液标准曲线,如图3所示,回归方程为y=0.1563x-0.0022,R2=0.9994。
取2mL扇贝裙边发酵液,加入2mL10%(W/V)的TAC溶液,于旋涡混合仪上混合均匀,静置5min,然后在3500r/min下离心15min,取离心上清液全部转移至5.0mL容量瓶中,并用5%的TAC定容至刻度摇匀,然后取3.0mL上述溶液置于另一管中,加入双缩脲试剂2.0mL,于旋涡混合仪上混合均匀,静置5min,2000r/min离心10min,取上清液于540nm处测定吸光值,以对照标准曲线求得样品溶液中的多肽浓度c(mg/mL),进而求得样品中多肽浓度。
空白对照:3mL蒸馏水加2mL双缩脲试剂,涡旋混合仪上混合均匀,静置4min,2000r/min离心10min,取上清液于540nm测定吸光值。
采用改良的Cushman紫外比色法进行扇贝裙边发酵液ACE抑制率的测定。取100ul5.0mmol/l的HHL溶液和40ul离心后的扇贝裙边发酵液混合,于37℃水浴中保温10min,加入0.1U/mL的ACE酶液20ul,混匀后于37℃恒温水浴中反应35min。从水浴锅中取出,向反应体系中加入200ul 1mol/l的HCI以终止反应,再加入1.2mL冷冻乙酸乙酯提取产生的马尿酸,旋涡震荡混匀后,以3500r/min离心5min,吸取1.0mL的乙酸乙酯层,在90℃烘箱中经1小时烘干冷却后加入4mL的蒸馏水充分溶解,漩涡混合后于228nm处测吸光值。平行对照管除在反应前先加入200ul 1mol/l的HCI以终止反应外,其余成分、操作步骤同反应管,吸光度值重复3次取平均值,进而根据公式计算扇贝裙边发酵液ACE抑制率,结果如图4-10所示。
公式:扇贝裙边发酵液ACE抑制率(%)=(B-A)/(B-C)
式中:A—扇贝裙边发酵液和ACE酶同时与HHL反应的吸光值,即实验组;
B—不含扇贝裙边发酵液时,ACE和HHL反应的吸光度值,即对照组;
C—扇贝裙边发酵液和ACE酶都不存在时的吸光度值,即空白组。
由图4-10可知,菌株BN-30发酵扇贝裙边48h时,多肽含量达到221mg/g,ACE抑制率达到77.11%,且多肽含量与ACE抑制率呈一定的正相关性。这是由于在发酵初期,纳豆菌在适宜的营养条件下生长繁殖会产生多种蛋白酶,从而分解扇贝裙边蛋白产生多肽,且部分多肽具有抗高血压的作用,随着多肽含量的增加,ACE抑制率也随之增加。但在发酵后期,多肽含量和ACE抑制率随着发酵时间增大而降低,这可能是发酵物中营养物质降低,导致纳豆菌利用分解扇贝裙边生成的小分子多肽作为碳源,ACE抑制肽也可能被蛋白酶分解,进而导致ACE抑制率也降低。
实施例4菌株BN-30的鉴定
提取菌株BN-30的基因组做模板,如图11所示。用细菌的通用引物(27F:5’AGAGTTTGATCCTGGCTCAG 3’;SEQ ID NO:1;1492R:5’GGTTACCTTGTTACGACTT 3’;SEQ ID NO:2)进行PCR扩增,经1%的琼脂糖电泳检测显示,在1500bp左右有一明显条带,如图12所示,说明已经扩增出该菌株基因组的16SrDNA片段。经测序得到的片段长度为1429bp,如SEQ IDNO:3所示。
将菌株BN-30的序列在NCBI中用Blast进行比对,其同源性较高的菌株均属于芽孢杆菌属,说明筛选出的菌株BN-30是芽孢杆菌属,枯草芽孢杆菌种,纳豆菌亚种,Bacillussubtilis subsp.natto。将菌株BN-30命名为Bacillus subtilis subsp.natto ACE-IP,Bacillus subtilis subsp.natto ACE-IP,并进行保藏,保藏编号为CCTCC NO:M 2018081,保藏日期为2018年02月02日,保藏单位为中国典型培养物保藏中心,位于中国,武汉,武汉大学;根据序列比对结果建立菌株BN-30的***发育树,如图13所示。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 青岛大学
<120> 纳豆菌及其在发酵扇贝裙边制备ACE抑制肽中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> Artificial
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1429
<212> DNA
<213> Bacillus subtilis subsp. natto
<400> 3
taatgcaagt cgagcggaca gatgggagct tgctccctga tgttagcggc ggacgggtga 60
gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg gggctaatac 120
cggatggttg tttgaaccgc atggttcaaa cataaaaggt ggcttcggct accacttaca 180
gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg 240
tagccgacct gagagggtga tcggccacac tgggactgag acacggccca gactcctacg 300
ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg 360
agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag taccgttcga 420
atagggcggt accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc 480
cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag ggctcgcagg 540
cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg 600
gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa atgcgtagag 660
atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg ctgaggagcg 720
aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg 780
ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag cactccgcct 840
ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900
gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga 960
caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc atggttgtcg 1020
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag 1080
ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg 1140
ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggaca 1200
gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt ctcagttcgg 1260
atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg 1320
ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380
acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaaggtg 1429
Claims (2)
1.枯草芽孢杆菌纳豆亚种(Bacillus subtilis subsp. natto)ACE-IP在发酵扇贝裙边制备ACE抑制肽中的应用,其特征在于,所述枯草芽孢杆菌纳豆亚种(Bacillus subtilis subsp. natto)ACE-IP的保藏编号为CCTCC NO:M 2018081。
2.根据权利要求1所述的枯草芽孢杆菌纳豆亚种ACE-IP发酵扇贝裙边的发酵物用于制备降血压功能性食品。
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发酵法制备蛤蜊ACE抑制肽的菌种筛选;陈应运等;《核农学报》;20171231;第31卷(第10期);参见摘要,结果与分析,第1986页左栏第1段 * |
纳豆菌液态发酵蛤蜊制备ACE 抑制肽的工艺研究;陈应运等;《核农学报》;20161231;第30卷(第11期);参见摘要,材料与方法,第2189页 * |
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