CN110699272B - 一种纳豆枯草芽孢杆菌以及生产mk-7的方法 - Google Patents
一种纳豆枯草芽孢杆菌以及生产mk-7的方法 Download PDFInfo
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- CN110699272B CN110699272B CN201910593690.9A CN201910593690A CN110699272B CN 110699272 B CN110699272 B CN 110699272B CN 201910593690 A CN201910593690 A CN 201910593690A CN 110699272 B CN110699272 B CN 110699272B
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Abstract
本申请公开一种纳豆枯草芽孢杆菌ST‑1008,保藏编号为CGMCC No.17894。本申请还提供生产MK‑7的方法,所述方法包括在培养基中培养所述纳豆枯草芽孢杆菌CGMCC No.17894以使在所述菌株细胞内和所述培养基中生产MK‑7,以及从所述菌株细胞内和所述培养基中回收并提纯MK‑7。本申请制备出的MK‑7克服了化学合成MK‑7的缺点;发酵产量可以到达300mg/L,对菌粉进行提纯,收率可以达到98%。本申请还提供一种MK‑7制剂、一种MK‑7纯品预混剂和一种MK‑7纯品。
Description
技术领域
本申请涉及但不限于生物领域,尤其涉及但不限于一种新的纳豆枯草芽孢杆菌CGMCC No.17894,利用该菌株发酵生产维生素K2(MK-7)的方法以及该菌株在制备维生素K2(MK-7)中的用途。
背景技术
维生素K2,也称甲基萘醌(Menaquinone),通常用MK来表示。它由一组化合物组成,共有14种形式,差别在于侧链的长短不一,代表性的分子是MK-4和MK-7。其中MK-7具备功能广泛、活性强大、半衰期长、安全等特点,主要对细胞的生长代谢及防止心脑血管和肾脏血管的钙化中起着非常重要的作用。美国国立卫生研究院(NIH)膳食补充剂办公室(ODS)评价维生素K2(MK-7)为革命性和多能性的维生素。维生素K2(MK-7)具有促进凝血、改善动脉硬化的作用。现在我国已进入老龄化社会,骨质疏松患者发病率较高。大量资料研究表明,维生素K2(MK-7)缺乏可导致老年人髋骨骨折和骨密度降低。维生素K2(MK-7)缺乏,血清未羧化骨钙蛋白水平降低,血清羧化骨钙蛋白水平也可能降低,从而可能导致老年人骨密度降低发生髋骨骨折危险。而且维生素K2(MK-7)可以用于治疗骨质疏松、心脑血管钙化、癌症、糖尿病、肾病、老年痴呆的药物,维生素K2(MK-7)具有健康保健的广阔应用前景。
通过“168国居民65岁以前死于心血管病与生物统计学,社会经济状况、性别、运动、大量营养元素及维生素K的相关性分析”(2016Cureus 8(8))表明摄入维生素K2(MK-7)日平均量<5μg/2000kcal的国家(n=70)男性和女性队列,过早的CVD死亡率是摄入维生素K2(MK-7)日平均量>24μ/2000kcal的国家(n=72)的2.2倍。因此,补充维生素K2(MK-7)是健康必须的。天然食物中维生素K2(MK-7)含量极低,基本无法通过食物摄取足量的维生素K2(MK-7)。
多年来,各国都在寻找维生素K2(MK-7)的生产途径。共有几个方面:(1)化学合成法:该方法需要用到有机溶剂,不仅对环境、操作人员不够友好,而且所得产品中有有机溶剂残留,产品中顺反式结构的维生素K2(MK-7)共存;(2)从固体发酵纳豆中提取:纳豆中维生素K2(MK-7)含量极低,没有市场竞争力;(3)用天然纳豆菌液体发酵的方法获得:该方法周期长,产量低。
基于维生素K2(MK-7)的生产与需求现状,世界市场需要更高效率的维生素K2(MK-7)生产方法。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
为了克服现有技术的不足和满足市场需求,本发明的目的在于提供一种能以高产率生产纯反式结构的维生素K2(MK-7)的方法。本申请提供了一种。
具体地,在一个方面中,本申请提供了一种纳豆枯草芽孢杆菌(Bacillussubtilis natto)ST-1008,保藏编号为CGMCC No.17894。
本申请人经过多年大量深入细致地研究,从野生型纳豆枯草芽孢杆菌诱变出一种新的纳豆枯草芽孢杆菌(Bacillus subtilis natto)ST-1008,CGMCC No.17894。本申请人从市售的6种不同来源新鲜纳豆中分离菌株,根据不同的菌落形态、菌落颜色、菌体生长速度进行分离纯化后进行发酵实验,获得维生素K2(MK-7)产生菌。以维生素K2(MK-7)产生菌作为出发菌株。对该菌株进行紫外诱变,紫外诱变26代后得到形态变异株,维生素K2(MK-7)产量提高20倍。以上形态变异以及维生素K2(MK-7)产量经数代传代培养较稳定。该菌种代号为ST-1008,菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏号为:CGMCCNo.17894,保藏日期为:2019年06月05日。
本申请提供的新菌株CGMCC No.17894,具有以下微生物学特性:革兰氏阳性菌,芽孢中间生,菌宽1μm-2μm,长3μm-5μm。在LB琼脂培养基上,菌落铺展、均匀、灰黄色、菌落直径0.3cm-0.5cm,无色素,菌体培养10小时出现芽孢。在LB液体培养基中,接种30分钟开始萌发;1小时,菌体成长串无明显分隔,长度约11μm-15μm;2小时,菌体成长串有明显分隔,每个长串由8-12个菌体串联而成,长度约为24μm-36μm;2.5小时,长串菌体开始断裂为单个菌体。
在本申请的另一个方面中,提供利用该新菌种来发酵生产维生素K2(MK-7)的方法,可以较高产率生产维生素K2(MK-7)。
本申请提供一种维生素K2的生产方法,所述方法包括在培养基中培养所述纳豆枯草芽孢杆菌CGMCC No.17894以使在所述菌株细胞内和所述培养基中生产维生素K2,以及从所述菌株细胞内和所述培养基中回收并提纯维生素K2,其中所述维生素K2为MK-7。
在本申请中,所述培养基可以是本领域常规或者已知的培养基。所述培养基含有碳源、氮源物质。还可以向培养基中加入有机物质或者无机物质以促进微生物的生长和提高维生素K2(MK-7)的产率。
在本申请中,所述培养基含有0.2重量%-20重量%碳源物质和0.2重量%-20重量%氮源物质,其中,所述碳源物质选自葡萄糖、蔗糖、麦芽糖、果糖和甘油中的任一种或更多种;所述氮源物质选自酵母粉、蛋白胨(Tryptone)、黄豆粉、鹰嘴豆粉、牛肉浸膏和酵母提取物(Yeast extract)中的任一种或更多种。
在本申请中,所述培养基还含有0.001重量%-0.5重量%无机物质,所述无机物质选自磷酸盐、镁盐和钠盐中的任一种或更多种;
优选地,所述无机物质选自磷酸二氢钠、磷酸氢二钠、磷酸二氢钾、磷酸氢二钾、硫酸镁、氯化镁和氯化钠中的任一种或更多种。
在本申请中,所述培养基还含有促萌发物质和前体物质,所述促萌发物质选自硼酸、维生素B1、硫酸铜和硫酸锰中的任一种或更多种;所述前体物质选自茄尼醇、乙醇、甲萘醌和叶绿醇中的任一种或更多种。
由于所述菌是枯草芽孢杆菌,培养过程有芽孢,所以促进芽孢萌发的元素定义为促萌发物质。
在本申请中,在所述培养过程中,按所述培养基的重量计,加入0.001%~0.5%的所述促萌发物质;在所述培养过程中,优选地在菌体对数生长期至稳定期,按所述培养基的重量计,加入0.003%~5%的所述前体物质。
在本申请中,所述培养在需氧条件下进行,培养温度为35-50℃,培养时间为10-48h,pH为5.5-8.0,压力为0.03-0.08MPa。
所述培养条件可以在本领域的常规或已知条件下进行。所述的发酵可以在需氧条件下,温度可以为35-50℃,优选38-42℃。培养时间10-48小时,优选16-24小时。在所述发酵过程中优选在接种的时候加入促萌发物质硼酸、维生素B1、硫酸铜、硫酸锰,优选其浓度(占培养基)0.001%~0.1%。在所述发酵过程中优选在菌体生长前期加入,其浓度(占培养基)为0.003%~5%。
所述的发酵过程可以在本领域常规或已知的装置和条件下进行,例如可以使用摇瓶在本领域常规或已知的转速下进行;也可以在常规的发酵罐中进行,例如5L罐、5T发酵罐。
在本申请中,回收并提纯维生素K2包括以下步骤:
(1)从所述培养过程所得培养液分离出发酵菌体和发酵上清液;
(2)发酵菌体进行干燥;
(3)将第(2)步所得菌粉用有机溶剂萃取,萃取所得溶液进行减压蒸发,去掉溶剂;
(4)将第(3)步所得物用色谱柱脱色、脱过氧化值;
(5)将第(4)步所得物用色谱柱脱酸,得维生素K2产品。
在本申请中,在步骤(1)中,所述分离选自陶瓷膜分离和离心机分离中的一种或两种,优选地陶瓷膜分离。
在本申请中,在步骤(2)中,所述干燥选自喷雾干燥、冷冻干燥和减压真空干燥中的任一种或更多种,优选地喷雾干燥;
所述干燥优选地在避光条件下进行。
在本申请中,在步骤(3)中,所述有机溶剂选自石油醚、乙醇、甲醇、二氯甲烷、异丙醇、乙酸乙酯中的任一种或更多种,优选石油醚和乙酸乙酯。
在本申请中,在步骤(4)中,所述色谱柱的填料为硅胶。
在本申请中,在步骤(5)中,所述色谱柱的填料为氧化铝。
在步骤(4)中所用柱填料为硅胶,所用有机溶剂是本领域常规或已知的有机溶剂,例如石油醚、乙醇,乙酸乙酯等,优选石油醚,并用常规的减压回收的方法回收有机溶剂。在步骤(5)中所用柱填料为氧化铝,所用有机溶剂是本领域常规或已知的有机溶剂,例如石油醚、乙醇,乙酸乙酯等,优选石油醚,并用常规的减压回收的方法回收有机溶剂。
采用本发明的菌种CGMCC No.17894及本发明的发酵方法,周期短16-24小时,产量高200-300mg/L,可以解决食物中维生素K2(MK-7)含量不足的问题。mg/L是指MK-7含量和发酵液总体积的比例。
在本申请的另外的方面中,还提供所述纳豆枯草芽孢杆菌CGMCC No.17894用于制备维生素K2的用途。
在本申请的另外的方面中,本申请还提供一种MK-7制剂,包含MK-7和MK-6,MK-6为MK-7的2重量%-7重量%,所述制剂呈油剂或者粉剂的形式。
在本申请中,所述油剂包含0.1重量%-20重量%的MK-7、80重量%-99重量%的载体和0.1重量%-1重量%的抗氧化剂。
在本申请中,所述载体选自大豆油、玉米油、葵花籽油、橄榄油和中链甘油三酯中的任一种或更多种;
所述抗氧化剂选自迷迭香、阿魏酸、生育酚和棉酚中的任一种或更多种。
在本申请中,按重量计,所述粉剂包含0.1重量%-20重量%的MK-7、55重量%-75重量%的吸附剂、10重量%-15重量%的溶媒、5重量%-10重量%的粉化剂、3重量%-5重量%的乳化剂和0.2重量%-0.5重量%的抗氧化剂。
在本申请中,所述吸附剂选自变性淀粉、抗性糊精、微晶纤维素和膳食纤维中的任一种或更多种;
所述溶媒选自大豆油、玉米油、葵花籽油、橄榄油和中链甘油三酯中的任一种或更多种;
所述粉化剂选自酪蛋白酸钠、酪蛋白磷酸肽和磷酸化二淀粉磷酸酯中的任一种或更多种;
所述粉化剂选自辛烯基琥珀酸淀粉钠和辛烯基琥珀酸酯化淀粉中的任一种或两种;
所述乳化剂选自单甘油脂肪酸酯、双甘油脂肪酸酯、阿拉伯胶、大豆磷脂、辛癸酸甘油酯、单棕榈酸酯、聚氧乙烯山梨醇酐、硬脂酰乳酸钠、硬脂酰乳酸钙和双乙酰酒石酸单甘油酯中的任一种或更多种;
所述抗氧化剂选自抗坏血酸棕榈酸酯、异抗坏血酸、异抗坏血酸钠、生育酚、谷胱甘肽、枸橼酸、乙二胺四乙酸(EDTA)和类胡萝卜素中的任一种或更多种。
本申请还提供所述MK-7制剂用于食品或保健品中的用途。
在本申请的又一方面中,本申请还提供一种MK-7纯品预混剂,所述MK-7纯品预混剂包含0.1重量%-25重量%的MK-7、74.5重量%-99.7重量%的稀释剂和0.2重量%-0.5重量%的抗氧化剂。
在本申请中,所述稀释剂选自变性淀粉、抗性糊精、微晶纤维素和膳食纤维中的任一种或更多种;
所述抗氧化剂选自抗坏血酸棕榈酸酯、异抗坏血酸、异抗坏血酸钠、生育酚、谷胱甘肽、枸橼酸、EDTA和类胡萝卜素中的任一种或更多种。
本申请还提供所述MK-7纯品预混剂用于食品或保健品中的用途。
在本申请的另外的方面中,本申请提供一种MK-7纯品,包含98重量%-99.9重量%的MK-7和0.1重量%-2重量%的MK-6。
本申请还提供所述MK-7纯品用于制备治疗骨质疏松、心脑血管钙化、肾病患者血管硬化、肿瘤、肌肉痉挛、神经系统、糖尿病或牛皮癣的药物中的用途。
在本申请中,术语MK-7纯品预混剂指的是将MK-7纯品,加入稀释剂、抗氧化剂后经过过筛、混合、干燥等工序,得到的粉末。
本申请的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本申请而了解。本申请的目的和其他优点可通过在说明书、权利要求书中所特别指出的结构来实现和获得。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚明白,下文中将对本申请的实施例进行详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互任意组合。
实施例1
从市售纳豆中分离产维生素K2(MK-7)的纳豆枯草芽孢杆菌
将纳豆(购自日本生物科技公司)用无菌水溶解、稀释后涂抹在固体LB培养基平板上,于37℃恒温培养24小时,平板表面长出白色菌落,用接种针挑取有拉丝现象。把白色菌落转移到LB斜面培养基上,37℃培养24小时,用接种环接种于发酵培养基(100ml三角瓶装量20ml,培养基配方:葡萄糖2%,甘油4%,黄豆粉3%,硫酸镁0.01%,磷酸氢二钾0.01%,氯化钠0.5%,丝氨酸0.08%),37℃,270rpm振摇48小时,加入等体积乙酸乙酯萃取,分层后,乙酸乙酯层干燥得浸膏,用高效液相分析,挑选与标准品有相同保留时间的样品所对应的微生物,最终鉴定出产维生素K2(MK-7)的纳豆枯草芽孢杆菌。该微生物具有以下微生物学特性:革兰氏阳性菌,芽孢中间生,菌宽0.2um,长1-2um。在LB琼脂培养基上,菌落白色,突出于培养基表面,菌落表面褶皱,菌落粘可拉丝,菌落直径0.3-0.5cm,无色素,菌体培养10小时出现芽孢。
实施例2
产维生素K2(MK-7)的菌种诱变
以实施例1所得的产维生素K2(MK-7)的菌株为出发菌株进行紫外诱变,经过36代诱变得变异株ST-1008。该微生物具有以下微生物学特性:革兰氏阳性菌,芽孢中间生,菌宽0.5um,长2-3um。在LB琼脂培养基上,菌落铺展、均匀、灰黄色、菌落直径0.3-0.5cm,无色素,菌体培养10小时出现芽孢。在LB液体培养基中,接种30分钟开始萌发;1小时,菌体成长串无明显分隔,长度约11-15um;2小时,菌体成长串有明显分隔,每个长串由8-12个菌体串联而成,长度约为24-36um;2.5小时,长串菌体开始断裂为单个菌体。该菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市中关村北一桥13号,保藏号为:CGMCCNo.17894。
实施例3
种子制备
接种实施例所得CGMCC No.17894菌种,至装有50ml种子培养基(见表1),于38-42℃,150-300rpm振摇培养箱培养3-16小时,即得待接种的菌种。
表1:种子培养基配方:
成分 | 含量g% |
葡萄糖 | 1.0 |
Tryptone | 1.0 |
Yeast extract | 0.5 |
硫酸镁 | 0.05 |
NaCl | 1.0 |
纯水 | 100ml |
pH | 7.0 |
实施例4
5L罐发酵
将实施例3所得种子按照接种量占发酵培养基量的20%接种至发酵生产基础培养基(见表2)中,42℃培养,培养30分钟后开始流加补入补料培养基(见表3),每半小时加入补料培养基100ml,持续到20小时,共补入2L。接种时加入促萌发物质0.001%硼酸、0.001%维生素B1、0.001%硫酸铜;培养到4小时,加入前体物质0.05%乙醇。培养过程pH值自然,溶解氧浓度控制在30%以上。发酵周期24小时。高效液相法测定发酵产量为200mg/L,具体高效液相测定条件如表2。
表2进行高效液相色谱法的条件
表3:基础培养基
表4:补料培养基
注:在表1、表3和表4中,g%指的是每100毫升的溶剂中添加的溶质克数
将所得发酵液用陶瓷膜进行固液分离,所得菌体浓缩液进行真空干燥,干燥后的菌粉,按照菌粉:石油醚或乙醇的1:3重量比加入乙醇或石油醚进行提取。提取后,分离有机相,有机相减压回收,得含有微生物K2的粗产物。粗产物用硅胶作为柱填料,用石油醚作为流动相,进行脱色。脱色后的粗品,经过氧化铝柱,用石油醚做流动相脱掉酸值。得维生素K2(MK-7),收率为80%。
表5硅胶柱洗脱条件
表6氧化铝柱洗脱条件
实施例5
与实施例4不同之处在于,接种时加入促萌发物质0.05%硼酸、0.001%维生素B1、0.05%硫酸铜、0.05%硫酸锰。发酵产量260mg/L。
实施例6
与实施例4不同之处在于,接种时加入促萌发物质0.1%硼酸、0.1%维生素B1、0.1%硫酸铜、0.1%硫酸锰。发酵产量220mg/L。
实施例7
与实施例5的不同之处在于,培养到4小时,加入前体物质0.05%茄尼醇,发酵产量280mg/L。
实施例8
与实施例7不同之处在于,培养到4小时,加入前体物质0.05%茄尼醇、0.05%的乙醇。发酵产量300mg/L。
实施例9
与实施例8不同之处在于,干燥后的菌粉,按照1:3重量比加入乙酸乙酯进行提取。维生素K2(MK-7),收率为90%。
实施例10
与实施例8不同之处在于,干燥后的菌粉,按照1:3重量比加入乙酸乙酯进行提取。维生素K2(MK-7)成品收率为98%。
通过本申请发酵方法获得的维生素K2,克服了化学合成维生素K2的缺点:产生大量异构体、副产物多、产率低、带来环境污染等问题,且其异戊二烯侧链多是顺式结构,活性较低。通过本申请发酵方法获得的维生素K2,发酵产量可以到达300mg/L,对菌粉进行提纯,收率可以达到98%,并且经结构鉴定与全反式维生素K2,即MK-7氢谱数据完全一致。
实施例11
K2(MK-7)2g、大豆油120g、变性淀粉743g、酪蛋白酸钠100g、辛烯基琥珀酸淀粉钠20g、单甘油脂肪酸酯10g、抗坏血酸棕榈酸酯5g。
取K2(MK-7)加入大豆油搅拌均匀,使完全溶解后,继续加入抗坏血酸棕榈酸酯、单甘油脂肪酸酯、辛烯基琥珀酸淀粉钠、酪蛋白酸钠,继续搅拌均匀后,加入变性淀粉,搅拌均匀后,过80目筛,80℃干燥2小时,即得2000ppm的维生素K2(MK-7)粉末1kg。
实施例12
K2(MK-7)2g、大豆油988g、迷迭香10g
取K2(MK-7)加入迷迭香,再加入大豆油搅拌溶解均匀后,即得2000ppm的维生素K2(MK-7)油1Kg。
实施例13
MK-7的粉剂和油剂的性能
表7实施例11的MK-7粉剂与现有MK-7粉剂的性能比较
表8实施例12的MK-7油剂与现有MK-7油剂的性能比较
MK-7的粉剂和油剂以及现有粉剂和油剂的稳定性根据《国家卫生和计划生育委员会公告2016年8号》进行测定,酸价和过氧化值分别根据GB5009.229-2016第一法和GB5009.227-2016第一法进行测定。
实施例14
K2(MK-7)2g、抗坏血酸棕榈酸酯2g、微晶纤维素996g
取K2(MK-7)加入抗坏血酸棕榈酸酯混合均匀,过80目筛后,用微晶纤维素倍比递增5次后,再过80目筛,加入剩余的微晶纤维素,混合均匀后,过80目筛,80℃干燥2小时,即得2000ppm的维生素K2(MK-7)预混剂1kg。
实施例15
MK-7纯品预混剂的性能
表9实施例14的MK-7纯品预混剂和现有MK-7预混剂的性能比较
实施例14的纯品预混剂与现有纯品预混剂的稳定性根据《国家卫生和计划生育委员会公告2016年8号》进行测定。
虽然本申请所揭露的实施方式如上,但所述的内容仅为便于理解本申请而采用的实施方式,并非用以限定本申请。任何本申请所属领域内的技术人员,在不脱离本申请所揭露的精神和范围的前提下,可以在实施的形式及细节上进行任何的修改与变化,但本申请的专利保护范围,仍须以所附的权利要求书所界定的范围为准。
Claims (19)
1.一种纳豆枯草芽孢杆菌ST-1008,保藏编号为CGMCC No.17894。
2.一种MK-7的生产方法,所述方法包括在培养基中培养权利要求1所述的纳豆枯草芽孢杆菌以使在所述纳豆枯草芽孢杆菌细胞内和所述培养基中生产MK-7,以及从所述纳豆枯草芽孢杆菌细胞内和所述培养基中回收并提纯MK-7。
3.根据权利要求2所述的生产方法,其中,以所述培养基的重量计,所述培养基含有0.2%-20%碳源物质、0.2%-20%氮源物质、促萌发物质和前体物质,其中,所述碳源物质选自葡萄糖、蔗糖、麦芽糖、果糖和甘油中的任一种或更多种;所述氮源物质选自酵母粉、蛋白胨、黄豆粉、鹰嘴豆粉、牛肉浸膏和酵母提取物中的任一种或更多种;所述促萌发物质选自硼酸、维生素B1、硫酸铜、硫酸锰和丝氨酸中的任一种或更多种;所述前体物质选自茄尼醇、乙醇、甲萘醌和叶绿醇中的任一种或更多种。
4.根据权利要求2所述的生产方法,其中,以所述培养基的重量计,所述培养基还含有0.001%-0.5%无机物质,所述无机物质选自磷酸盐、镁盐和钠盐中的任一种或更多种。
5.根据权利要求4所述的生产方法,其中,所述无机物质选自磷酸二氢钠、磷酸氢二钠、磷酸二氢钾、磷酸氢二钾、硫酸镁、氯化镁和氯化钠中的任一种或更多种。
6.根据权利要求3所述的生产方法,其中,在所述培养过程中,按所述培养基的重量计,加入0.001%~0.5%的所述促萌发物质。
7.根据权利要求6所述的生产方法,其中,在所述培养过程中,在菌体对数生长期至稳定期,按所述培养基的重量计,加入0.003%~5%的所述前体物质。
8.根据权利要求2所述的生产方法,其中,所述培养在需氧条件下进行,培养温度为35-50℃,培养时间为10-48h,pH为5.5-8.0,压力为0.03-0.08MPa。
9.根据权利要求2至8中任一项所述的生产方法,其中,所述回收并提纯MK-7包括以下步骤:
(1)从所述培养过程所得培养液分离出发酵菌体和发酵上清液;
(2)发酵菌体进行干燥;
(3)将第(2)步所得干燥后的菌体用有机溶剂萃取,萃取所得溶液进行减压蒸发,去掉溶剂;
(4)将第(3)步所得物用色谱柱脱色、脱过氧化值;
(5)将第(4)步所得物用色谱柱脱酸,得MK-7。
10.根据权利要求9所述的生产方法,其中,在步骤(1)中,所述分离选自陶瓷膜分离和离心机分离中的一种或两种。
11.根据权利要求10所述的生产方法,其中,在步骤(1)中,所述分离为陶瓷膜分离。
12.根据权利要求9所述的生产方法,其中,在步骤(2)中,所述干燥选自喷雾干燥、冷冻干燥和减压真空干燥中的任一种或更多种。
13.根据权利要求12所述的生产方法,其中,在步骤(2)中,所述干燥为喷雾干燥。
14.根据权利要求12所述的生产方法,其中,在步骤(2)中,所述干燥在避光条件下进行。
15.根据权利要求9所述的生产方法,其中,在步骤(3)中,所述有机溶剂选自石油醚、乙醇、甲醇、二氯甲烷、异丙醇和乙酸乙酯中的任一种或更多种。
16.根据权利要求15所述的生产方法,其中,在步骤(3)中,所述有机溶剂选自石油醚和乙酸乙酯中的任一种或两种。
17.根据权利要求9所述的生产方法,其中,在步骤(4)中,所述色谱柱的填料为硅胶。
18.根据权利要求9所述的生产方法,其中,在步骤(5)中,所述色谱柱的填料为氧化铝。
19.权利要求1所述的纳豆枯草芽孢杆菌用于制备MK-7的用途。
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