CN116042738A - 肠膜明串珠菌的应用及方法 - Google Patents
肠膜明串珠菌的应用及方法 Download PDFInfo
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Abstract
本发明公开了肠膜明串珠菌的应用及方法,涉及微生物技术领域。肠膜明串珠菌能够用于制备植酸,且能够改善因高脂高糖饮食导致的非酒精性脂肪肝、肝脏重量增加、血脂升高、血糖升高、血清脂多糖水平升高、血清胰岛素水平升高、炎症反应、肝脏中甘油三酯水平升高,抑制促炎因子脂多糖的释放。
Description
技术领域
本发明涉及微生物技术领域,尤其是涉及肠膜明串珠菌的应用及方法。
背景技术
植酸(Phytic acid)又被称为肌醇六磷酸,是一种含磷有机酸类化合物。先前研究表明植酸能与多价金属离子(如钙离子、镁离子等)螯合成复合物,进而影响矿物质离子的吸收和利用,被视为一种抗氧化剂。然而,随着研究的深入,发现小分子的植酸并不会拮抗矿物质的吸收和利用,且其毒性极低。近年来,大量研究表明植酸具有独特的生理学活性,如抗氧化、抗肿瘤、免疫调节和减肥等,是一种天然的营养成分。目前,植酸已经作为抗氧化剂、保鲜剂和稳定剂广泛应用于食品领域,如油脂食品、饮料和新鲜果蔬等。
当前,植酸的生产仍依赖于传统化学法从植物种子、籽粒或果壳中提取出来,即原料经过粉碎后,通过酸浸、过滤、中和等获取植酸钙,再通过酸化、过滤、中和、离子交换树脂、浓缩、脱色等制备植酸,可见传统法生产植酸的工艺存在过程繁琐、耗时较长、环境污染等不足,因此其可持续和环保性受到制约。微生物发酵法即生物合成是近年来新兴的技术,其具有条件温和、环境污染小、原料成本低等优点,已广泛应用于多种高价值平台化合物的生物合成,如乳酸、D-葡萄糖二酸等。这提示开发经济、环保、高效的植酸生物合成途径是解决其现有制备工艺问题的重要策略之一,然而目前尚未有微生物发酵合成植酸的报道。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明提出肠膜明串珠菌的应用,能够用于制备植酸。
本发明还提供一种植酸的制备方法。
根据本发明的第一方面实施例肠膜明串珠菌在制备植酸中的应用。
根据本发明的一些实施例,所述应用通过从肠膜明串珠菌的发酵液中分离纯化得到植酸实现。
根据本发明的一些实施例,所述肠膜明串珠菌包括保藏号为GDMCC No:61720的肠膜明串珠菌。
根据本发明的第二方面实施例的一种植酸的制备方法,通过对肠膜明串珠菌的发酵液进行分离、纯化得到。
根据本发明的一些实施例,所述肠膜明串珠菌包括保藏号为GDMCC No:61720的肠膜明串珠菌。
植酸可通过常规植酸分离纯化工艺从发酵液中制备得到植酸。
根据本发明的一些实施例,所述纯化包括离子交换树脂法。所述离子交换树脂法通过阴离子交换树脂吸附溶液中的植酸根离子,然后采用碱性洗脱剂进行洗脱,以除去氯离子等阴离子杂质,之后采用阳离子树脂吸附溶液中的Na+、Ca2+及Mg2+等阳离子杂质,从而达到纯化的目的。使用的阴离子树脂包括但不限于330、717、D315、D318、D301。
根据本发明的一些实施例,制备所述发酵液的方法包括:将所述肠膜明串珠菌接种到培养基中,在30℃~37℃条件下培养,即得发酵液。
根据本发明的一些实施例,所述发酵的时间为12h~96h。优选为24h~72h。进一步优选为36h~60h。
根据本发明的一些实施例,所述肠膜明串珠菌的接种密度为106CFU/mL~1010CFU/mL。优选为107CFU/mL~109CFU/mL。
根据本发明的一些实施例,所述发酵的温度为25℃~40℃。优选为35℃~40℃。进一步优选为34℃~37℃。
根据本发明的一些实施例,所述培养基包括MRS培养基、Rogosa培养基、LMM培养基。可以理解的是,其他公知的乳酸菌用培养基或培养液等均适用。
根据本发明的一些实施例,所述制备方法还包括纯化后处理。所述纯化后处理包括浓缩、脱色中的至少一种。
根据本发明的第三方面实施例的的保藏号为GDMCC No:61720的肠膜明串珠菌在A1至A9任一项中的应用,
A1:制备防治非酒精性脂肪肝的产品;
A2:制备预防和/或减少体重增加的产品;
A3:制备预防和/或减少肝脏重量增加的产品;
A4:制备降血脂的产品;
A5:制备降血糖的产品;
A6:制备降低血清中脂多糖水平的产品;
A7:制备降低血清胰岛素水平的产品;
A8:制备改善炎症症状的产品;
A9:制备降低肝脏甘油三酯水平的产品。
根据本发明的一些实施例,所述产品针对由摄入高脂和/或高糖引起的非酒精性脂肪肝、肝脏重量增加、血脂升高、血糖升高、血清脂多糖水平升高、血清胰岛素水平升高、炎症反应、肝脏中甘油三酯水平升高。
根据本发明的一些实施例,所述降血脂包括降低血清总胆固醇、血清总甘油三酯、肝脏甘油三酯的水平中的至少一种。
根据本发明的一些实施例,所述降血糖包括降低血清葡萄糖的水平。
根据本发明的一些实施例,所述改善炎症症状包括降低肝脏白介素6的水平。
根据本发明的一些实施例,所述产品包括食品、保健品或药物。
根据本发明的一些实施例,所述产品还包括药学、食品或保健品上可接受的辅料。
根据本发明的一些实施例,所述药物的剂型包括片剂、胶囊剂、颗粒剂、丸剂中的至少一种。
本发明至少具有如下有益效果:
当前传统法提取植酸具有复杂、环境污染、时间长的缺点,而保藏号为GDMCC No:61720的肠膜明串珠菌能够通过发酵以环保、经济、高效的生物合成高水平的植酸。保藏号为GDMCC No:61720的肠膜明串珠菌能够改善因高脂高糖饮食导致的非酒精性脂肪肝、肝脏重量增加、血脂升高、血糖升高、血清脂多糖水平升高、血清胰岛素水平升高、炎症反应、肝脏中甘油三酯水平升高,抑制促炎因子脂多糖的释放。
本发明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,或者通过实施本发明而了解。
附图说明
图1是本发明一实施例的肠膜明串珠菌的发酵液的液相色谱图;
图2是本发明一实施例的肠膜明串珠菌的肌醇磷酸盐代谢的示意图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
在本发明的描述中,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、***、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下述实施例中的肠膜明串珠菌(Leuconostoc mesenteroides)I1/53,于2021年6月11日保藏于广州市先烈中路100号大院59号楼5楼广东省微生物研究所的广东省微生物菌种保藏中心,保藏编号为GDMCC No:61720。
C57小鼠,雄性,6~7周龄,由珠海百试通生物科技有限公司提供。
正常小鼠饲料和高糖高脂饲料(含有蔗糖30%和脂肪35%),均由北京科澳协力饲料有限公司提供。
植酸由上海麦克林生化科技有限公司提供。
实施例1(肠膜明串珠菌I1/53发酵液中植酸的鉴定)
将活性肠膜明串珠菌I1/53接种于10mL MRS培养基中37℃培养48h(接种量为1×109CFU);随后将发酵液于4℃和8000rpm条件下离心10min,取上清50μL,加入750μL预冷(4℃)的乙腈中,然后超声3min,并于4℃和13000rpm条件下离心15min;取600μL上清用超高压液相三重四级杆飞行时间质谱仪进行非靶标代谢组学分析。通过Progenesis QI V2.0软件收取色谱和质谱信息,并通过一系列质控操作,包括降噪、峰对齐、去零值等,将获得的高质量色谱和质谱信息在公共数据库对比分析,并根据二级质谱信息进行精准鉴定。
其中,色谱柱是ACQUITY UPLC BEH C18(100×2.1mm,1.7μm);参数设置为:柱温40℃,流动相流速0.35mL/min,进样量2.0μL。流动相A是纯水,流动相B是乙腈;洗脱梯度:0min98.0%A,6.5min 50%A,10min 98%A,总检测时长是12.5min。
质谱参数设置为:正离子模式下,M/Z为50-1200,碰撞电压为35V;去溶剂和锥性气体是氮气,毛细管压力2.5千瓦伏,锥性气体流速为40L/h,离子源温度为120℃。
鉴定结果如图1所示。共检测到1069个代谢物,其中,植酸的保留时间为3.6412,质荷比659.8614。进一步通过液相色谱和标准品定量检测结果显示,发酵液中植酸含量为36.6mg/L。
实施例2
本实施例通过PromethlON和NovaSeq6000平台测定肠膜明串珠菌I1/53的全基因组,具体如下:
利用Hipure Bacterial DNAKit试剂盒(由上海美吉生物医药科技有限公司提供)提取肠膜明串珠菌I1/53的DNA,并通过Nanodrop2000和Qubit 3.0荧光计分析DNA的浓度和纯度;通过SQK-LSK109试剂盒(由上海美吉生物医药科技有限公司提供)进行DNA文库构建,并测序;随后对测序结果进行质控去除低质量的片段、基因组装配、结构分析,并在公共数据库包括蛋白质家族数据库、基因序列数据库、同源序列数据库和KEGG数据库进行功能分析。
分析结果显示肠膜明串珠菌I1/53的基因组大小为2044872bps,GC含量为37.5%,编码DNA序列(CDS)有2027条,16个人RNA操纵子和71个转运RNA基因。
肠膜明串珠菌I1/53基因组功能分析显示共有2205个代谢通路,通过与代谢组联合分析显示,共有5条代谢通路是两种组学的交集,其中肌醇磷酸盐代谢(inositolphosphate metabolism)是合成植酸的途径,如图2所示。
实施例3
本实施例通过针对由高糖高脂诱导的非酒精性脂肪肝小鼠模型的相关动物实验评估肠膜明串珠菌I1/53及其代谢物植酸的生理功能。步骤如下:
将24只C57小鼠随机分为4组,即正常组,模型组,肠膜明串珠菌I1/53干预组和植酸干预组,每组6只。灌胃,每天1次,肠膜明串珠菌给药量是1*109CFU/只,植酸给药量是100mg/kg·bw,实验期间,正常组给予正常小鼠饲料,其他三组给予高糖高脂饲料,所有小鼠自由饮水,进行为期8周的实验;实验结束时禁食12h后,称重,麻醉后取血清、肝脏组织,其中,肝脏称重后,利用酶联免疫吸附测定试剂盒(武汉酶免生物科技有限公司)测定血清中胰岛素、葡萄糖、脂多糖和胆固醇(包括总胆固醇和甘油三酯)的含量、肝脏中甘油三酯和白细胞介素6的含量。
检测结果如表1所示,与模型组相比,肠膜明串珠菌干预组和植酸干预组小鼠体重、肝脏组织、血清胰岛素、葡萄糖、脂多糖、总胆固醇和甘油三酯含量均明显降低,此外,肝脏甘油三酯和白细胞介素6含量在肠膜明串珠菌干预组和植酸干预组也明显降低。这说明肠膜明串珠菌I1/53和其代谢物植酸均具有缓解非酒精性脂肪肝的作用。
表1肠膜明串珠菌活菌制剂和植酸具有缓解非酒精性脂肪肝的作用
注:*表示P<0.05;**表示P<0.01;***表示P<0.001。
上面结合附图对本发明实施例作了详细说明,但本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。
Claims (10)
1.肠膜明串珠菌在制备植酸中的应用。
2.根据权利要求1所述的应用,所述肠膜明串珠菌包括保藏号为GDMCC No:61720的肠膜明串珠菌。
3.一种植酸的制备方法,其特征在于,通过对肠膜明串珠菌的发酵液进行分离纯化得到。
4.根据权利要求3所述的制备方法,其特征在于,所述纯化包括离子交换树脂法。
5.根据权利要求3所述的制备方法,其特征在于,制备所述发酵液的方法包括:将所述肠膜明串珠菌接种到培养基中,在25℃~40℃条件下培养,即得发酵液。
6.根据权利要求5所述的制备方法,其特征在于,所述发酵的时间为12h~96h。
7.根据权利要求5所述的制备方法,其特征在于,所述肠膜明串珠菌的接种密度为106CFU/mL~1010CFU/mL。
8.根据权利要求5所述的制备方法,其特征在于,所述培养基包括MRS培养基、Rogosa培养基、LMM培养基。
9.保藏号为GDMCC No:61720的肠膜明串珠菌在A1至A9任一项中的应用,其特征在于,
A1:制备防治非酒精性脂肪肝的产品;
A2:制备预防和/或减少体重增加的产品;
A3:制备预防和/或减少肝脏重量增加的产品;
A4:制备降血脂的产品;
A5:制备降血糖的产品;
A6:制备降低血清中脂多糖水平的产品;
A7:制备降低血清胰岛素水平的产品;
A8:制备改善炎症症状的产品;
A9:制备降低肝脏甘油三酯水平的产品。
10.根据权利要求9所述的应用,其特征在于,所述所述产品针对由摄入高脂和/或高糖引起的非酒精性脂肪肝、肝脏重量增加、血脂升高、血糖升高、血清脂多糖水平升高、血清胰岛素水平升高、炎症反应或肝脏中甘油三酯水平升高。
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