CN115161237B - 一株能降解脂多糖并抑制α-葡萄糖苷酶的凝结芽孢杆菌及其应用 - Google Patents
一株能降解脂多糖并抑制α-葡萄糖苷酶的凝结芽孢杆菌及其应用 Download PDFInfo
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- CN115161237B CN115161237B CN202210833845.3A CN202210833845A CN115161237B CN 115161237 B CN115161237 B CN 115161237B CN 202210833845 A CN202210833845 A CN 202210833845A CN 115161237 B CN115161237 B CN 115161237B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株能降解脂多糖并抑制α‑葡萄糖苷酶的凝结芽孢杆菌SA12及其应用。所述SA12菌株已于2022年6月6日保存于广东省菌种保藏中心,保藏编号:GDMCC NO:62518。本发明提供的凝结芽孢杆菌SA12分离自桑葚酵素,具有较强的降解脂多糖能力,抑制α‑葡萄糖苷酶能力和DPPH自由基能力,在4h对80EU/mL的脂多糖去除率高达到91.55%,同时,能够通过抑制引起糖尿病的关键酶α‑葡萄糖苷酶来实现缓解糖尿病。本发明提供的凝结芽孢杆菌SA12不仅能清除脂多糖从而达到抗炎效果,还能缓解糖尿病,为降解脂多糖和抑制α‑葡萄糖苷酶提供一种新的微生物菌株。
Description
技术领域
本发明属于微生物技术领域。更具体地,涉及一株能降解脂多糖并抑制α-葡萄糖苷酶的凝结芽孢杆菌及其应用。
背景技术
脂多糖是革兰氏阴性菌外膜上最主要的成分,也被称为内毒素,是炎症的强效刺激物。研究发现,Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路是脂多糖所介导的信号传导通路中最重要的下游通路,TLR4受体可识别并结合脂多糖,进而激活NF-κB信号通路诱导炎症因子的释放,导致炎症的发生,引起组织损伤,如细菌感染、神经***疾病、心血管病、肾功能衰竭、代谢综合征和内毒素败血症等。脂多糖结合蛋白(LBP)和白细胞分化抗原14(CD14)这两个辅助因子可使脂多糖有效刺激炎症反应。当脂多糖结合TRL4受体后可激活NF-κB信号因子,启动和调节肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)和白细胞介素-6(IL-6)等炎症因子表达,上调NLRP3炎症小体的表达,分泌释放众多内源性生物活性炎症因子,引起全身炎症反应综合征等。
糖尿病是一类因遗传或后天环境所引起的内分泌***疾病,同时也是一种威胁人类生命与健康的疾病。在2型糖尿病患者的肠道菌群中,革兰氏阴性菌相对丰度较高,而革兰氏阴性菌外膜的主要化合物是脂多糖,革兰氏阴性菌死亡后在肠道中持续产生脂多糖,脂多糖能强烈刺激胰岛素抵抗的关键诱导因子的释放。脂多糖结合TLR-4激活的信号级联对能够抑制胰岛中胰岛素,但对TLR-4缺陷小鼠胰岛素分泌无抑制作用。当脂多糖与CD14结合后,可作为载体触发高脂肪摄食引起的肥胖/胰岛素抵抗,且CD14基因敲除小鼠对饮食诱导的肥胖和相关疾病如肝脏胰岛素抵抗具有抵抗力。而α-葡萄糖苷酶作为小肠黏膜上调节血糖的关键酶,可以通过影响葡萄糖的释放和吸收来调节餐后血糖。因此,可通过抑制α-葡萄糖苷酶的活性、降低多糖含量来避免高脂肪摄食引起的肥胖/胰岛素抵抗和延缓肠道对碳水化合物的吸收,从而达到抑制糖尿病患者餐后血糖的升高。
现有研究中有大部分都是通过对α-葡萄糖苷酶的抑制来缓解糖尿病,目前也仅有双歧乳杆菌(Bifidobacterium)F-35、鼠李糖乳杆菌(Lactobacillus rhamnosus)GG、植物乳杆菌(Lactobacillus plantarum)以及嗜酸乳杆菌NM(Lactobacillus acidophilus)等能抑制α-葡萄糖苷酶。但在现有技术公开的微生物仅能抑制α-葡萄糖苷酶,并不具有降解脂多糖的功能,如现有技术公开了一株凝结芽孢杆菌JA845对α-葡萄糖苷酶的抑制率为39.51%,其抑制率还有待提高。可见,具有能抑制α-葡萄糖苷酶以及能降解脂多糖的微生物菌株还是太少,到目前为止还是缺乏能够抑制α-葡萄糖苷酶、同时还具有降解脂多糖的微生物菌株。因此,为了得到更多的具有抑制α-葡萄糖苷酶及降解脂多糖的微生物菌株,有必要筛选出更多的新的、效率更好的多功能微生物菌株,充实微生物菌种资源库。
发明内容
本发明要解决的技术问题是克服上述问题的缺陷和不足,提供一株能降解脂多糖并缓解糖尿病的凝结芽孢杆菌及其应用。
本发明的目的是提供一株凝结芽孢杆菌(Weizmannia coagulans)SA12菌株。
本发明另一目的是提供所述凝结芽孢杆菌SA12菌株的应用。
本发明再一目的是提供一种具有降解脂多糖和/或抑制α-葡萄糖苷酶的制剂。
本发明再一目的是提供一种降解脂多糖和/或抑制α-葡萄糖苷酶的方法。
本发明上述目的通过以下技术方案实现:
本发明提供一株凝结芽孢杆菌(Weizmannia coagulans)SA12菌株,分离于自然发酵的桑葚残渣,该菌株于2022年6月6日保藏于广东省微生物菌种保藏中心(GDMCC),保藏编号为:GDMCC NO:62518。本发明研究显示SA12菌株具有较强的清除DPPH自由基能力,对DPPH自由基清除率达到37.95%;具有高效降解脂多糖的能力,对脂多糖清除率达到了91.55%;同时,SA12菌株还能抑制α-葡萄糖苷酶,对α-葡萄糖苷酶的抑制效果与酶抑制剂阿卡波糖相比达到42.59%,能有效降低炎症反应,对糖尿病有缓解作用。
本发明提供凝结芽孢杆菌SA12菌株和/或其发酵液在降解脂多糖和/或在制备脂多糖降解菌剂、在抑制α-葡萄糖苷酶和/或在制备α-葡萄糖苷酶抑制剂、在清除DPPH自由基或在制备清除DPPH自由基的制剂中的应用、在制备缓解糖尿病药物中的应用。
本发明提供一种具有降解脂多糖和/或抑制α-葡萄糖苷酶的制剂,含有凝结芽孢杆菌SA12菌株和/或其发酵液。
优选地,所述SA12菌株的添加量为0.1~10%。
更优选地,SA12菌株的添加量为0.2%
优选地,所述发酵液为去除菌体的上清液。
优选地,所述发酵条件为:30~40℃,10~15h,150~200r/min。
更优选地,所述发酵条件为:37℃,12h,180r/min。
本发明提供一种降解脂多糖和/或抑制α-葡萄糖苷酶的方法,采用凝结芽孢杆菌SA12菌株和/或其发酵液对脂多糖和/或α-葡萄糖苷酶进行处理。
本发明具有以下有益效果:
本发明提供一株凝结芽孢杆菌(Weizmannia coagulans)SA12菌株,从桑葚酵素在自然发酵中分离得到一株产酸能力和清除DPPH自由基能力较强的SA12菌株,SA12菌株对DPPH自由基清除率达到37.95%。本研究显示,SA12菌株具有高效降解脂多糖的能力,在添加凝结芽孢杆菌SA12中的脂多糖含量从原始浓度80U/mL降到了6.76U/mL,对脂多糖清除率达到了91.55%。表明凝结芽孢杆菌SA12具有较强的清除脂多糖能力,能够有效降低由脂多糖诱导的炎症反应。同时,凝结芽孢杆菌SA12还具有抑制α-葡萄糖苷酶的能力,以常用的α-葡萄糖苷酶抑制剂—阿卡波糖作为阳性对照(阳性对照对α-葡萄糖苷酶的抑制率接近100%),SA12菌株对α-葡萄糖苷酶的抑制率为42.59%,能够通过抑制引起糖尿病的关键酶α-葡萄糖苷酶来实现缓解糖尿病。本发明提供的凝结芽孢杆菌SA12不仅能清除脂多糖从而达到抗炎效果,还能缓解糖尿病,为降解脂多糖和抑制的α-葡萄糖苷酶提供一种新的微生物菌株。
附图说明
图1为菌株菌落图;
图2为菌株进化树;
图3为菌株DPPH清除率;
图4为菌株脂多糖清除率;
图5为菌株α-葡萄糖苷酶抑制率。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
MRS液体培养基:蛋白胨10g/L,酵母提取物5g/L,牛肉浸膏10g/L,无水葡萄糖20g/L,磷酸氢二钾2g/L,无水乙酸钠5g/L,柠檬酸三铵2g/L,硫酸镁0.1g/L,硫酸锰0.05g/L,吐温80 1g/L,用蒸馏水配制而成。
MRS固体培养基:蛋白胨10g/L,酵母提取物5g/L,牛肉浸膏10g/L,无水葡萄糖20g/L,磷酸氢二钾2g/L,无水乙酸钠5g/L,柠檬酸三铵2g/L,硫酸镁0.1g/L,硫酸锰0.05g/L,吐温80 1g/L,琼脂,15g/L,用蒸馏水配制而成。
TSB培养基:胰蛋白胨17g/L,大豆蛋白胨3g/100L,氯化钠5g/L,K2HPO4 2.5g/L,葡萄糖2.5g/L,用蒸馏水配制而成。
实施例1菌株的筛选和鉴定
(1)菌株分离
将从市面上选取新鲜桑葚冲洗干净,装入发酵罐并加入不同浓度的糖水,进行避光自然发酵。称取适量的上述发酵残渣碾碎,加入锥形瓶中的MRS液体培养基,置于37℃摇床中培养48h。培养后从培养基中吸取1mL混合菌液,用无菌水进行梯度稀释,在MRS固体平板上涂布10-2~10-6五个稀释度,并在37℃培养48h。培养结束后,从平板上随机挑取不同单菌落在MRS固体培养基上进行划线纯化,在37℃培养48h。得到的纯培养菌种用20%甘油冲洗,吸取到甘油管中,在-20℃冰箱保存备用。
(2)形态学鉴定
将菌株接种于培养基平板上37℃倒置培养24h,观察其菌落形态。菌株在培养基培养24h的菌落形态如图1所示,菌落呈现不透明乳白色,表面光滑湿润,菌株通过革兰氏染色呈阳性,经测定该菌的适宜生长温度范围37~50℃,适宜生长pH范围6~7。
(3)细菌的分子生物学鉴定
采用SDS法对上述纯化后的细菌进行DNA提取,使用通用引物27f和1492r对细菌保守序列进行扩增。再将扩增产物送至测序公司进行测序,并将得到的序列进行拼接,将拼接后的序列在NCBI数据库进行比对,得到多种不同细菌,根据多次重复比对结果,其中有22株都为同一菌属,其16S rRNA基因序列与凝结芽孢杆菌(Weizmannia coagulans)(注:凝结芽孢杆菌现已经更新拉丁文名称,由原来的Bacillus coagulans更新为Weizmanniacoagulans,本发明则采用最新的拉丁文名称进行命名和保藏)同源性最高,其中五株菌的***发育树如图2所示。因此,将这同一菌属的菌株鉴定为凝结芽孢杆菌(Weizmanniacoagulans),命名为SA1~SA22。
将上述鉴定出的多株凝结芽孢杆菌进行产酸能力和清除DPPH自由基能力的测定,通过测定筛选出清除DPPH自由基能力较好的凝结芽孢杆菌,DPPH清除率结果如图3所示,其中SA12菌株对DPPH自由基清除率达到37.95%,显著优于其他凝结芽孢杆菌。因此,确定性能优良的凝结芽孢杆菌SA12,后续实验均采用SA12菌株进行,并将凝结芽孢杆菌(Weizmannia coagulans)SA12于2022年6月6日保藏于广东省微生物菌种保藏中心(GDMCC),菌种保藏号为GDMCC NO:62518,保藏地址为:广东广州市越秀区先烈中路100号。
实施例2菌株SA12的降解脂多糖实验
由于MRS培养基对鲎试剂动态显色法测定脂多糖含量方法有干扰,因此选择TSB培养基进行实验。采用实施例1中分离得到的凝结芽孢杆菌SA12以0.2%的接种量接入灭菌后的TSB培养基中,在37℃中180r/min摇床中培养12h,备用。从培养好的凝结芽孢杆菌SA12菌液中吸取5mL菌液置于去热源的离心管中,离心4000r/min,10min,吸取离心后的上清液置于去热源的试管中,菌泥用无脂多糖的无菌水重悬3次洗去残留培养基,并用无脂多糖的无菌水补足至5mL。
实验处理组为去除凝结芽孢杆菌SA12菌体的上清液;对照组为没有接入凝结芽孢杆菌SA12菌液的空白TSB培养基。
分别取菌液上清液置于TSB培养基内,准备TSB空白培养基,各加入1mL60EU的脂多糖中,在37℃条件下分别反应0h、4h。分别取0h反应后溶液和4h反应后溶液,分别稀释200倍、400倍以及800倍稀释液。然后各取0.1mL的稀释后的溶液,加到除热原微孔板内,每一浓度加3孔,再分别加入0.1mL鲎试剂,采用中速振摇10s混匀后,将微孔板放入已预热好的内毒素自动分析仪ELx808中进行检测,同时采用0.005EU/mL、0.05EU/mL、0.5EU/mL和5EU/mL浓度的标准内毒素建立标准曲线,每一浓度内毒素溶液至少3个平行孔,阴性对照平行2孔,检测的结果取三个平行处理的结果平均值。再根据制作得到的标准曲线y=-0.3039x+2.8422,R2=0.998,y为时间(s),x为质量浓度,单位为EU/mL,计算内毒素浓度。
结果如图4所示,在0h时TSB培养基(未去除脂多糖)和60EU的脂多糖总共含脂多糖80U/mL。在0~4h期间,在对照组TSB中脂多糖含量从原始浓度80U/mL降低到63.24U/mL,降低了16.76U/mL,而实验组凝结芽孢杆菌SA12上清液组中的脂多糖含量从对照组中的原始浓度80U/mL变成了6.76U/mL,以变化量比原始浓度计算得到清除率,发现凝结芽孢杆菌SA12的脂多糖清除率达到91.55%。结果表明凝结芽孢杆菌SA12的发酵上清液可以显著降解脂多糖。
实施例3菌株SA12抑制α-葡萄糖苷酶活性实验
实验取培养至对数期的凝结芽孢杆菌SA12菌液50μL与100μL的α-葡萄糖苷酶溶液(1U/mL)混合,在37℃下培养10min,再进一步加入底物5mM的对硝基苯基-α-D-吡喃葡萄糖苷,再在37℃培养30min。最后加入1mL0.1M碳酸钠,使反应停止。
以不加凝结芽孢杆菌的等体积α-葡萄糖苷酶溶液作为对照组;以不加底物的α-葡萄糖苷酶溶液作为空白组;以常用的α-葡萄糖苷酶抑制剂阿卡波糖为阳性对照组(control)。以上每组做三个平行试验,并通过测定每组的吸光值并计算抑制率。
抑制率计算如下:
其中:An:为SA12菌株样品在400nm下测定的吸光值;
AB:为空白组的吸光值;
AC:为对照组的吸光值。
结果如图5所示,在阿卡波糖阳性对照组中对α-葡萄糖苷酶的抑制率为100%,而使用SA12菌株对α-葡萄糖苷酶的抑制率达到了42.59%,表明凝结芽孢杆菌SA12菌株可以抑制α-葡萄糖苷酶,从而能够缓解糖尿病,可作为血糖控制的高效菌株。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1. 一株凝结芽孢杆菌(Weizmannia coagulans)SA12菌株,其特征在于,该菌株于2022年6月6日保藏于广东省微生物菌种保藏中心(GDMCC),保藏编号为:GDMCC NO:62518。
2.权利要求1所述凝结芽孢杆菌SA12菌株和/或其发酵液在制备脂多糖降解菌剂中的应用。
3.权利要求1所述凝结芽孢杆菌SA12菌株和/或其发酵液在制备α-葡萄糖苷酶抑制剂中的应用。
4.权利要求1所述凝结芽孢杆菌SA12菌株和/或其发酵液制备清除DPPH自由基的制剂中的应用。
5.权利要求1所述凝结芽孢杆菌SA12菌株和/或其发酵液在制备缓解糖尿病药物中的应用。
6.一种具有降解脂多糖和/或抑制α-葡萄糖苷酶的制剂,其特征在于,含有权利要求1所述凝结芽孢杆菌SA12菌株和/或其发酵液。
7.根据权利要求6所述制剂,其特征在于,所述SA12菌株的添加量为0.1~10%。
8.根据权利要求6所述制剂,其特征在于,所述发酵条件为:30~40℃,10~15h,150~200r/min。
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CN113425747A (zh) * | 2021-07-26 | 2021-09-24 | 吉林省农业科学院 | 凝结芽孢杆菌ja845在制备具有降血糖功能的酶活性抑制剂中的应用 |
CN113444668A (zh) * | 2021-07-26 | 2021-09-28 | 吉林省命之元生物科技有限公司 | 一株降血糖作用的凝结芽孢杆菌及其应用 |
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WO2018023003A1 (en) * | 2016-07-29 | 2018-02-01 | Isothrive Llc | Optimized individualized prebiotic compositions |
CN111094576A (zh) * | 2017-09-13 | 2020-05-01 | 丹尼斯科美国公司 | 用于增加芽胞杆菌属中蛋白质产生的经修饰的5′-非翻译区(utr)序列 |
CN113425747A (zh) * | 2021-07-26 | 2021-09-24 | 吉林省农业科学院 | 凝结芽孢杆菌ja845在制备具有降血糖功能的酶活性抑制剂中的应用 |
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