CN112625968B - 一株缓解内毒素感染的粘膜乳杆菌及应用 - Google Patents
一株缓解内毒素感染的粘膜乳杆菌及应用 Download PDFInfo
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- CN112625968B CN112625968B CN202011623705.0A CN202011623705A CN112625968B CN 112625968 B CN112625968 B CN 112625968B CN 202011623705 A CN202011623705 A CN 202011623705A CN 112625968 B CN112625968 B CN 112625968B
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- endotoxin
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Abstract
本发明公开了一株缓解内毒素感染的粘膜乳杆菌及应用,属于微生物技术领域。本发明通过大量实验筛选获得了一种具有缓解内毒素感染的粘膜乳杆菌CCFM1142,该菌株能够降低内毒素感染宿主血液中促炎因子的含量,增加抑炎因子的浓度,及稳定肠道菌群组成。通过进一步考察菌株在食物和药物组合中的应用,实验表明,含粘膜乳杆菌CCFM1142的食品或药物可用于制备预防和/或治疗内毒素感染,具有广泛的应用前景。
Description
技术领域
本发明涉及一株缓解内毒素感染的粘膜乳杆菌及应用,属于微生物技术领域。
背景技术
内毒素,又称脂多糖,是革兰氏阴性菌细胞壁的重要组成组分。内毒素结构稳定,主要由多糖抗原、核心多糖和类脂A(lipid A)三部分组成。类脂A是脂化的葡萄胺二糖,具有致热作用,是革兰氏阴性细菌内毒素的主要毒性成分。一般情况下,内毒素只有当细菌死亡溶解或用人工方法破坏菌细胞后才释放出来,而内毒素结构稳定,只有长时间的高温处理或以强碱、强酸或强氧化剂辅助高温处理才能破坏其生物活性。
正常机体肠腔内含有大量的肠道细菌,肠粘膜能够选择性地吸收营养成分,并阻止微生物及其有害的代谢物进入。然而,当机体处在应激状态下或过量使用抗生素时,肠道菌群紊乱,释放出内毒素,过量的内毒素能够增加肠道通透性,导致一系列免疫失调,从而引起肠道疾病;而且内毒素通过肠屏障进入血液循环,作用于机体的巨噬细胞、中性粒细胞、内皮细胞、血小板、补体系统和凝血系统等,引起微循环紊乱而导致内毒素休克;内毒素还有加重抑郁症的可能性。已有研究报道,每年至少有75万例患者发生内毒素感染休克,其中超过21万人因此丧命。
目前,针对内毒素感染的治疗原则多主张减少内毒素的产生和吸收及改善内毒素引起的微循环障碍。专利CN102406869A公开了一种采用大黄和芍丹等中药降低内毒素水平的应用方法;有研究发现,多粘菌素能特异性结合内毒素中活性部位类脂A,从而中和或者灭活其毒性(吕根法、卫国等,“多粘菌素B拈抗内毒素的体外作用研究”,《第三军医大学学报》,2004,26,14:1252-1254);CN106334540A、CN1864755等专利发明了用于血液灌流的内毒素吸附剂,应用于清除机体内内毒素。虽然,传统的中药对内毒素具有一定的清除作用,但疗效并不显著;抗生素并不能有效地清除内毒素,反而具有一定的负面影响;血液净化法对内毒素有着良好的清除作用,但费用较高,且临床数据不足。因此,急需安全有效且成本较低的缓解内毒素感染的方法。
发明内容
[技术问题]
本发明要解决的技术问题是提供一株可缓解内毒素感染的粘膜乳杆菌(Lactobacillus mucosae)
[技术方案]
为解决本发明的技术问题,本发明提供了一株粘膜乳杆菌(Lactobacillusmucosae)CCFM1142,所述粘膜乳杆菌CCFM1142已于2020年8月21日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61160,保藏地址为广州市先烈中路100号大院59号楼5楼。
所述粘膜乳杆菌CCFM1142是从来源于湖南麻阳地区的猪粪样本中分离得到的,该菌株经测序分析,将测序得到的序列在NCBI中进行核酸序列比对(扩增得到的16S rDNA的核苷酸序列如SEQ ID NO.1所示),结果显示菌株为粘膜乳杆菌,命名为粘膜乳杆菌(Lactobacillus mucosae)CCFM1142。
所述粘膜乳杆菌CCFM1142在MRS培养基上的菌落呈现乳白色、表面光滑、圆形凸起。
本发明还提供了所述的粘膜乳杆菌CCFM1142在制备预防和/或治疗内毒素感染的产品中的应用。
在一种实施方式中,所述预防和/或治疗内毒素感染包括如下功能:
(1)降低内毒素感染的哺乳动物血清中的LPS含量;
(2)显著改善内毒素感染的哺乳动物结肠的病理损伤;
(3)显著降低内毒素感染的哺乳动物血清中的促炎因子水平;
(4)显著提升内毒素感染的哺乳动物血清中的抗炎因子水平;
(5)显著提升内毒素感染的哺乳动物粪便中的短链脂肪酸含量;
(6)显著降低了Anaeroplasma、Negativibacillus和Eubacterium_xylanophilum_group等肠道微生物的相对丰度。
在一种实施方式中,所述产品中粘膜乳杆菌CCFM1142的活菌数不低于1×106CFU/mL或1×106CFU/g。
在一种实施方式中,所述产品中粘膜乳杆菌CCFM1142的活菌数不低于1×1010CFU/mL或1×1010CFU/g。
在一种实施方式中,所述产品包括功能性食品、保健品或药品。
本发明还提供含有所述粘膜乳杆菌CCFM1142的食品或药物。
在一种实施方式中,所述药物含有上述粘膜乳杆菌CCFM1142、药物载体和/或药用辅料。
本一种实施方式中,所述食品包括但不限于含有上述粘膜乳杆菌CCFM1142的乳制品、豆制品、果蔬制品或胶囊制品;或使用含上述粘膜乳杆菌CCFM1142的发酵剂生产得到的乳制品、豆制品、果蔬制品或胶囊制品。
本发明还提供了含有所述粘膜乳杆菌CCFM1142的发酵剂。
在一种实施方式中,所述发酵剂的制备方法为:将上述粘膜乳杆菌CCFM1142接种到培养基中,于35~37℃下培养至少16h,得到培养液;将培养液离心,收集菌体;将菌体重悬,得到液体发酵剂。
在一种实施方式中,所述方法采用细胞保护剂对菌体进行重悬,再将重悬后的菌悬液冻干处理,获得固体发酵剂。
在一种实施方式中,所述培养基为MRS培养基。
在一种实施方式中,用生理盐水将菌体重悬。
[有益效果]
本发明筛选出了一株粘膜乳杆菌(Lactobacillus mucosae)CCFM1142,此粘膜乳杆菌(Lactobacillus mucosae)CCFM1142具有缓解内毒素感染的作用,具体体现在:
(1)显著降低内毒素感染小鼠血清中的LPS含量;
(2)显著改善内毒素感染小鼠结肠的病理损伤;
(3)显著降低内毒素感染小鼠血清中的促炎因子水平;
(4)显著提升内毒素感染小鼠血清中的抗炎因子水平;
(5)显著提升内毒素感染小鼠粪便中的短链脂肪酸含量
(6)显著降低了Anaeroplasma、Negativibacillus和Eubacterium_xylanophilum_group等肠道微生物的相对丰度。
因此,粘膜乳杆菌CCFM1142在制备预防和/或治疗腹泻的产品(如食品或药品等)中,具有巨大的应用前景。
生物材料保藏
一株粘膜乳杆菌(Lactobacillus mucosae)CCFM1142,分类学命名为Lactobacillus mucosae,已于2020年8月21日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61160,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1:小鼠血清中的内毒素含量;
图2:小鼠结肠的病理损伤情况;
图3:小鼠血清中的炎症因子水平;
图4:小鼠粪便中的短链脂肪酸含量;
图5:小鼠粪便中Anaeroplasma的水平;
图6:小鼠粪便中Negativibacillus的水平;
图7:小鼠粪便中Eubacterium_xylanophilum_group的水平。
具体实施方式
实施例1:粘膜乳杆菌CCFM1142的筛选、鉴定、培养和保存
1、筛选
取1g来源于湖南怀化市麻阳苗族自治县的健康人粪便样本,梯度稀释后涂布于MRS固体培养基中,置于37℃厌氧环境中培养72h,观察并记录菌落形态;挑取表面湿润、有凸起的、白色泛黄的菌落在MRS固体培养基上划线,于37℃厌氧的条件下进行纯化培养,重复此操作3次,获得纯化后的单菌落;挑取单菌落在MRS固体培养基上划线,37℃厌氧培养36h,对所得菌落进行革兰氏染色,记录菌落的形态,并根据《常见细菌系统鉴定手册》考察菌株的生理生化特性,保留革兰氏阴性、菌落呈凸起且白色泛黄状、过氧化氢酶阴性的菌株。
2、鉴定
提取筛选得到的菌株的基因组,将菌株的16S rDNA进行扩增和测序(扩增得到的16S rDNA的核苷酸序列如SEQ ID NO.1所示),将获得的序列在NCBI-Blast中进行核酸序列比对,结果显示菌株为粘膜乳杆菌,命名为粘膜乳杆菌(Lactobacillus mucosae)CCFM1142;
其中,16S rDNA扩增所用引物如下:
27F:5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO.2);
1492R:5’-TACGGCTACCTTGTTACGACTT-3’(SEQ ID NO.3);
16S rDNA扩增程序如下:
95℃ 5min;35个循环(95℃ 30s;55℃ 30s;72℃ 2min);72℃ 10min。
表1菌株的生理生化特性
实验项目 | 结果 | 实验项目 | 结果 |
过氧化氢酶实验 | - | 甘露糖 | + |
接触酶实验 | - | 海藻糖 | - |
葡萄糖 | + | 棉子糖 | + |
果糖 | + | 麦芽糖 | + |
蔗糖 | + | 松三糖 | + |
注:“-”表示阴性,“+”表示阳性。
3、培养
挑取粘膜乳杆菌CCFM1142的单菌落接入MRS固体培养基上,37℃培养48h后,观察粘膜乳杆菌CCFM1142在MRS固体培养基上的菌落特征。观察可得,粘膜乳杆菌CCFM1142在MRS固体培养基上的菌落突起,呈光滑、圆形、乳白色、半透明状,直径为1~2mm。
4、保存
挑取粘膜乳杆菌CCFM1142的单菌落接入MRS液体培养基中,于37℃厌氧的条件下培养24h,得到菌液;将菌液置于离心管中3000rpm离心10min收集菌体;在菌体中加入灭菌后的PBS缓冲溶液后置于离心管中3000rpm离心10min进行洗涤,得到洗涤后的菌体,重复此操作3次,在所得菌体中加入灭菌后的30%(v/v)甘油后-80℃保存于甘油管中。
实施例2:粘膜乳杆菌CCFM1142菌液的制备
(1)从甘油管中蘸取粘膜乳杆菌CCFM1142的菌液在MRS固体培养基上划线,厌氧环境下37℃培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,厌氧环境下37℃培养48h进行活化培养,重复此操作3次,得到活化后的菌液。
(2)将步骤(1)得到的活化后的菌液按照按2%(v/v)的接种量接种至MRS液体培养基中,37℃培养24h后得到发酵液,将发酵液离心收集菌体,用生理盐水重悬菌体,并调整活菌数5×109CFU/mL,制成菌悬液。
实施例3:粘膜乳杆菌CCFM1142对LPS感染后小鼠血清LPS的影响
56只6周龄、18~20g的C57BL/6J雄性小鼠,经过一周的适应期之后,随机分成4组:对照组,LPS造模组,CCFM1142组和FZJTZ26M3组,每组8只。其中,对照组每天腹腔注射0.2mL生理盐水并灌胃0.2mL保护剂(保护剂的制备方法为:将130g/L的脱脂乳,105℃高压灭菌10min;将20g/L的蔗糖溶液与20g/L的海藻糖溶液在115℃高压灭菌20min;将三种溶液以1:1:1的体积比混合即为保护剂);LPS造模组每天腹腔注射0.2mL的LPS溶液(LPS溶于生理盐水制成终浓度为0.15mg/kg的LPS溶液)并灌胃0.2mL保护剂;CCFM1142组和FZJTZ26M3组每天腹腔注射0.2mL的LPS溶液,并分别灌胃0.2mL粘膜乳杆菌CCFM1142和粘膜乳杆菌FZJTZ26M3(菌液浓度:5×109CFU/mL)的菌悬液,其中,粘膜乳杆菌FZJTZ26M3分离自浙江台州健康人粪便,其16S rDNA序列如SEQ ID NO.4所示。保护剂和菌悬液均在腹腔注射1小时后灌胃。实验进行5天后处死所有小鼠,取血后采用ELISA试剂盒(上海酶联生物科技有限公司)测定LPS的含量。
实验结果如图所示,经过连续5天的腹腔注射LPS,小鼠血液中的LPS含量明显增高。通过粘膜乳杆菌CCFM1142干预显著降低了小鼠血清中的LPS含量,降低了47.60%;粘膜乳杆菌FZJTZ26M3干预后降低了27.82%。
实施例4:粘膜乳杆菌CCFM1142对LPS感染后小鼠的结肠形态的影响
56只6周龄、18~20g的C57BL/6J雄性小鼠,经过一周的适应期之后,随机分成4组:对照组,LPS造模组,CCFM1142组和FZJTZ26M3组,每组8只。其中,对照组每天腹腔注射0.2mL生理盐水并灌胃0.2mL保护剂(保护剂的制备方法为:将130g/L的脱脂乳,105℃高压灭菌10min;将20g/L的蔗糖溶液与20g/L的海藻糖溶液在115℃高压灭菌20min;将三种溶液以1:1:1的体积比混合即为保护剂);LPS造模组每天腹腔注射0.2mL的LPS溶液(LPS溶于生理盐水制成0.15mg/kg的LPS溶液)并灌胃0.2mL保护剂;CCFM1142组和FZJTZ26M3组每天腹腔注射0.2mL的LPS溶液,并分别灌胃0.2mL粘膜乳杆菌CCFM1142和粘膜乳杆菌FZJTZ26M3(活菌数:5×109CFU/mL)的菌悬液,其中,粘膜乳杆菌FZJTZ26M3分离自浙江台州健康人粪便(16S序列)。保护剂和菌悬液均在腹腔注射1小时后灌胃。实验进行5天后处死所有小鼠,取出结肠,经多聚甲醛固定后,参照已有研究方法,制作结肠病理切片,通过扫片仪器进行扫描并保存图片。采用Dieleman的评分系统对各组结肠组织切片进行组织损伤评分,组织损伤评分包括炎症程度、病变深度、隐窝破坏和病变范围四个方面。
通过观察结肠病理切片,造模组的小鼠结肠粘膜比较完整、轮廓清晰,肠道上皮连续,肠上皮细胞排列规整,但隐窝结构局部变形,黏膜固有层肿胀且出现炎症细胞浸润及淋巴滤泡。进一步对切片进行病理评分,结果显示,对照组为2.25±1.2583,造模组为7.5±1.2909,说明造模组确有炎症。灌胃粘膜乳杆菌CCFM1142显著降低了LPS对肠上皮的破坏,炎性细胞减少,隐窝结构增加,且在粘膜乳杆菌CCFM1142组中观察到大量杯状细胞的存在,说明它能够降低肠道的通透性,最大程度的恢复肠道结构的完整度。
实施例5:粘膜乳杆菌CCFM1142对LPS感染后小鼠的血清中炎症相关细胞因子的影响
56只6周龄、18~20g的C57BL/6J雄性小鼠,经过一周的适应期之后,随机分成4组:对照组,LPS造模组,CCFM1142组和FZJTZ26M3组,每组8只。其中,对照组每天腹腔注射0.2mL生理盐水并灌胃0.2mL保护剂(保护剂的制备方法为:将130g/L的脱脂乳,105℃高压灭菌10min;将20g/L的蔗糖溶液与20g/L的海藻糖溶液在115℃高压灭菌20min;将三种溶液以1:1:1的体积比混合即为保护剂);LPS造模组每天腹腔注射0.2mL的LPS溶液(LPS溶于生理盐水制成0.15mg/kg的LPS溶液)并灌胃0.2mL保护剂;CCFM1142组和FZJTZ26M3组每天腹腔注射0.2mL的LPS溶液,并分别灌胃0.2mL粘膜乳杆菌CCFM1142和粘膜乳杆菌FZJTZ26M3(活菌数:5×109CFU/mL)的菌悬液,其中,粘膜乳杆菌FZJTZ26M3分离自浙江台州健康人粪便。保护剂和菌悬液均在腹腔注射1小时后灌胃。实验进行5天后处死所有小鼠,取血后采用ELISA试剂盒(上海酶联生物科技有限公司)测定TNF-α、IFN-γ、IL-6和IL-10的含量。
实验结果如图所示,与空白组相比,LPS的注射使造模组TNF-α、IFN-γ、IL-6和IL-10四种炎症相关的细胞因子水平显著提高。粘膜乳杆菌CCFM1142干预后,促炎因子TNF-α、IFN-γ和IL-6的含量均显著降低(43.97%、30.93%和34.24%),抑炎因子IL-10的含量则显著升高(19.22%)。
实施例6:粘膜乳杆菌CCFM1142对LPS感染后小鼠的肠道短链脂肪酸含量的影响
56只6周龄、18~20g的C57BL/6J雄性小鼠,经过一周的适应期之后,随机分成4组:对照组,LPS造模组,CCFM1142组和FZJTZ26M3组,每组8只。其中,对照组每天腹腔注射0.2mL生理盐水并灌胃0.2mL保护剂(保护剂的制备方法为:将130g/L的脱脂乳,105℃高压灭菌10min;将20g/L的蔗糖溶液与20g/L的海藻糖溶液在115℃高压灭菌20min;将三种溶液以1:1:1的体积比混合即为保护剂);LPS造模组每天腹腔注射0.2mL的LPS溶液(LPS溶于生理盐水制成0.15mg/kg的LPS溶液)并灌胃0.2mL保护剂;CCFM1142组和FZJTZ26M3组每天腹腔注射0.2mL的LPS溶液,并分别灌胃0.2mL粘膜乳杆菌CCFM1142和粘膜乳杆菌FZJTZ26M3(活菌数:5×109CFU/mL)的菌悬液,其中,粘膜乳杆菌FZJTZ26M3分离自浙江台州健康人粪便。保护剂和菌悬液均在腹腔注射1小时后灌胃。实验进行5天后处死所有小鼠。在处死小鼠之前收集每只小鼠的粪便。
参照已有研究方法,测定小鼠粪便中短链脂肪酸的含量。操作步骤具体如下:称取50mg粪便样品放置于2ml离心管中,加入500μL饱和氯化钠溶液,震荡至无明显块状物;均质后,加入40μL的硫酸溶液(0.5ml浓硫酸与5ml水混合)酸化,漩涡震荡仪震荡30s,混匀;在通风橱中加入1000μL乙醚用以萃取短链脂肪酸,漩涡震荡仪震荡30s;4℃条件下12000×g离心15min,取上层乙醚相,加入到装有0.25g无水硫酸钠的2ml离心管中,静置15min,再次以同样的条件离心,目的是除水。离心后取上清加入气相小瓶,上机分析。采用GC-MS分析乙酸、丙酸、异丁酸、丁酸、异戊酸及戊酸等短链脂肪酸的含量。
GC-MS采用Rtx-Wax柱,柱长30m,内径0.25μm;载气为氦气,流速2mL/min;进样体积1μL,分流比为10:1;进样温度为240℃,升温程序为:起始温度100℃,以7.5℃/min的升温速率升至140℃,再以60℃/min的升温速率升至200℃,在200℃下保持3min,离子化温度为220℃;分析方法为全扫描模式(质荷比扫描范围33-110)检测各短链脂肪酸,选取各分析物标准品的特征离子进行定量分析。
连续五天的LPS注射,使得六种短链脂肪酸的含量显著下降;当采用粘膜乳杆菌CCFM1142干预后,粪便中的乙酸、丙酸、异丁酸、丁酸、异戊酸和戊酸含量分别升高了4.31、4.63、2.08、14.14、1.93和1.99倍。粘膜乳杆菌CCFM1142表现出的免疫调节作用可能与其上调短链脂肪酸的含量存在一定的联系。
实施例7:粘膜乳杆菌CCFM1142对LPS感染后小鼠的肠道菌群的影响
56只6周龄、18~20g的C57BL/6J雄性小鼠,经过一周的适应期之后,随机分成4组:对照组,LPS造模组,CCFM1142组和FZJTZ26M3组,每组8只。其中,对照组每天腹腔注射0.2mL生理盐水并灌胃0.2mL保护剂(保护剂的制备方法为:将130g/L的脱脂乳,105℃高压灭菌10min;将20g/L的蔗糖溶液与20g/L的海藻糖溶液在115℃高压灭菌20min;将三种溶液以1:1:1的体积比混合即为保护剂);LPS造模组每天腹腔注射0.2mL的LPS溶液(LPS溶于生理盐水制成0.15mg/kg的LPS溶液)并灌胃0.2mL保护剂;CCFM1142组和FZJTZ26M3组每天腹腔注射0.2mL的LPS溶液,并分别灌胃0.2mL粘膜乳杆菌CCFM1142和粘膜乳杆菌FZJTZ26M3(活菌数:5×109CFU/mL)的菌悬液,其中,粘膜乳杆菌FZJTZ26M3分离自浙江台州健康人粪便。保护剂和菌悬液均在腹腔注射1小时后灌胃。实验进行5天后处死所有小鼠。在处死小鼠之前收集每只小鼠的粪便。
使用粪便DNA提取试剂盒对小鼠粪便中的细菌基因组进行提取,对提取得到的基因组DNA的V3-V4区进行特异性PCR扩增(上游引物341F:CCTAYGGGRBGCASCAG;下游引物806R:GGACTACNNGGGTATCTAAT),16S rDNA测序,分析肠道菌群变化,分析结果见图4。
由图5~7可知,与对照组小鼠相比,造模组小鼠肠道的Anaeroplasma、Negativibacillus和Eubacterium_xylanophilum_group丰度分别升高了2.96、4.17和3.25倍;与造模组小鼠相比,CCFM1142组小鼠肠道的Anaeroplasma、Negativibacillus和Eubacterium_xylanophilum_group丰度显著降低。
可见,粘膜乳杆菌CCFM1142可有效改善内毒素感染小鼠肠道中某些菌属的丰度。
实施例8:含粘膜乳杆菌CCFM1142的发酵剂的制备
MRS培养基:胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、醋酸钠5g、柠檬酸氢二铵2g、磷酸氢二钾2g、七水硫酸镁0.5g、吐温80 1ml、一水合硫酸锰0.25g,水定容至1000ml,调节pH至6.5,在119-123℃条件下灭菌15-25min。
保护剂:100g/L-150g/L脱脂奶粉、100g/L-150g/L麦芽糊精、140g/L-160g/L海藻糖。
将粘膜乳杆菌CCFM1142接种到MRS培养基中,37℃厌氧条件下培养18-20h,收集菌体,用保护剂重悬菌体细胞,使菌浓度达到1010CFU/mL,然后悬浮液在37℃厌氧条件下培养50-70min,获得液态发酵剂。
可选地,对制备获得的液态发酵剂干燥处理,获得固态发酵剂;所述干燥是在-15~-20℃预冻8-14h后进行真空冷冻干燥。
实施例9:粘膜乳杆菌CCFM1142制备益生菌乳饮料
将原料乳脱脂奶在95℃热杀菌20min,然后冷却至4℃,再加入实施例1筛选的粘膜乳杆菌CCFM1142或实施例8制备的发酵剂,使菌体浓度达到106CFU/mL以上,在4℃冷藏保存即得到含粘膜乳杆菌CCFM1142活菌的乳饮料。
实施例10:粘膜乳杆菌CCFM1142制备豆奶
采用软水浸泡大豆,水量为原大豆量三倍体积,在温度80℃下浸泡1~2h,再去除大豆皮。接着,沥去浸泡水,另加沸水磨浆,并在温度高于80℃的条件下保温10~15min。浆体用150目滤膜过滤后接着进行离心,得到的离心液即为粗豆奶,再将它加热到温度140~150℃,然后将热的粗豆奶迅速导入真空冷却室进行抽真空,所述粗豆奶中的异味物质随着水蒸汽迅速排出。经过真空脱气后,将其温度降至37℃左右,再接入实施例1筛选的粘膜乳杆菌CCFM1142或实施例8制备的发酵剂,使粘膜乳杆菌CCFM1142浓度达到106CFU/mL以上,在4℃冷藏保存即得到含粘膜乳杆菌CCFM1142活菌的豆奶。
实施例11:粘膜乳杆菌CCFM1142制造果蔬饮料
选用新鲜蔬菜,例如黄瓜、胡萝卜、甜菜、芹菜或圆白菜中的一种或多种,洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2s后,立即降温到37℃左右,再接入本发明的粘膜乳杆菌CCFM1142发酵剂,使其浓度达到106CFU/mL以上,在4℃冷藏保存即得到含粘膜乳杆菌CCFM1142活菌的果蔬饮料。
实施例12:粘膜乳杆菌CCFM1142制备药物
本发明的粘膜乳杆菌CCFM1142在MRS培养基上培养24h,在温度4℃与4000r/min的条件下离心20min,用PBS冲洗两次,再加入以最后得到含粘膜乳杆菌CCFM1142的粉剂重量计4%脱脂奶粉和6%乳糖混合10min,再加入无菌2%氯化钙和3%海藻酸钠,同时以150r/min搅拌10min,再静止固化30min,最后清洗过滤,得到的滤液进行冷冻干燥20h,得到含粘膜乳杆菌CCFM1142的粉剂,把这种粉剂装入目前市场上销售的药用微胶囊,得到所述的胶囊制品。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一株缓解内毒素感染的粘膜乳杆菌及应用
<130> BAA201500A
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1409
<212> DNA
<213> Lactobacillus mucoase
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ggggataaca tttggaaaca gatgctaata ccgcataaca atttgaatcg catgattcaa 60
atttaaaaga tggcttcggc tatcactttg ggatggacct gcggcgcatt agcttgttgg 120
tagggtaacg gcctaccaag gctgtgatgc gtagccgagt tgagagactg atcggccaca 180
atggaactga gacacggtcc atactcctac gggaggcagc agtagggaat cttccacaat 240
gggcgcaagc ctgatggagc aacaccgcgt gagtgaagaa gggtttcggc tcgtaaagct 300
ctgttgttag agaagaacgt gcgtgagagc aactgttcac gcagtgacgg tatctaacca 360
gaaagtcacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc 420
cggatttatt gggcgtaaag cgagcgcagg cggtttgata agtctgatgt gaaagccttt 480
ggcttaacca aagaagtgca tcggaaactg tcagacttga gtgcagaaga ggacagtgga 540
actccatgtg tagcggtgga atgcgtagat atatggaaga acaccagtgg cgaaggcggc 600
tgtctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac 660
cctggtagtc catgccgtaa acgatgagtg ctaggtgttg gagggtttcc gcccttcagt 720
gccgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa 780
aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agctacgcga 840
agaaccttac caggtcttga catcttgcgc caaccctaga gatagggcgt ttccttcggg 900
aacgcaatga caggtggtgc atggtcgtcg tcagctcgtg tcgtgagatg ttgggttaag 960
tcccgcaacg agcgcaaccc ttgttactag ttgccagcat tcagttgggc actctagtga 1020
gactgccggt gacaaaccgg aggaaggtgg ggacgacgtc agatcatcat gccccttatg 1080
acctgggcta cacacgtgct acaatggacg gtacaacgag tcgcgaactc gcgagggcaa 1140
gctaatctct taaaaccgtt ctcagttcgg actgcaggct gcaactcgcc tgcacgaagt 1200
cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1260
acaccgcccg tcacaccatg agagtttgca acacccaaag tcggtggggt aacccttcgg 1320
ggagctagcc gcctaaggtg gggcagatga ttagggtgaa gtcgtaacaa ggtagccgta 1380
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tacggctacc ttgttacgac tt 22
<210> 4
<211> 1464
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<213> Lactobacillus mucosae
<400> 4
gatccctctc tgcaccttag gcggctagct ccccgaaggg ttaccccacc gactttgggt 60
gttgcaaact ctcatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgc 120
ggcatgctga tccgcgatta ctagcgattc cgacttcgtg caggcgagtt gcagcctgca 180
gtccgaactg agaacggttt taagagatta gcttgccctc gcgagttcgc gactcgttgt 240
accgtccatt gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ctgacgtcgt 300
ccccaccttc ctccggtttg tcaccggcag tctcactaga gtgcccaact gaatgctggc 360
aactagtaac aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 420
gacgacgacc atgcaccacc tgtcattgcg ttcccgaagg aaacgcccta tctctagggt 480
tggcgcaaga tgtcaagacc tggtaaggtt cttcgcgtag cttcgaatta aaccacatgc 540
tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt caaccttgcg gtcgtactcc 600
ccaggcggag tgcttaatgc gttagctgcg gcactgaagg gcggaaaccc tccaacacct 660
agcactcatc gtttacggca tggactacca gggtatctaa tcctgttcgc tacccatgct 720
ttcgagcctc agcgtcagtt gcagaccaga cagccgcctt cgccactggt gttcttccat 780
atatctacgc attccaccgc tacacatgga gttccactgt cctcttctgc actcaagtct 840
gacagtttcc gatgcacttc tttggttaag ccaaaggctt tcacatcaga cttatcaaac 900
cgcctgcgct cgctttacgc ccaataaatc cggataacgc ttgccaccta cgtattaccg 960
cggctgctgg cacgtagtta gccgtgactt tctggttaga taccgtcact gcgtgaacag 1020
ttgctctcac gcacgttctt ctctaacaac agagctttac gagccgaaac ccttcttcac 1080
tcacgcggtg ttgctccatc aggcttgcgc ccattgtgga agattcccta ctgctgcctc 1140
ccgtaggagt atggaccgtg tctcagttcc attgtggccg atcagtctct caactcggct 1200
acgcatcaca gccttggtag gccgttaccc taccaacaag ctaatgcgcc gcaggtccat 1260
cccaaagtga tagccgaagc catcttttaa atttgaatca tgcgattcaa attgttatgc 1320
ggtattagca tctgtttcca aatgttatcc cccgctttgg ggcaggttac ctacgtgtta 1380
ctcacccgtc cgccactcgc tggtaaacca acgtcaagtc cgtgcaagca cgtccaatca 1440
gttgggccaa cgcgttcgac tgca 1464
<210> 5
<211> 17
<212> DNA
<213> 人工序列
<400> 5
cctaygggrb gcascag 17
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 6
ggactacnng ggtatctaat 20
Claims (9)
1.一株粘膜乳杆菌(Lactobacillus mucoase)CCFM1142,已于2020年8月21日保藏在广东微生物菌种保藏中心,保藏编号为 GDMCC No. 61160。
2.含有权利要求1所述粘膜乳杆菌CCFM1142的微生物制剂。
3.权利要求1所述的粘膜乳杆菌CCFM1142在制备缓解内毒素感染的产品中的应用。
4.根据权利要求3所述的应用,其特征在于,所述产品具有如下至少一种功能:
(1)降低内毒素感染的哺乳动物血清中的LPS含量;
(2)改善内毒素感染的哺乳动物结肠的病理损伤;
(3)降低内毒素感染的哺乳动物血清中的促炎因子水平;
(4)提升内毒素感染的哺乳动物血清中的抗炎因子水平;
(5)提升内毒素感染的哺乳动物粪便中的短链脂肪酸含量;
(6)降低内毒素感染的哺乳动物Anaeroplasma、Negativibacillus和Eubacterium_xylanophilum_group中至少一种肠道微生物的相对丰度。
5.根据权利要求3所述的应用,其特征在于,所述产品为食品、药品或保健品。
6.根据权利要求4或5所述的应用,其特征在于,所述产品中粘膜乳杆菌的活菌数不低于1×106CFU/g。
7.含有权利要求1所述的粘膜乳杆菌CCFM1142的药物,其特征在于,还含有药物载体和/或药用辅料。
8.含有权利要求1所述的粘膜乳杆菌CCFM1142的食品。
9.根据权利要求8所述的食品,其特征在于,所述食品为含有权利要求1所述的粘膜乳杆菌CCFM1142的乳制品、豆制品或果蔬制品。
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