CN116058469A - 一种发酵粘液乳杆菌iob802发酵后生元粉的制备方法及其应用 - Google Patents

一种发酵粘液乳杆菌iob802发酵后生元粉的制备方法及其应用 Download PDF

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CN116058469A
CN116058469A CN202310199983.5A CN202310199983A CN116058469A CN 116058469 A CN116058469 A CN 116058469A CN 202310199983 A CN202310199983 A CN 202310199983A CN 116058469 A CN116058469 A CN 116058469A
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fermented
iob802
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lactobacillus
mucilaginosus
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梁武
付田民
王海宽
韩雪梅
刘成琪
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Tianjin Inncorigin Biological Technology Co ltd
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Abstract

本发明提出一种发酵粘液乳杆菌IOB802发酵后生元粉的制备方法及其应用,发酵粘液乳杆菌IOB802发酵后生元粉能增加小鼠成骨细胞活性和骨更新率、促进小鼠骨骼生长,增强骨骼强度,同时可以改善小鼠免疫力的相关指标,为改善机体免疫力、增强体质提供了一定的药理学依据,同时为开发发酵粘液乳杆菌IOB802发酵后生元粉的新产品提供了思路。

Description

一种发酵粘液乳杆菌IOB802发酵后生元粉的制备方法及其应用
技术领域
本发明涉及微生物技术领域,尤其是一种发酵粘液乳杆菌IOB802发酵后生元粉及其在儿童青少年骨骼增长方面的应用。
背景技术
身高的发育状况是判断儿童身体是否健康的一个标准,其影响着儿童成长过程及成年后的方方面面。因此,在儿童生长发育的关键时期,增强体质、调节免疫、改善骨骼生长发育状况是家长十分关心的问题,也是全社会重点关注的焦点;此外,长久以来骨骼伤由于其病程长、恢复周期长而给患者带来较大的痛苦折磨,而传统药物在长期服用过程中带来了较大的代谢压力和身体损伤,因此亟需对于骨骼生产促进、生病恢复,且对身体代谢压力较小的产品。
领域内公知,骨骼生长需要多种营养的配合支持,有研究表明,肠道微生物可以影响宿主的生长发育,通过诱导胰岛素生长因子(IGF-1)促进骨骼的生长和重塑,目前IGF-1已被证明是一种对骨骼生长有作用的激素,随着肠道有益菌定殖的增加,血清中IGF-1的含量显著增高,另外,微生物代谢产物短链脂肪酸(SCFA)可诱导IGF-1,从而影响青少年以及成人骨伤患者的骨骼生长发育和健康。然而,领域内缺少优质的食品和药品,能够具备菌群调节和促进儿童骨骼发育的多重功效。
因此,开发出一种既能促进儿童骨骼和骨伤患者的骨骼生长发育,又能调节肠道菌群,增强体质的益生菌后生元粉,对社会具有重要意义。
发明内容
发酵粘液乳杆菌( Limosilactobacillus fermentum)IOB802,菌株已于2021年8月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,其保藏编号为CGMCC No.23120。
第一方面,本发明提出一种发酵粘液乳杆菌IOB802后生元粉的制备方法,具体步骤为:
(1)菌种活化;
(2)发酵参数的确定和发酵完成;
(3)将发酵物进行烘干,烘干水分含量控制在≤10%,得到发酵粘液乳杆菌IOB802发酵后生元粉;
(4)后生元粉活性成分测定;
(5)后生元粉功能验证。
进一步地,所述步骤(1)中,菌种活化操作中斜面种子的培养时间为14-28h,活化种子的种龄为0-24h。
进一步地,所述步骤(1)中,菌种活化操作中斜面种子的培养时间为26h,活化种子的种龄为24h。
进一步地,所述步骤(2)中,发酵原料可以为大豆粉或胚芽米粉。
进一步地,所述步骤(2)中,发酵原料为大豆粉时的发酵条件参数为:料水比为1:1-1:2,接种量为1×106-5×107CFU/mL,培养时间为24-40h。
进一步地,所述步骤(2)中,发酵原料为大豆粉时的发酵条件参数为:料水比为1:2,接种量为5×107CFU/mL,培养时间为32h。
进一步地,所述步骤(2)中,发酵原料为胚芽米粉时的发酵条件参数为:料水比为1:0.5-1.1.25,接种量为1×106-5×107CFU/mL,培养时间为24-36h。
进一步地,所述步骤(2)中,发酵原料为胚芽米粉时的发酵条件参数为:料水比为1:0.75,接种量为5×107CFU/mL,培养时间为28h。
进一步地,所述步骤(4)中,具体检测方法苯酚-硫酸法检测多糖。
进一步地,所述步骤(5)中,具体的实验步骤为:
(a)取1月龄、体重25±2g的SPF级昆明雄鼠若干只,随机分成4组:空白组、实验1组、实验2组、实验3组,每组10只;
(b)鼠房控制温度(22 ± 2) ℃,相对湿度40% -60% ,照明时间12 h,分笼饲养,自由饮水,定期清洗鼠笼,适应5天后开始实验,5天后空白组、实验组分别对小鼠进行灌胃,每日一次,持续灌胃4周;
(c)空白组灌胃生理盐水,实验1组灌胃发酵粘液乳杆菌IOB802发酵大豆粉后生元粉,实验2组灌胃发酵粘液乳杆菌IOB802发酵胚芽米粉后生元粉;
(d)4周后对小鼠进行相关指标检测。
进一步地,所述步骤(4)中,所述相关指标为:小鼠的体重、毛色、粪便、关节、体长、骨密度情况,小鼠血清胰岛素生长因子(IGF-1)水平、小鼠骨钙素(OCN)水平。
第二方面,本发明提供一种根据上述发酵粘液乳杆菌IOB802发酵后生元粉的制备方法所得到的发酵粘液乳杆菌IOB802发酵后生元粉。
第三发面,本发明提供一种发酵粘液乳杆菌IOB802发酵后生元粉在骨骼增长相关药物制备中的应用。
有益效果:
(1)本发明通过实验得到发酵粘液乳杆菌IOB802菌种活化的最佳参数,得到其发酵大豆粉和胚芽米的分别最佳发酵参数,为本领域内发酵粘液乳杆菌IOB802菌种的潜在功能研究提供了创造性的发酵条件控制思路和方法。
(2)本发明所得发酵粘液乳杆菌IOB802发酵后生元粉,经动物实验验证,显著促进了细胞因子IGF-1表达水平上升,促进骨骼的生长和重塑,使血清中OCN表达水平上升,促进成骨细胞活性和骨更新率增加,进而促进骨骼的生长,增加骨密度,增强骨骼强度,因此,该发酵所得后生元粉具有微生物本身带来的营养消化吸收促进以及骨骼生长发育、再生增强的双重功效,有望成为不带来身体负担的骨骼促生长优质产品,极具市场机制。
附图说明
图1:本发明的种子生长曲线;
图2:本发明的小鼠的精神状态;
图3:本发明的不同组别小鼠体重变化;
图4:本发明的不同组别小鼠血清IGF-1含量变化;
图5:本发明的不同组别小鼠血清OCN含量变化;
图6:本发明的小鼠股骨重构照片;
图7:本发明的不同组别小鼠股骨长度和骨重变化;
图8:本发明的不同组别小鼠骨密度变化。
发酵粘液乳杆菌IOB802分类命名为发酵粘液乳杆菌( Limosilactobacillusfermentum),已于2021年8月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,其保藏编号为CGMCC No.23120。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:发酵粘液乳杆菌IOB802菌粉的制备
发酵粘液乳杆菌IOB802菌粉的制备方法如下: (1)菌株活化:将菌株按1:20接种于活化培养基中,37±2℃,静置,密闭,培养24h,即 得一级种子液,将一级种子按1:25接种于活化培养基中,37±2℃,静置,密闭,培养24h,即得二级种子液; 一级培养基为蛋白胨10g,酵母浸粉10g,葡萄糖10g,蒸馏水1000ml,将上述组分混合,并调节溶液pH为7.0-7.2,然后121℃灭菌25min。(2)发酵:按照1:50的接种量,将培养好的二级种子液接种到灭好菌的发酵液培养基中,发酵罐搅拌转速100r/min,37℃,密闭培养30h,即得发酵液;发酵液培养基为:蛋白胨10g,酵母浸粉10g,葡萄糖6g,硫酸锰1g ,七水硫酸镁1g,并调节溶液pH为7.0-7.2,然后121℃灭菌25min。(3)菌粉:发酵液培养基离心,收集菌泥;将菌泥冷冻干燥,即可获得发酵粘液乳杆菌IOB802菌粉。
实施例2:发酵粘液乳杆菌IOB802发酵大豆粉后生元粉的制备。
1.种龄的确定
将冻存管中的菌种接种至斜面培养基中,并放置到37℃±2℃培养,观察不同培养时间的菌落形态,菌体特征,结果如表1所示,培养要求种子个体均匀,形态一致,粗壮,培养时间为26h的种龄较符合条件,所以确定种子的最佳培养时间为26h。
表1 不同培养时间种子的生长情况
2.活化培养基种龄的确定
取一环新鲜的斜面菌种接种于装有25mL 2%大豆粉的瓶子中,37±2℃,密闭静置培养,于不同培养时间取样镜检,测定活菌浓度,结果如图1所示,由图所知,培养时间24h,菌体处于对数生长期的后期,此时菌体活力旺盛,浓度较高,故选择活化种子的最佳种龄为24h。
3. 发酵培养基的确定
采用发酵粘液乳杆菌IOB802在固体培养基上能获得的最高活菌数作为菌种生长活力的判断指标。如表2-4所示,最佳料水比为1:2,斜面种子的最佳培养时间为26h,活化种子的最佳种龄为24h,最佳料水比为1:2,最佳的接种量为5×107CFU/mL,最佳培养时间为32h。
表2 料水比的确定
表3 接种量的确定
表4 培养时间的确定
4. 干燥产品
按照最佳的发酵条件进行实验,最终获得发酵样品,将培养后的发酵物进行烘干,烘干温度为55±2℃,烘干水分含量控制在≤10%,即可获得发酵粘液乳杆菌IOB802发酵后生元粉。
实施例:3:发酵粘液乳杆菌IOB802发酵胚芽米粉后生元粉的制备。
考虑到胚芽米料水比太大的话,灭菌后黏度会比较大。所以在料水比上,本发明均衡活菌和料水比、灭菌后黏度三个指标进行衡量。
种龄的确定、活化培养基种龄的确定同实施例1相同。
如表5-7所示,综合选择,1:0.75的料水比最佳。基于1:0.75的料水比,最佳的接种量为5×107CFU/mL,最佳培养时间为28h。
表5 料水比的确定
表6 接种量的确定
表7 培养时间的确定
实施例4:发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌菌粉的成分测定
后生元成分检测,具体检测成分及方法为:多糖的检测方法为SN/T 24260-2015出口植物源食品中粗多糖的测定苯酚-硫酸法,如表8所示,发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉的成分得到鉴定。
表8 营养成分检测结果
实施例5:发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉对骨骼生长发育的改善作用
(1)实验材料:
实验鼠:1月龄SPF级昆明雄鼠,体重25±2g。
实验材料:发酵粘液乳杆菌IOB802发酵大豆粉后生元粉、发酵粘液乳杆菌IOB802发酵胚芽米粉后生元粉、发酵粘液乳杆菌IOB802菌粉。
(2)小鼠建模与分组:取1月龄、体重25±2g的SPF级昆明雄鼠若干只,随机分成4组(每组10只):空白组、实验1组、实验2组、实验3组。空白组灌胃生理盐水,实验1组灌胃发酵粘液乳杆菌IOB802发酵大豆粉后生元粉,实验2组灌胃发酵粘液乳杆菌IOB802发酵胚芽米粉后生元粉,实验3组灌胃发酵粘液乳杆菌IOB802菌粉。鼠房控制温度(22 ± 2) ℃,相对湿度40% ~ 60% ,照明时间 12 h。分笼饲养,自由饮水,定期清洗鼠笼,适应5天后开始实验。5天后空白组、实验组分别对小鼠进行灌胃,每日一次,持续灌胃4周。其中实验组样品现用现配。4周后颈椎脱臼处死小鼠进行相关指标检测。
(3)观察并记录小鼠的体重、毛色、粪便、关节、体长情况,分别记录0天、7天、14天、28天的情况。
小鼠体重的测定。实验开始后,每周同一时间对小鼠体重进行测定。
小鼠血清胰岛素生长因子(IGF-1)和小鼠骨钙素(OCN)水平测定。分别于实验第0、7、14、28天通过采血,测定血清中IGF-1水平和小鼠骨钙素(OCN)水平。
采血结束处死,分离小鼠双侧股骨,左侧股骨密封-20℃保存,右侧股骨在多聚甲醛固定保存待检。分离小鼠股骨,进行以下指标检测:a.X线骨密度仪检测和分析骨密度;b.记录骨长、骨重;
(4)实验结果:
(a)如图2所示,通过观察小鼠的活动和精神情况,空白组、实验组的小鼠活动正常,精神状态良好。表明小鼠食用发酵粘液乳杆菌IOB802发酵大豆粉后生元粉、发酵胚芽米后生元粉和发酵粘液乳杆菌IOB802菌粉后,小鼠精神状态较好。
(b)如图3所示,小鼠体重:实验期间小鼠的死亡率为零,各组小鼠在实验期间均有所增加,且增加趋势一致,并且相同时间,空白组和实验组小鼠体重接近,没有显著差异。
(c)如图4所示,小鼠血清IGF-1含量:与空白组相比,经发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉灌胃小鼠后,血清中IGF-1含量呈现上升趋势,说明实验1组、实验2组、实验3组均能够诱导血清中IGF-1表达水平上升,促进骨骼的生长和重塑,且实验1组和实验2组效果优于实验3组。
(d)如图5所示,小鼠血清OCN含量:与空白组相比,经发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉灌胃小鼠后,血清中OCN含量增加,说明实验1组、实验2组和实验3组均能够使血清中OCN表达水平上升,促进成骨细胞活性和骨更新率增加,进而促进骨骼的生长,且实验1组和实验2组效果优于实验3组。
(e)小鼠股骨长度和骨重测量:如图6和图7所示,与空白组相比,经发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉干预小鼠,小鼠骨长增长的长度比较明显,且骨重也明显增加,说明实验1组、实验2组和实验3组均能够促进骨骼生长发育,增强骨骼强度,且实验1组和实验2组效果优于实验3组。
(g)小鼠骨密度:如图8所示,与空白组相比,小鼠经口服灌胃发酵粘液乳杆菌IOB802发酵后生元粉和发酵粘液乳杆菌IOB802菌粉后,经X线骨密度仪检测,小鼠骨密度明显增加,说明实验1组、实验2组和实验3组均能够增加骨密度,提高骨骼强度,且实验1组和实验2组效果优于实验3组。
综上所述,本实验研究了发酵粘液乳杆菌IOB802发酵后生元粉对小鼠骨骼生长发育的影响。结果表明,发酵粘液乳杆菌IOB802发酵后生元粉能增加小鼠成骨细胞活性和骨更新率、促进小鼠骨骼生长,增强骨骼强度,同时可以改善小鼠免疫力的相关指标,为改善机体免疫力、增强体质提供了一定的药理学依据,同时为开发发酵粘液乳杆菌IOB802发酵后生元粉的新产品提供了思路。
本发明可用其他的不违背本发明的精神或主要特征的具体形式来概述。因此,无论从哪一点来看,本发明的上述实施方案都只能认为是对本发明的说明而不能限制本发明,权利要求书指出了本发明的范围,而上述的说明并未指出本发明的范围,因此,在与本发明的权利要求书相当的含义和范围内的任何改变,都应认为是包括在本发明的权利要求书的范。

Claims (13)

1.一种发酵粘液乳杆菌IOB802后生元粉的制备方法,其特征在于:
具体步骤为:
(1)发酵粘液乳杆菌(Limosilactobacillus fermentum)IOB802,(保藏编号CGMCCNo.23120)的活化;
(2)发酵参数的确定和发酵完成;
(3)将发酵物进行烘干,烘干水分含量控制在≤10%,得到发酵粘液乳杆菌IOB802发酵后生元粉;
(4)后生元粉活性成分测定;
(5)后生元粉功能验证。
2.根据权利要求1所述的制备方法,其特征在于:所述步骤(1)中,菌种活化操作中斜面种子的培养时间为14-28h,活化种子的种龄为0-24h。
3.根据权利要求2所述的制备方法,其特征在于:所述步骤(1)中,菌种活化操作中斜面种子的培养时间为26h,活化种子的种龄为24h。
4.根据权利要求3所述的制备方法,其特征在于:所述步骤(2)中,发酵原料为大豆粉或胚芽米粉。
5.根据权利要求4所述的制备方法,其特征在于:所述步骤(2)中,发酵原料为大豆粉时的发酵条件参数为:料水比为1:1-1:2,接种量为1×106-5×107CFU/mL,培养时间为24-40h。
6.根据权利要求5所述的制备方法,其特征在于:所述步骤(2)中,发酵原料为大豆粉时的发酵条件参数为:料水比为1:2,接种量为5×107CFU/mL,培养时间为32h。
7.根据权利要求4所述的制备方法,其特征在于:所述步骤(2)中,发酵原料为胚芽米粉时的发酵条件参数为:料水比为1:0.5-1.1.25,接种量为1×106-5×107CFU/mL,培养时间为24-36h。
8.根据权利要求7所述的制备方法,其特征在于:所述步骤(2)中,发酵原料为胚芽米粉时的发酵条件参数为:料水比为1:0.75,接种量为5×107CFU/mL,培养时间为28h。
9.根据前述权利要求任一项所述的制备方法,其特征在于:所述步骤(4)中,具体检测方法苯酚-硫酸法检测多糖。
10.根据权利要求9所述的制备方法,其特征在于:所述步骤(5)中,具体的实验步骤为:
(a)取1月龄、体重25±2g的SPF级昆明雄鼠若干只,随机分成4组:空白组、实验1组、实验2组、实验3组,每组10只;
(b)鼠房控制温度(22 ± 2) ℃,相对湿度40% -60% ,照明时间12 h,分笼饲养,自由饮水,定期清洗鼠笼,适应5天后开始实验,5天后空白组、实验组分别对小鼠进行灌胃,每日一次,持续灌胃4周;
(c)空白组灌胃生理盐水,实验1组灌胃发酵粘液乳杆菌IOB802发酵大豆粉后生元粉,实验2组灌胃发酵粘液乳杆菌IOB802发酵胚芽米粉后生元粉,实验3组灌胃发酵粘液乳杆菌IOB802菌粉;
(d)4周后对小鼠进行相关指标检测。
11.根据权利要求10所述的制备方法,所述步骤(4)中,所述相关指标为:小鼠的体重、毛色、粪便、关节、体长、骨密度情况,小鼠血清胰岛素生长因子(IGF-1)水平、小鼠骨钙素(OCN)水平。
12.一种根据权利要求1-11任一项发酵粘液乳杆菌IOB802发酵后生元粉的制备方法所得到的发酵粘液乳杆菌IOB802发酵后生元粉。
13.一种发酵粘液乳杆菌IOB802发酵后生元粉在促进骨骼增长相关药物制备中的应用。
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