CN116410901B - 一种长双歧杆菌冻干粉剂的制备方法及应用 - Google Patents
一种长双歧杆菌冻干粉剂的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及微生物或酶技术领域,具体公开了一种长双歧杆菌冻干粉剂的制备方法及应用。该冻干粉剂是长双歧杆菌经培养后加入冻干保护剂冷冻干燥得到的,发明人制备了一种海藻糖聚合物,海藻糖经聚合后其蛤壳构象经轴向连接形成刚性骨架,能够将益生菌更好地保护起来,并且海藻糖聚合物的稳定性也有所提升,将海藻糖聚合物应用到冻干保护剂以制备长双歧杆菌冻干粉剂,能够提升菌体稳定性,延长活性。
Description
技术领域
本发明涉及微生物或酶技术领域,具体涉及一种长双歧杆菌冻干粉剂的制备方法及应用。
背景技术
结直肠癌(colorectal cancer,CRC)是一种常见的消化道恶性肿瘤多发于乙状结肠和直肠的交界处,即所谓的大肠癌。目前,针对CRC治疗效果最好、研究较多的是外科和放疗的综合治疗,包括术前放疗、术中放疗、术后放疗、“三明治”式放疗。化学治疗为主的化疗药物包括:伊利替康、5-氟尿嘧啶、卡培他滨、奥沙利铂等多种药物。然而化疗最常见的副作用就是胃肠道反应,表现为严重腹泻、恶心、呕吐、腹痛等。针对腹泻这一并发症,通常是由于化疗后肠道内菌群紊乱和肠粘膜损伤导致的,肠道粘膜屏障是机体内外联系的重要界面,若在各种病理因素作用下,肠粘膜受损,则引起肠内外多种炎性失调,尤其是溃疡性结肠炎和感染性结肠炎。此外,化疗在增加肠道炎症的同时,不仅削弱屏障功能,还伴随着肠道菌群组成改变,是菌群多样性下降、抗炎细菌减少,可经脑肠轴影响大脑炎症和功能。
有研究表明,作为天然益生菌的长双歧杆菌同益生元、短链脂肪酸、多不饱和脂肪酸和酚类等可调节肠道菌群,促进肠道内稳态,恢复肠道内菌群的多样性。长双歧杆菌可能的作用机制包括与肠粘膜上皮细胞紧密结合,提高黏膜防御能力,促进上皮细胞分泌粘液使其形成黏液层,形成保护层阻止细菌的侵入和栖生菌易位,增强吞噬细胞对病原菌的吞噬以及非T细胞依赖性IgA的分泌等方面,从而减少肠道炎症反应。此外,长双歧杆菌还可以分解乳糖、纤维素等食物成分,产生有益物质如乳酸和醋酸,促进肠道健康。
中国专利申请201910504466.8公开了一种影响伊立替康所致小鼠腹泻和肠道菌群的富硒益生菌,建立伊立替康所致腹泻小鼠模型,通过长期灌胃给予富硒益生菌,评价其对伊立替康所致小鼠腹泻及肠道菌群的影响。实验组小鼠每天灌胃给予富硒益生菌,测定小鼠体重、腹泻等级、小鼠免疫指标、肠道菌群以及回肠组织结构。结果表明,富硒益生菌能够抑制伊立替康所致的体重下降和免疫功能降低;降低小鼠腹泻发生率和平均腹泻等级,缓解伊立替康所致的小鼠腹泻;减少与β-D-葡糖醛酸糖苷酶相关的梭菌、大肠杆菌和拟杆菌含量,增加肠道内双歧杆菌含量;缓解伊立替康导致的小鼠肠道粘膜、细胞和腺体的损伤。
中国专利202010622073.X公开了一种可提高双歧杆菌耐酸性的冻干保护剂及其应用,属于微生物技术领域。该发明提供了一种可显著提高双歧杆菌冻干存活率和耐酸性的冻干保护剂,此冻干保护剂的成分包含成分A和成分B,其中,成分A为海藻酸钠、酪蛋白酸钠或羧甲基纤维素钠中的一种或一种以上,成分B为鼠李糖、木糖、阿拉伯糖、甘露糖、山梨糖醇或棉子糖中的一种或一种以上;使用此冻干保护剂制备双歧杆菌冻干粉时,双歧杆菌的冻干存活率高达65.68%,并且,将使用此冻干保护剂制备得到的双歧杆菌冻干粉用pH为3.0的模拟胃液处理2h后,双歧杆菌存活率高达36.92%。
然而长双歧杆菌作为益生菌调节肠道健康时必须是足量且有活性的才能发挥作用,然而双歧杆菌制品普遍存在着不能常温保存、保质期短等缺陷。另外,双歧杆菌不能抵抗人体胃部的低pH,人服用双歧杆菌后,难以保证有足够多的活菌能够到达人体肠道而发挥其益生作用,使其作用发挥和应用受限。因此,双歧杆菌活菌制剂的制备及储存稳定性研究十分重要。与冷冻产品以及液态双歧杆菌制剂相比,冻干菌粉易于处理并且活菌保存期较长,但在冻干的过程中,细菌会因渗透休克、形成胞内冰晶和再结晶而造成细胞膜损伤,导致细菌的冻干存活率降低,因此需要利用冻干保护剂来减少菌体的活性损失。通常选用的冻干保护剂为可溶性物质,如海藻糖、蔗糖、脱脂奶粉等。多糖类的氢键等可以与微生物细胞膜上的糖蛋白等结合,使细胞的功能蛋白、多糖等相对稳定;多糖类可以在微生物细胞膜周围形成玻璃化的保护壳,保护益生菌的空间结构稳定。然而海藻糖作为一种多糖粘度较高,这导致其分散性较差,这会影响其与其他保护剂的复配也会影响其对益生菌的保护性。在本发明中,发明人合成了一种海藻糖聚合物以改善海藻糖作为一种小分子而具有高粘度或更复杂的流体流动性能,使得其分散性更佳、稳定性更好,发明人将该聚合物应用到长双歧杆菌冻干粉剂的制备中以改善结肠癌患者放疗后出现的腹泻等肠胃不适等。
发明内容
有鉴于现有技术中的上述缺陷,本发明所要解决的技术问题是提供一种分散性佳、稳定性好的海藻糖聚合物应用到长双歧杆菌冻干粉剂的制备中以改善结肠癌患者放疗后出现的腹泻等肠胃不适等。
尽管冷冻干燥可以大大降低水分含量,但是在冷冻干燥过程中环境温度较低,会导致一系列的负面影响,如菌体细胞膜破裂、细胞内的部分蛋白质变性、细胞脱水,进而导致益生菌细胞活力的下降,甚至菌体的死亡。因此需要采取相应的保护措施,其中添加冷冻干燥保护剂是一个行之有效的方法,常用的保护剂:如甘油、海藻糖、改性牛奶及乳糖等,保护剂的存在可以使益生菌更加有效抵御在冷冻干燥过程时由工艺引起的失活或死亡,从而减少冷冻干燥过程中菌体的死亡率,增加益生菌的包埋率。海藻糖又称为漏芦糖、蕈糖,是由两分子葡萄糖通过α,α-1,1糖苷键连接而成的一种非还原性双糖,海藻糖的羟基可以替代水分子与细菌的细胞膜和蛋白质相互作用形成氢键,在脱水过程中维持乳酸菌细胞结构及功能的稳定性;还可以在微生物细胞膜周围形成玻璃化的保护壳,保护益生菌的空间结构稳定。然而海藻糖单体由于具有独特的流体流动性和高粘度,导致将其配制成保护剂时其分散性较差,会产生聚集,这会影响其最终对益生菌的保护效果。本发明中,发明人制备了一种海藻糖聚合物,海藻糖经聚合后其蛤壳构象经轴向连接形成刚性骨架,能够将益生菌更好地保护起来,并且海藻糖聚合物的稳定性也有所提升,这可能是由于聚合后局部浓度增加带来的簇状糖苷效应或连接到疏水骨架上的亲水糖侧链的非离子表面活性剂特性,这能够帮助提升其分散性,将海藻糖聚合物应用到冻干保护剂以制备长双歧杆菌冻干粉剂,能够提升菌体稳定性,延长活性。
本发明的技术方案:
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌接种于固体培养基中,恒温培养后将所得菌落接种于液体培养基中继续恒温培养得到种子液;
S2将步骤S1得到的种子液接种于液体培养基中,恒温培养后得到菌液,将菌液离心,收集菌泥;
S3将步骤S2中得到的菌泥重悬于生理盐水中,用1~3wt%的氢氧化钠水溶液调节pH至6~7后,加入冻干保护剂后在无菌条件下混合均匀,冷冻干燥,得到长双歧杆菌冻干粉剂。
进一步的,所述步骤S1中长双歧杆菌的接种量为2~3wt%,恒温培养的条件为37℃,培养10~30h。所述长双歧杆菌为SX-1326,保藏编号CGMCC No.19853;也可以是市售产品,例如,长双歧杆菌(Bifidobacterium longum),菌株保藏编号CICC 6186,中国工业微生物菌种保藏管理中心。
进一步的,所述步骤S1中固体培养基为BBL固体培养基,其组成为牛肉蛋白胨15g/L,无水葡萄糖20g/L,氯化钠5g/L,酵母粉2g/L,番茄浸粉5g/L,可溶性淀粉0.5g/L,L-半胱氨酸盐酸盐0.5g/L,肝浸粉2g/L,吐温-80 1g/L,琼脂15g/L,纯水1L。
进一步的,所述步骤S1、S2中液体培养基为MRS液体培养基,其组成为:酵母粉5g/L,无水乙酸钠5g/L,牛肉膏10g/L,无水葡萄糖20g/L,牛肉蛋白胨10g/L,七水合硫酸镁0.1g/L,三水合磷酸氢二钾2.6g/L,一水合硫酸锰0.05g/L、柠檬酸氢二铵2g/L、吐温-801g/L、L-半胱氨酸盐酸盐1g/L,纯水1L。
进一步的,所述步骤S2中种子液的接种量为3~4%(v/v),恒温培养的条件为37℃,培养10~20h。
进一步的,所述步骤S2中离心转速为8000~10000rpm,离心时间为5~10min。
进一步的,所述步骤S3中菌泥与生理盐水的质量比为1:1~5。
进一步的,所述步骤S3中菌泥与冻干保护剂的质量比为1:2~5。
进一步的,所述冻干保护剂包括脱脂奶粉、海藻糖聚合物、谷氨酸钠、水。
所述冻干保护剂的制备方法,包括如下步骤:
X1称取氢氧化钠5~8重量份、海藻糖6~10重量份加入到80~120重量份纯水中,搅拌混合均匀,再加入4-氯-3-甲基苯乙烯0.2~0.8重量份、四甲基硫酸氢铵0.5~0.8重量份,加入完毕后在20~30℃下搅拌16~24h,反应结束后加6~8mol/L的稀盐酸调节pH至中性,再倒入1500~2000重量份的正己烷中,降温至0~5℃有沉淀析出,过滤,残余物经减压浓缩至干得到甲基苯乙烯取代海藻糖;
X2称取步骤X1中的甲基苯乙烯取代海藻糖2~3重量份加入到80~160重量份的乙酸中,再加入三氟甲磺酸0.8~1.6重量份、乙酰氯0.5~1重量份,室温下搅拌1~3h,反应结束后倒入乙醚中,降温至0~5℃,有沉淀出现,过滤,残余物经酸洗、碱洗、水洗后冷冻干燥得到白色产物,得到乙酰海藻糖取代物;
X3称取步骤X1中的乙酰海藻糖取代物1~2重量份、偶氮二异丁腈0.001~0.003重量份,加入到35~80重量份纯水和65~100重量份N,N-二甲基甲酰胺的混合溶液中,在氩气气氛下加热至80~100℃进行聚合,搅拌16~32h后将体系冷却至室温,混合物浸泡在纯水中经透析(<3500Da)2~3d后冷冻干燥,得到乙酰海藻糖聚合物;将乙酰海藻糖聚合物加入40~80重量份三氯甲烷、20~40重量份甲醇的混合溶液中,再加入乙酸钠0.05~0.5重量份,加热至60~65℃下搅拌4~6h,反应结束后降至室温,将反应液离心后收集下层沉淀后溶于水中,加入稀盐酸调节pH至中性后再进行透析(<3500Da)2~3d后冷冻干燥得到海藻糖聚合物;
X4称取脱脂奶粉4~8重量份、步骤X3中的海藻糖聚合物8~12重量份、谷氨酸钠0.5~2重量份加入到30~40重量份纯水中,经灭菌处理后得到冻干保护剂。
进一步的,所述步骤S3中冷冻干燥的过程为先于﹣40~﹣30℃下进行预冻1~2h,升温至﹣30~﹣20℃升华10~20h,继续升温至﹣20~﹣10℃升华10~20h,再在20~30℃下升华5~10h;真空度为7~10Pa。
本发明还提供了一种长双歧杆菌冻干粉剂由上述方法制备而成。
与现有技术相比,本发明的有益效果:
(1)本发明所述的长双歧杆菌冻干粉剂,耐酸性良好,且经过冻干粉等工艺操作后菌株活性仍然较为理想、保存期长;
(2)在制备本发明所述的长双歧杆菌冻干粉的过程中,由于采用了特定的冻干保护剂,使制为冻干粉后长双歧杆菌保持了较高的存活率;
(3)长双歧杆菌会抑制有害菌,进而维护肠道菌群生态平衡,形成生物屏障,抑制有害菌对肠道的入侵,并且可改善结直肠癌化疗后产生的肠道菌群失调、腹泻等现象。
具体实施方式
下面,通过具体实施例对本发明的技术方案进行详细说明,但是应该明确提出这些实施例用于举例说明,但是不解释为限制本发明的范围。
本发明实施例中部分原料的参数如下:
长双歧杆菌(Bifidobacterium longum),菌株保藏编号CICC 6186,中国工业微生物菌种保藏管理中心。
4-氯-3-甲基苯乙烯,4-Chlor-3-methyl-styrol,CAS:1835-79-6。
对照例1
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌3g接种于100g BBL固体培养基中,37℃恒温培养24h后将所得菌落接种于MRS液体培养基中继续37℃恒温培养12h得到种子液;
S2将步骤S1得到的种子液3mL接种于100mLMRS液体培养基中,37℃恒温培养12h后得到菌液,将菌液在9500rpm下离心10min,,收集菌泥;
S3将步骤S2中得到的菌泥1g重悬于5g生理盐水中,用3wt%的氢氧化钠水溶液调节pH至6.5后,加入冻干保护剂3g后在无菌条件下混合均匀进行冷冻干燥,先于﹣40℃下进行预冻1h,升温至﹣30℃升华10h,继续升温至﹣20℃升华10h,再在25℃下升华5h,真空度为10Pa,最后得到长双歧杆菌冻干粉剂。
所述冻干保护剂的制备方法,包括如下步骤:
称取脱脂奶粉5g、海藻糖8g、谷氨酸钠2g加入到35mL纯水中,经115℃灭菌处理15min后得到冻干保护剂。
实施例1
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌3g接种于100g BBL固体培养基中,37℃恒温培养24h后将所得菌落接种于MRS液体培养基中继续37℃恒温培养12h得到种子液;
S2将步骤S1得到的种子液3mL接种于100mLMRS液体培养基中,37℃恒温培养12h后得到菌液,将菌液在9500rpm下离心10min,收集菌泥;
S3将步骤S2中得到的菌泥1g重悬于5g生理盐水中,用3wt%的氢氧化钠水溶液调节pH至6.5后,加入冻干保护剂3g后在无菌条件下混合均匀进行冷冻干燥,先于﹣40℃下进行预冻1h,升温至﹣30℃升华10h,继续升温至﹣20℃升华10h,再在25℃下升华5h,真空度为10Pa,最后得到长双歧杆菌冻干粉剂。
所述冻干保护剂的制备方法,包括如下步骤:
X1称取氢氧化钠6.4g、海藻糖6.8g加入到120mL纯水中,搅拌混合均匀,再加入4-氯-3-甲基苯乙烯0.6g、四甲基硫酸氢铵0.68g,加入完毕后在25℃下搅拌20h,反应结束后加6mol/L的稀盐酸调节pH至中性,再倒入2L正己烷中,降温至0℃有沉淀析出,过滤,残余物经45℃,﹣0.9MPa减压浓缩至干得到甲基苯乙烯取代海藻糖;
X2称取步骤X1中的甲基苯乙烯取代海藻糖3g加入到100mL乙酸中,再加入三氟甲磺酸0.85g、乙酰氯0.6g,室温下搅拌3h,反应结束后倒入500mL乙醚中,降温至0℃,有沉淀出现,过滤,残余物经1mol/L稀盐酸洗、5wt%碳酸氢钠水溶液洗、纯水洗后﹣30℃,10Pa下冷冻干燥48h得到乙酰海藻糖取代物;
X3称取步骤X1中的乙酰海藻糖取代物2g、偶氮二异丁腈0.0015g,加入到40mL纯水和80mLN,N-二甲基甲酰胺的混合溶液中,在氩气气氛下加热至90℃进行聚合,搅拌24h后将体系冷却至室温,混合物浸泡在水中经透析(<3500Da)2d后﹣30℃,10Pa下冷冻干燥48h,得到乙酰海藻糖聚合物;将乙酰海藻糖聚合物加入30mL三氯甲烷、30mL甲醇的混合溶液中,再加入乙酸钠0.2g,加热至65℃下搅拌4h,反应结束后降至室温,将反应液经8000rpm离心10min后收集下层沉淀溶于100mL纯水中,加入1mol/L稀盐酸调节pH至中性后再进行透析(<3500Da)2d后﹣30℃,10Pa下冷冻干燥48h得到海藻糖聚合物;
X4称取脱脂奶粉5g、步骤X3中的海藻糖聚合物8g、谷氨酸钠2g加入到35mL纯水中,经115℃灭菌处理15min后得到冻干保护剂。
实施例2
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌3g接种于100g BBL固体培养基中,37℃恒温培养24h后将所得菌落接种于MRS液体培养基中继续37℃恒温培养12h得到种子液;
S2将步骤S1得到的种子液3mL接种于100mLMRS液体培养基中,37℃恒温培养12h后得到菌液,将菌液在9500rpm下离心10min,收集菌泥;
S3将步骤S2中得到的菌泥1g重悬于5g生理盐水中,用3wt%的氢氧化钠水溶液调节pH至6.5后,加入冻干保护剂3g后在无菌条件下混合均匀进行冷冻干燥,先于﹣40℃下进行预冻1h,升温至﹣30℃升华10h,继续升温至﹣20℃升华10h,再在25℃下升华5h,真空度为10Pa,最后得到长双歧杆菌冻干粉剂。
所述冻干保护剂的制备方法,包括如下步骤:
称取脱脂奶粉5g加入到45mL纯水中,经115℃灭菌处理15min后得到冻干保护剂。
实施例3
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌3g接种于100g BBL固体培养基中,37℃恒温培养24h后将所得菌落接种于MRS液体培养基中继续37℃恒温培养12h得到种子液;
S2将步骤S1得到的种子液3mL接种于100mLMRS液体培养基中,37℃恒温培养12h后得到菌液,将菌液在9500rpm下离心10min,收集菌泥;
S3将步骤S2中得到的菌泥1g重悬于5g生理盐水中,用3wt%的氢氧化钠水溶液调节pH至6.5后,加入冻干保护剂3g后在无菌条件下混合均匀进行冷冻干燥,先于﹣40℃下进行预冻1h,升温至﹣30℃升华10h,继续升温至﹣20℃升华10h,再在25℃下升华5h,真空度为10Pa,最后得到长双歧杆菌冻干粉剂。
所述冻干保护剂的制备方法,包括如下步骤:
称取脱脂奶粉5g、谷氨酸钠2g加入到43mL纯水中,经115℃灭菌处理15min后得到冻干保护剂。
实施例3
一种长双歧杆菌冻干粉剂的制备方法,包括如下步骤:
S1将长双歧杆菌3g接种于100g BBL固体培养基中,37℃恒温培养24h后将所得菌落接种于MRS液体培养基中继续37℃恒温培养12h得到种子液;
S2将步骤S1得到的种子液3mL接种于100mLMRS液体培养基中,37℃恒温培养12h后得到菌液,将菌液在9500rpm下离心10min,收集菌泥;
S3将步骤S2中得到的菌泥1g重悬于5g生理盐水中,用3wt%的氢氧化钠水溶液调节pH至6.5后,加入冻干保护剂3g后在无菌条件下混合均匀进行冷冻干燥,先于﹣40℃下进行预冻1h,升温至﹣30℃升华10h,继续升温至﹣20℃升华10h,再在25℃下升华5h,真空度为10Pa,最后得到长双歧杆菌冻干粉剂。
所述冻干保护剂的制备方法,包括如下步骤:
称取谷氨酸钠2g加入到48mL纯水中,经115℃灭菌处理15min后得到冻干保护剂。
测试例1
对对照例及实施例所制备的长双歧杆菌冻干粉剂进行保存稳定性实验,样品分别放在25℃保存,分别在30d、120d取出样品依次梯度稀释到10-9,涂BBL培养基,放在厌氧箱中37℃培养48h后计数,每个样品做三个平行。存活率=V2/V1×100%,V1为保存/冻干前的总活菌数;V2为保存/冻干后的总活菌数。具体测试结果见表1.
表1长双歧杆菌冻干粉剂保存稳定性实验结果表
实验方案 | 30d后存活率/% | 120d后存活率/% |
对照例1 | 64.12 | 16.10 |
实施例1 | 78.58 | 59.13 |
实施例2 | 45.12 | 7.89 |
实施例3 | 34.10 | 6.54 |
实施例4 | 31.28 | 5.17 |
与冷冻产品和液态双歧杆菌制剂相比,长双歧杆菌冻干菌粉的保存条件和保存时间都具有明显的优势,但随着保存时间的延长,活菌数会随着时间的推移而降低。FAO/WHO建议食品益生菌活菌数应在(1.0×106~1.0×107)CFU/g以上,当长双歧杆菌冻干菌粉的活菌数低于此标准时,就失去了其益生作用,所以有必要考察冻干菌粉的保存稳定性。保护剂中蛋白类如脱脂奶粉的保护机理为蛋白类可以在细胞外形成一层致密的蛋白保护层,通过蛋白保护层阻止细胞与外界的接触,降低氧、营养物等与菌体接触的几率,进而减少菌体体内的代谢,最终达到稳定益生菌在冷冻干燥过程的活性。保护剂中海藻糖的作用机理是海藻糖的羟基可以替代水分子与细菌的细胞膜和蛋白质相互作用形成氢键,在脱水过程中维持乳酸菌细胞结构及功能的稳定性;还可以在微生物细胞膜周围形成玻璃化的保护壳,保护益生菌的空间结构稳定。然而海藻糖单体由于具有独特的流体流动性和高粘度,导致将其配制成保护剂时其分散性较差,会产生聚集,这会影响其最终对益生菌的保护效果。当冻干保护剂的成分较为单一时,其保护作用明显较差,这可能是由于不同保护剂的保护机制不尽相同,将多种保护剂联用能够增强保护效果。而在实施例1中将海藻糖单体经取代、聚合制备得到一种海藻糖聚合物,海藻糖经聚合后其蛤壳构象经轴向连接形成刚性骨架,能够将益生菌更好地保护起来,并且海藻糖聚合物的稳定性也有所提升,这可能是由于聚合后局部浓度增加带来的簇状糖苷效应或连接到疏水骨架上的亲水糖侧链的非离子表面活性剂特性,这能够帮助提升其分散性,因此将其应用到冻干保护剂中能够提升菌体稳定性,延长活性。
测试例2
为了模拟动物体内的胃肠道环境,配制人工胃液,对对照例及实施例所制备的长双歧杆菌冻干粉剂进行体外的耐酸性能的研究,样品经pH为3.0的模拟胃液37℃厌氧培养2h后进行存活率的检测。取培养后长双歧杆菌冻干粉剂进行活菌数的测定,计算存活率,存活率=V2/V1×100%,V1为保存/冻干前的总活菌数;V2为胃液处理2h的总活菌数。具体测试结果见表2。
表2长双歧杆菌冻干粉剂耐酸性实验结果表
实验方案 | 存活率/% |
对照例1 | 12.50 |
实施例1 | 38.21 |
实施例2 | 3.12 |
实施例3 | 5.13 |
实施例4 | 4.05 |
由于胃液的强酸性,大部分益生菌都难以存活,长双歧杆菌作为一种转性厌氧菌,对氧气、pH、温度、湿度等外界不良环境因素极为敏感,因此在胃液中很难存活,通过对长双歧杆菌进行保护可以一定程度地提升其在胃液下的存活率,从耐酸性实验结果可以看到,实施例1中长双歧杆菌的存活率最高,这可能是由于海藻糖经聚合后得到的海藻糖聚合物其蛤壳构象经轴向连接形成刚性骨架,能够将益生菌更好地保护起来,相较于其他保护剂其更不易被破坏,因此存活率较高。
测试例3
购买70只C57BL/6小鼠(SPF级,5-7周,体重20g左右。实验前适应性饲喂一周。饲养环境:相对湿度40~60%、温度22~26℃,供应SPF级小鼠饲料,并及时更换垫料及添加饮用水。严格按照无菌操作利用氧化偶氮甲烷/葡聚糖硫酸钠诱导法进行结直肠癌的造模。将小鼠随机分为7组,分别为空白对照组、伊利替康组及实验组,空白对照组除正常饲喂外每天仅灌胃纯水,伊利替康组采用伊利替康进行化疗,每天灌胃纯水,实验组则同时采用伊利替康进行化疗和长双歧杆菌冻干粉剂来进行辅助治疗,剂量为0.2g/10g,每组每天灌胃1次。期间,每天观察小鼠腹泻情况并进行评价。腹泻程度分为以下几种等级:0级正常:大便正常或没有,1级轻微腹泻:大便可见轻微湿软,2级中度腹泻:大便较湿且不成型且有中度肛门着色,3级重度腹泻:大便水样且有中度肛门着色。根据腹泻情况计算腹泻率,具体实验结果见表3.
表3长双歧杆菌冻干粉剂对伊立替康所致小鼠腹泻情况的影响
实验方案 | 腹泻率/% |
空白对照组 | 100 |
伊立替康组 | 100 |
对照例1 | 60 |
实施例1 | 40 |
实施例2 | 80 |
实施例3 | 70 |
实施例4 | 80 |
伊立替康及其代谢产物通过胆汁排泄到小肠中,其中一部分被吸收经肠肝循环返回肝脏。进入大肠的SN-38在肠道细菌产生的β-葡萄糖醛酸酶的作用下,重新转化为活性产物SN-38G,SN-38可破坏肠粘膜,并通过肠肝循环在体内蓄积,从而引发严重的迟发性腹泻。长双歧杆菌可以产生短链脂肪酸SCFAs,是肠道菌群维持肠道生态平衡的重要途径之一。短链脂肪酸主要包含乙酸、丙酸、丁酸等,是一类有机羧酸,常参与水和电解质的调节,可促进Na+和水的吸收,抑制Cl-的分泌,从而减少肠道腹泻的发生,SCFAs还可以通过上调紧密连接蛋白,表达增强细胞紧密连接、抑制肠道通透性等增强肠道机械性屏障功能。SCFAs可以减轻局部炎症反应的机制是通过下调NF-KB信号通路,减少肠道炎性因子IL-1、IL-6、TNF-α等的表达,减少中性粒细胞在炎症部位的趋化。从小鼠的腹泻情况可以看到,服用长双歧杆菌能够很好地改善有伊立替康导致的迟发性腹泻,不同冻干粉剂的改善情况不尽相同,这与益生菌的存活率有很大关系,实施例1中益生菌的存活率更高,因此其活性最佳,对于改善小鼠腹泻效果也最好。
Claims (6)
1.一种长双歧杆菌冻干粉剂的制备方法,其特征在于,包括如下步骤:
S1将长双歧杆菌接种于固体培养基中,恒温培养后将所得菌落接种于液体培养基中继续恒温培养得到种子液;
S2将步骤S1得到的种子液接种于液体培养基中,恒温培养后得到菌液,将菌液离心,收集菌泥;
S3将步骤S2中得到的菌泥重悬于生理盐水中,用1~3wt%的氢氧化钠水溶液调节pH至6~7后,加入冻干保护剂后在无菌条件下混合均匀,冷冻干燥,得到所述长双歧杆菌冻干粉剂;
所述冻干保护剂的制备方法,包括如下步骤:
X1将氢氧化钠、海藻糖加入到水中,混合均匀,再加入4-氯-3-甲基苯乙烯、四甲基硫酸氢铵,20~30℃下搅拌16~24h,反应结束后加稀盐酸调节pH至中性,再将混合液倒入正己烷中,降温至0~5℃有沉淀析出,过滤,残余物经减压浓缩至干得到甲基苯乙烯取代的海藻糖;
X2将步骤X1中的甲基苯乙烯取代海藻糖加入到乙酸中,再加入三氟甲磺、乙酰氯,室温下搅拌1~3h,反应结束后将混合液倒入乙醚中,降温至0~5℃,有沉淀出现,过滤,残余物经酸洗、碱洗、水洗后冷冻干燥得到白色产物,得到乙酰海藻糖取代物;
X3将步骤X2中的乙酰海藻糖取代物、偶氮二异丁腈加入到水和N,N-二甲基甲酰胺的混合溶液中,在氩气气氛下加热至80~100℃进行聚合,搅拌16~32h后将体系冷却至室温,混合物浸泡在水中经透析2~3d后冷冻干燥,得到乙酰海藻糖聚合物;将乙酰海藻糖聚合物加入三氯甲烷、甲醇的混合溶液中,再加入乙酸钠,加热至60~65℃下搅拌4~6h,反应结束后降至室温,将反应液离心后收集下层沉淀后溶于水中,加入稀盐酸调节pH至中性后再进行透析2~3d后冷冻干燥得到海藻糖聚合物;
X4将脱脂奶粉、步骤X3中的海藻糖聚合物、谷氨酸钠加入到纯水中,经灭菌处理后得到所述冻干保护剂。
2.如权利要求1所述的冻干粉剂的制备方法,其特征在于:所述步骤S1中长双歧杆菌的接种量为2~3wt%,恒温培养的条件为37℃,培养10~30h。
3.如权利要求1所述的冻干粉剂的制备方法,其特征在于:所述步骤S2中种子液的接种量为3~4%(v/v),恒温培养的条件为37℃,培养10~20h。
4.如权利要求1所述的冻干粉剂的制备方法,其特征在于:所述步骤S2中离心转速为8000~10000rpm,离心时间为5~10min。
5.如权利要求1所述的冻干粉剂的制备方法,其特征在于:所述步骤S3中菌泥与生理盐水的质量比为1:1~5。
6.如权利要求1所述的冻干粉剂的制备方法,其特征在于:所述步骤S3中菌泥与冻干保护剂的质量比为1:2~5。
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