CN113424965A - 一种更适合黄种人群的微生态制剂 - Google Patents
一种更适合黄种人群的微生态制剂 Download PDFInfo
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- CN113424965A CN113424965A CN202110724290.4A CN202110724290A CN113424965A CN 113424965 A CN113424965 A CN 113424965A CN 202110724290 A CN202110724290 A CN 202110724290A CN 113424965 A CN113424965 A CN 113424965A
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- bifidobacterium
- lactobacillus
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- drying
- fermentation liquor
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- A—HUMAN NECESSITIES
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- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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Abstract
本发明提供一种更适合黄种人群的微生态制剂,属于微生物制剂领域,由益生菌活菌、复合培养基、载体以及中药提取物组成,所述益生菌活菌包括乳杆菌发酵液、双歧杆菌发酵液、链球菌发酵液、芽孢杆菌发酵液和酵母菌发酵液;所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧杆菌;所述微生态制剂制备方法为:以第一培养基分别对各类菌种进行活化并扩大培养,再分别接种到第二培养基中分别发酵培养12‑24h,按活菌数量比例混合得到混合发酵液,加入载体后冻干,与所述中药提取物混合后制得;所述微生态制剂可以改善肠道菌群,维持微生态平衡,且分泌的β‑半乳糖苷酶填补了黄种人小肠分泌的不足。
Description
技术领域
本发明涉及功能性微生物制剂领域,具体涉及一种更适合黄种人群的微生态制剂。
背景技术
正常人体肠道内寄居着数量庞大、种类繁多的微生物,以细菌为主,统称为肠道菌群, 肠道菌群与人体之间的关系错综复杂,贯穿于人体各种生理活动和病理过程,已成为人体不 可或缺的一部分。正常情况下,肠道各菌种与宿主相互依存、相互制约,维持一种动态的生 态平衡一旦受到宿主及外环境变化的影响,平衡状态就会被打破,从而造成肠道菌群失调。 近年来,随着分子生物学技术在肠道菌群检测方面的应用,菌群失调与疾病的相关性研究及 其在发病机制中的作用逐渐引起了人们的广泛重视,因此,研究肠道菌群失调对人体健康的 重要作用无论是对于基础研究还是临床应用,均具有重要的意义。调节肠道菌群的益生保健 食品也具有非常好的社会和经济效益。
但肠道菌群跟人种有关,不同种的人的生活环境不同,饮食风俗习惯等都有差别,这种 差别会直接影响肠道内的菌群分布。
发明内容
针对上述问题,本发明提供一种更适合黄种人群的微生态制剂。
本发明的目的采用以下技术方案来实现:
一种更适合黄种人群的微生态制剂,由益生菌活菌、复合培养基、载体以及中药提取物 组成,所述益生菌活菌包括乳杆菌发酵液、双歧杆菌发酵液、链球菌发酵液、芽孢杆菌发酵 液和酵母菌发酵液,其中,所述乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌的活菌数量 比为100:(60-100):(10-30):(5-10):(1-5);
所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧 杆菌;
所述微生态制剂制备方法为:
以第一培养基分别对乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌进行活化并扩大培 养,再将培养后的菌种分别接种到第二培养基中,分别发酵培养12-24h,以各菌种发酵液按 活菌数量比例混合得到混合发酵液,加入冻干保护剂,混合均匀后再加入所述载体,所述载 体经浸泡后滤出,冷冻干燥制得负载的载体,再与所述中药提取物混合后制得所述微生态制 剂;其中,所述第一培养基的组成为大豆蛋白胨、牛肉蛋白粉、酵母膏、醋酸钠、葡萄糖、 吐温80、柠檬酸铵、硫酸镁和水;所述第二培养基包括所述第一培养基和大豆多肽粉。
优选的,所述中药提取物包括山药30份、苍术15份、砂仁10份、人参8份、当归8份、甘草4份、党参5份、茯苓8份、黄精4份、山楂10份,其制备方法是:
依次称取各组分,加入重量/体积比10倍的蒸馏水,浸泡4h后,并于100℃提取1h,过 滤,再向滤渣中加入重量/体积比8倍的蒸馏水,100℃提取1h后,过滤后合并滤液,离心,浓缩干燥制得所述中药提取物;
所述中药提取物与所述负载的载体的混合比例为(1-100):10。
优选的,所述乳杆菌为卷曲乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、詹氏乳杆菌、加氏乳 杆菌、唾液乳杆菌、干酪乳杆菌、发酵乳杆菌、植物乳杆菌、保加利亚乳杆菌中的至少一种。
优选的,所述链球菌包括嗜热链球菌、消化链球菌、粪链球菌中的至少一种。
优选的,所述芽孢杆菌包括凝结芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌中的至少一种。
优选的,所述冻干保护剂为甘油和低聚木糖,添加量分别为所述混合发酵液质量的0.1-1%。
优选的,所述大豆多肽粉的制备方法为:
s1、将经过清洗的大豆破碎粉磨,制得大豆匀浆,将所述大豆匀浆升温至80-90℃保温 5-30min,经高速冷冻离心后除去上层油层,干燥后制得大豆蛋白粉;
s2、在所述大豆蛋白粉中滴加浓度为0.2mol/L、pH为7.5的磷酸盐缓冲液,使所述大豆 蛋白粉的含水率提高到50-60%,再在120℃下加热10-20min灭菌,得到培养基;将活化好的 黑曲霉菌液接种至所述培养基中,所述黑曲霉菌液的浓度不小于106CFU/ml,接种量为4-8%, 接种完成后在35-37℃下发酵3-4d,待发酵结束后,加入蒸馏水,搅拌并浸泡后分离滤液, 将滤液浓缩干燥,制得多肽粉;
s3、称取3-(二乙基氨基)丙基硫代异氰酸酯并溶解在DMSO或DMF中,配制为质量分数10%的溶液,制得溶液A;将所述多肽粉以料液比1g/100ml超声分散在DMSO或DMF中, 得到悬液A,在室温和搅拌条件下,将所述悬液A缓慢滴加到等体积的所述溶液A中,添加 完毕后继续搅拌反应24-28h,反应完成后分离沉淀,沉淀依次以DMF、二氯甲烷和冷的乙醚 洗涤后冻干制得。
优选的,所述载体为改性蒙脱石。
优选的,所述改性蒙脱石的制备方法为:
将蒙脱石粉碎并筛分,得到粒径在500-1000筛目之间的微粉,将蒙脱石微粉按料液比 1g/100ml悬浮分散在蒸馏水中,得到悬液B,加入所述悬液B体积1-2%的3-(三乙氧基硅基) 丙基琥珀酸酐或3-氨基丙基三乙氧基硅烷,在60-70℃下搅拌回流12-24h,回流完成后滤出 沉淀,沉淀依次以乙醇和蒸馏水洗涤制得。
优选的,所述改性蒙脱石的制备方法还包括以下步骤:
(1)烯丙基改性6-氨基嘌呤制备
称取溴乙醇,在冰水浴条件下溶解在二氯甲烷中,加入三乙胺和甲基丙烯酰氯,在室温 条件下搅拌反应12-14h,反应完成后分离滤液,所述滤液依次经冰水洗涤、无水硫酸镁干燥 后蒸去二氯甲烷,得到酯化产物;称取6-氨基嘌呤和氢化钠并溶解在无水DMF中,充分搅 拌均匀后,加入所述酯化产物,在室温条件下搅拌反应12-14h,反应完成后分离滤液,并用 丙酮沉淀数次,真空干燥;其中,
所述溴乙醇与所述二氯甲烷、所述三乙胺、所述甲基丙烯酰氯的质量比例为1:(6-7): (0.1-0.15):(0.9-0.95);所述6-氨基嘌呤与所述氢化钠、所述无水DMF、所述酯化产物的 质量比例为1:(0.2-0.22):(10-12):(2.4-2.5);
(2)共聚产物制备
称取所述烯丙基改性6-氨基嘌呤和磺基甜菜碱甲基丙烯酸酯并溶解在无水DMSO中,充 分溶解后加入引发剂,经氮吹除氧后封闭反应体系,将反应体系置于70-75℃下恒温反应3-4d, 反应完成后冷却,分离滤液,并用丙酮沉淀数次,真空干燥制得共聚产物;其中,
所述烯丙基改性6-氨基嘌呤与所述磺基甜菜碱甲基丙烯酸酯、所述无水DMSO、所述引 发剂的质量比例为1:(1.0-1.1):(50-100):(0.07-0.09);
(3)包覆产物制备
称取所述共聚产物并溶解在热的蒸馏水中,加入经乙醇和蒸馏水洗涤后制得的沉淀,充 分分散后边搅拌边滴加与所述蒸馏水等体积的丙酮,过滤,滤饼真空干燥后制得,其中,
所述共聚产物与所述沉淀、所述蒸馏水的质量比例为1:1:1000。
本发明的有益效果为:
(1)本发明通过外源性的补充具有优势地位的双歧杆菌等益生菌,形成优势菌群,可以 同时通过竞争粘附机制阻止致病微生物粘附,抑制多种病原微生物的生长,并刺激免疫系统 维持肠道微生态平衡;双歧杆菌可定植在肠粘膜表面形成生物屏障,抵御大肠杆菌、痢疾杆 菌等致病菌和条件致病菌的入侵,双歧杆菌全菌及表面分子能增强机体的非特异性和特异性 免疫反应,提高K细胞和巨噬细胞的活性,提高局部或全身的抗感染和防御功能,其产生的 抗菌性物质可抑制外源致病菌和肠道内固有腐败细菌的生长繁殖,同时调节和协调其它肠道 菌群,促进肠道蠕动,减少致病菌粘附到肠粘膜的机会,降低肠道pH值和氧化还原电势, 对有害菌起到抑制作用;本发明所述微生态制剂一方面改善肠道菌群,维持微生态平衡,另 一方面其分泌的β半乳糖苷酶填补了黄种人小肠分泌的不足,改善肠胃对乳糖的吸收和吸收, 其还能醇解寡糖产生醋酸和乳酸,减少碱性物质对大肠粘膜的刺激,促进蠕动;
本发明所述中药提取物可以有效地促进肠道益生菌的生长,提高活菌进入肠道中的存活 率及定植能力,调节肠道微生物群的组成以恢复其体内平衡,从而更好的改善人体健康状况。
(2)本发明通过先对益生菌进行高密度的前期培养,直接冻干后可以最大程度保留菌种 的活力,同时可以与培养基、益生菌代谢产物以及细菌细胞素或类细胞素等活性物质一同负 载于载体之上,使得冻干后的益生菌在使用环境中可以很快活化并继续生长,快速刺激免疫 系统维持微生态平衡,减少用药时间,提高用药效率;所述冻干保护剂不仅保护益生菌菌体 的冻干活力,本发明所述冻干保护剂还添加有低聚木糖,可通过释放而增加有益的乳杆菌的 丰度,促进生长。
(3)本发明以大豆为底物,以黑曲霉为发酵菌发酵制备了大豆多肽粉,一方面,大豆多 肽粉作为培养基组分,为益生菌活菌提供氨基酸源,另一方面可以作为益生菌活菌的发酵促 进剂,提高活化速度,通过氨基N末端偶联硫代异氰酸酯提高多肽粉的疏水性,提高与菌体 的亲和性及其利用度,同时提高稳定性。
(4)蒙脱石不仅作为菌种载体,其还对乳杆菌形成的生物膜具有支持作用,可以促进包 括乳杆菌在内的益生菌在粘膜上的定植,促进益生菌种群的快速生长,同时抑制致病菌活性; 本发明还以具有良好生物亲和性的6-氨基嘌呤和超亲水性的磺酸甜菜碱两性表面活性剂为单 体,通过烯丙基单体的引发共聚制备得到具有两亲性的类膜聚合物载体,用作包覆载体,可 实现在温度和酸性pH环境下释放负载以降低外界环境因素对负载菌种的活性影响,进一步 提高稳定性。
具体实施方式
结合以下实施例对本发明作进一步描述。
实施例1
一种更适合黄种人群的微生态制剂,其制备方法为:
以第一培养基分别对乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌进行活化并扩大培 养,再将培养后的菌种分别接种到第二培养基中,分别发酵培养12-24h,以各菌种发酵液按 活菌数量比例100:70:20:10:1混合得到混合发酵液,加入冻干保护剂,混合均匀后再加 入所述载体,所述载体经浸泡后滤出,冷冻干燥制得负载的载体,所述载体为蒙脱石,再与 所述中药提取物混合后制得所述微生态制剂;其中,按质量份数计,所述第一培养基的组成 为:大豆蛋白胨10份、牛肉蛋白粉10份、酵母膏5份、醋酸钠5份、葡萄糖20份、吐温800.1份、柠檬酸铵2份、硫酸镁0.58份、水1000份;所述第二培养基在所述第一培养基的 组成基础上还包括大豆多肽粉12份;
所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧 杆菌,其接种量为1:1:1:1:1;
所述冻干保护剂为甘油和低聚木糖,添加量分别为所述混合发酵液质量的1%;
所述中药提取物按质量份数计,包括山药30份、苍术15份、砂仁10份、人参8份、当归8份、甘草4份、党参5份、茯苓8份、黄精4份、山楂10份,其制备方法是:
依次称取各组分,加入重量/体积比10倍的蒸馏水,浸泡4h后,并于100℃提取1h,过 滤,再向滤渣中加入重量/体积比8倍的蒸馏水,100℃提取1h后,过滤后合并滤液,离心,浓缩干燥制得所述中药提取物;
所述中药提取物与所述负载的载体的混合质量比例为1:10;
所述乳杆菌为卷曲乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌和植物乳杆菌,其接种量为1:1:1:1:1;
所述链球菌为嗜热链球菌;所述芽孢杆菌为枯草芽孢杆菌和地衣芽孢杆菌,其接种量为 1:1;
所述大豆多肽粉的制备方法为:
s1、将经过清洗的大豆破碎粉磨,制得大豆匀浆,将所述大豆匀浆升温至80-90℃保温 5-30min,经高速冷冻离心后除去上层油层,干燥后制得大豆蛋白粉;
s2、在所述大豆蛋白粉中滴加浓度为0.2mol/L、pH为7.5的磷酸盐缓冲液,使所述大豆 蛋白粉的含水率提高到50-60%,再在120℃下加热10-20min灭菌,得到培养基;将活化好的 黑曲霉菌液接种至所述培养基中,所述黑曲霉菌液的浓度不小于106CFU/ml,接种量为4-8%, 接种完成后在35-37℃下发酵3-4d,待发酵结束后,加入蒸馏水,搅拌并浸泡后分离滤液, 将滤液浓缩干燥,制得多肽粉;
s3、称取3-(二乙基氨基)丙基硫代异氰酸酯并溶解在DMSO或DMF中,配制为质量分数10%的溶液,制得溶液A;将所述多肽粉以料液比1g/100ml超声分散在DMSO或DMF中, 得到悬液A,在室温和搅拌条件下,将所述悬液A缓慢滴加到等体积的所述溶液A中,添加 完毕后继续搅拌反应24-28h,反应完成后分离沉淀,沉淀依次以DMF、二氯甲烷和冷的乙醚 洗涤后冻干制得。
实施例2
一种更适合黄种人群的微生态制剂,其制备方法为:
以第一培养基分别对乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌进行活化并扩大培 养,再将培养后的菌种分别接种到第二培养基中,分别发酵培养12-24h,以各菌种发酵液按 活菌数量比例100:70:20:10:1混合得到混合发酵液,加入冻干保护剂,混合均匀后再加 入所述载体,所述载体经浸泡后滤出,冷冻干燥制得负载的载体,所述载体为改性蒙脱石, 再与所述中药提取物混合后制得所述微生态制剂;其中,按质量份数计,所述第一培养基的 组成为:大豆蛋白胨10份、牛肉蛋白粉10份、酵母膏5份、醋酸钠5份、葡萄糖20份、吐温800.1份、柠檬酸铵2份、硫酸镁0.58份、水1000份;所述第二培养基在所述第一培养 基的组成基础上还包括大豆多肽粉12份;
所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧 杆菌,其接种量为1:1:1:1:1;
所述冻干保护剂为甘油和低聚木糖,添加量分别为所述混合发酵液质量的1%;
所述中药提取物包括山药30份、苍术15份、砂仁10份、人参8份、当归8份、甘草4份、党参5份、茯苓8份、黄精4份、山楂10份,其制备方法是:
依次称取各组分,加入重量/体积比10倍的蒸馏水,浸泡4h后,并于100℃提取1h,过 滤,再向滤渣中加入重量/体积比8倍的蒸馏水,100℃提取1h后,过滤后合并滤液,离心,浓缩干燥制得所述中药提取物;
所述中药提取物与所述负载的载体的混合质量比例为1:10;
所述乳杆菌为卷曲乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌和植物乳杆菌,其接种量为1:1:1:1:1;
所述链球菌为嗜热链球菌;所述芽孢杆菌为枯草芽孢杆菌和地衣芽孢杆菌,其接种量为 1:1;
所述大豆多肽粉的制备方法为:
s1、将经过清洗的大豆破碎粉磨,制得大豆匀浆,将所述大豆匀浆升温至80-90℃保温 5-30min,经高速冷冻离心后除去上层油层,干燥后制得大豆蛋白粉;
s2、在所述大豆蛋白粉中滴加浓度为0.2mol/L、pH为7.5的磷酸盐缓冲液,使所述大豆 蛋白粉的含水率提高到50-60%,再在120℃下加热10-20min灭菌,得到培养基;将活化好的 黑曲霉菌液接种至所述培养基中,所述黑曲霉菌液的浓度不小于106CFU/ml,接种量为4-8%, 接种完成后在35-37℃下发酵3-4d,待发酵结束后,加入蒸馏水,搅拌并浸泡后分离滤液, 将滤液浓缩干燥,制得多肽粉;
s3、称取3-(二乙基氨基)丙基硫代异氰酸酯并溶解在DMSO或DMF中,配制为质量分数10%的溶液,制得溶液A;将所述多肽粉以料液比1g/100ml超声分散在DMSO或DMF中, 得到悬液A,在室温和搅拌条件下,将所述悬液A缓慢滴加到等体积的所述溶液A中,添加 完毕后继续搅拌反应24-28h,反应完成后分离沉淀,沉淀依次以DMF、二氯甲烷和冷的乙醚 洗涤后冻干制得;
所述改性蒙脱石的制备方法为:
将蒙脱石粉碎并筛分,得到粒径在500-1000筛目之间的微粉,将蒙脱石微粉按料液比 1g/100ml悬浮分散在蒸馏水中,得到悬液B,加入所述悬液B体积1-2%的3-(三乙氧基硅基) 丙基琥珀酸酐或3-氨基丙基三乙氧基硅烷,在60-70℃下搅拌回流12-24h,回流完成后滤出 沉淀,沉淀依次以乙醇和蒸馏水洗涤制得。
实施例3
一种更适合黄种人群的微生态制剂,其制备方法为:
以第一培养基分别对乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌进行活化并扩大培 养,再将培养后的菌种分别接种到第二培养基中,分别发酵培养12-24h,以各菌种发酵液按 活菌数量比例100:70:20:10:1混合得到混合发酵液,加入冻干保护剂,混合均匀后再加 入所述载体,所述载体经浸泡后滤出,冷冻干燥制得负载的载体,所述载体为蒙脱石,再与 所述中药提取物混合后制得所述微生态制剂;其中,按质量份数计,所述第一培养基的组成 为:大豆蛋白胨10份、牛肉蛋白粉10份、酵母膏5份、醋酸钠5份、葡萄糖20份、吐温800.1份、柠檬酸铵2份、硫酸镁0.58份、水1000份;所述第二培养基在所述第一培养基的 组成基础上还包括大豆多肽粉12份;
所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧 杆菌,其接种量为1:1:1:1:1;
所述冻干保护剂为甘油和低聚木糖,添加量分别为所述混合发酵液质量的1%;
所述中药提取物包括山药30份、苍术15份、砂仁10份、人参8份、当归8份、甘草4份、党参5份、茯苓8份、黄精4份、山楂10份,其制备方法是:
依次称取各组分,加入重量/体积比10倍的蒸馏水,浸泡4h后,并于100℃提取1h,过 滤,再向滤渣中加入重量/体积比8倍的蒸馏水,100℃提取1h后,过滤后合并滤液,离心,浓缩干燥制得所述中药提取物;
所述中药提取物与所述负载的载体的混合质量比例为1:10;
所述乳杆菌为卷曲乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌和植物乳杆菌,其接种量为1:1:1:1:1;
所述链球菌为嗜热链球菌;所述芽孢杆菌为枯草芽孢杆菌和地衣芽孢杆菌,其接种量为 1:1;
所述大豆多肽粉的制备方法为:
s1、将经过清洗的大豆破碎粉磨,制得大豆匀浆,将所述大豆匀浆升温至80-90℃保温 5-30min,经高速冷冻离心后除去上层油层,干燥后制得大豆蛋白粉;
s2、在所述大豆蛋白粉中滴加浓度为0.2mol/L、pH为7.5的磷酸盐缓冲液,使所述大豆 蛋白粉的含水率提高到50-60%,再在120℃下加热10-20min灭菌,得到培养基;将活化好的 黑曲霉菌液接种至所述培养基中,所述黑曲霉菌液的浓度不小于106CFU/ml,接种量为4-8%, 接种完成后在35-37℃下发酵3-4d,待发酵结束后,加入蒸馏水,搅拌并浸泡后分离滤液, 将滤液浓缩干燥,制得多肽粉;
s3、称取3-(二乙基氨基)丙基硫代异氰酸酯并溶解在DMSO或DMF中,配制为质量分数10%的溶液,制得溶液A;将所述多肽粉以料液比1g/100ml超声分散在DMSO或DMF中, 得到悬液A,在室温和搅拌条件下,将所述悬液A缓慢滴加到等体积的所述溶液A中,添加 完毕后继续搅拌反应24-28h,反应完成后分离沉淀,沉淀依次以DMF、二氯甲烷和冷的乙醚 洗涤后冻干制得;
所述改性蒙脱石的制备方法为:
(1)将蒙脱石粉碎并筛分,得到粒径在500-1000筛目之间的微粉,将蒙脱石微粉按料 液比1g/100ml悬浮分散在蒸馏水中,得到悬液B,加入所述悬液B体积1%的3-(三乙氧基硅 基)丙基琥珀酸酐或加入所述悬液B体积1.6%的3-氨基丙基三乙氧基硅烷,在60-70℃下搅拌 回流12-24h,回流完成后滤出沉淀,沉淀依次以乙醇和蒸馏水洗涤制得初产物;
(2)烯丙基改性6-氨基嘌呤制备
称取溴乙醇4g,在冰水浴条件下溶解在20ml的二氯甲烷中,加入0.4g三乙胺和3.7g甲 基丙烯酰氯,在室温条件下搅拌反应12-14h,反应完成后分离滤液,所述滤液依次经冰水洗 涤、无水硫酸镁干燥后蒸去二氯甲烷,得到酯化产物;称取6-氨基嘌呤2g和氢化钠0.4g,并 溶解在50ml无水DMF中,充分搅拌均匀后,加入5g所述酯化产物,在室温条件下搅拌反 应12-14h,反应完成后分离滤液,并用丙酮沉淀数次,真空干燥制得烯丙基改性6-氨基嘌呤;
(3)共聚产物制备
称取0.8g所述烯丙基改性6-氨基嘌呤和0.85g磺基甜菜碱甲基丙烯酸酯并溶解在40ml 无水DMSO中,充分溶解后加入0.06g的偶氮二异丁腈,经氮吹除氧后封闭反应体系,将反 应体系置于75℃下恒温反应3d,反应完成后冷却,分离滤液,并用丙酮沉淀数次,真空干燥 制得共聚产物;
(4)包覆产物制备
称取所述共聚产物并溶解在热的蒸馏水中,加入所述初产物,充分分散后边搅拌边滴加 与所述蒸馏水等体积的丙酮,过滤,滤饼真空干燥后制得,其中,所述共聚产物与所述沉淀、 所述蒸馏水的质量比例为1:1:1000。
实验例
1、建立高血脂症大鼠动物模型
选用8周龄,体重200-225g的雄性大鼠50只,适应性饲养3天后,随机分为2组(正常组:10只,高脂模型组:40只),采用高脂饲料喂养法来建造高脂血症大鼠动物模型,正 常组进食正常基础饲料,高脂模型组进食高脂饲料,饮用经紫外线消毒的自来水,喂养时间30天,30天后禁食12h,眼眶取血检测大鼠血清中总胆固醇、甘油三酯和低密度脂蛋白胆固醇的含量,其结果呈现显著差异,具体见表1。
表1
含量(mmol/L) | 总胆固醇 | 甘油三酯 | 低密度脂蛋白胆固醇 |
正常组 | 1.42 | 0.76 | 1.72 |
高脂模型组 | 2.38 | 0.82 | 1.69 |
随机将高脂模型组分为4组,模型组和3组治疗组,每组10只,治疗组喂养高脂饲料, 每天灌胃2次实施例1-3所述微生态制剂,正常组合模型组每天灌胃2次等量生理盐水,喂 养时间60天,60天后禁食12h,眼眶取血检测大鼠血清中总胆固醇、甘油三酯和低密度脂蛋 白胆固醇的含量,其结果呈现显著差异,具体见表2。
表2
最后应当说明的是,以上实施例仅用以说明本发明的技术方案,而非对本发明保护范围 的限制,尽管参照较佳实施例对本发明作了详细地说明,本领域的普通技术人员应当理解, 可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种更适合黄种人群的微生态制剂,其特征在于,由益生菌活菌、复合培养基、载体以及中药提取物组成,所述益生菌活菌包括乳杆菌发酵液、双歧杆菌发酵液、链球菌发酵液、芽孢杆菌发酵液和酵母菌发酵液,其中,所述乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌的活菌数量比为100:(60-100):(10-30):(5-10):(1-5);
所述双歧杆菌包括两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、长双歧杆菌和短双歧杆菌;
所述微生态制剂制备方法为:
以第一培养基分别对乳杆菌、双歧杆菌、链球菌、芽孢杆菌和酵母菌进行活化并扩大培养,再将培养后的菌种分别接种到第二培养基中,分别发酵培养12-24h,以各菌种发酵液按活菌数量比例混合得到混合发酵液,加入冻干保护剂,混合均匀后再加入所述载体,所述载体经浸泡后滤出,冷冻干燥制得负载的载体,再与所述中药提取物混合后制得所述微生态制剂;其中,所述第一培养基的组成为大豆蛋白胨、牛肉蛋白粉、酵母膏、醋酸钠、葡萄糖、吐温80、柠檬酸铵、硫酸镁和水;所述第二培养基包括所述第一培养基和大豆多肽粉。
2.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述中药提取物包括山药、苍术、砂仁、人参、当归、甘草、党参、茯苓、黄精和山楂,其制备方法是:
依次称取各组分,加入重量/体积比10倍的蒸馏水,浸泡4h后,并于100℃提取1h,过滤,再向滤渣中加入重量/体积比8倍的蒸馏水,100℃提取1h后,过滤后合并滤液,离心,浓缩干燥制得所述中药提取物;
所述中药提取物与所述负载的载体的混合比例为(1-100):10。
3.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述乳杆菌为卷曲乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、詹氏乳杆菌、加氏乳杆菌、唾液乳杆菌、干酪乳杆菌、发酵乳杆菌、植物乳杆菌、保加利亚乳杆菌中的至少一种。
4.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述链球菌包括嗜热链球菌、消化链球菌、粪链球菌中的至少一种。
5.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述芽孢杆菌包括凝结芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌中的至少一种。
6.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述冻干保护剂为甘油和低聚木糖,添加量分别为所述混合发酵液质量的0.1-1%。
7.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述载体为改性蒙脱石。
8.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述改性蒙脱石的制备方法为:
将蒙脱石粉碎并筛分,得到粒径在500-1000筛目之间的微粉,将蒙脱石微粉按料液比1g/100ml悬浮分散在蒸馏水中,得到悬液B,加入所述悬液B体积1-2%的3-(三乙氧基硅基)丙基琥珀酸酐或3-氨基丙基三乙氧基硅烷,在60-70℃下搅拌回流12-24h,回流完成后滤出沉淀,沉淀依次以乙醇和蒸馏水洗涤制得。
9.根据权利要求1所述的一种更适合黄种人群的微生态制剂,其特征在于,所述改性蒙脱石的制备方法还包括以下步骤:
(1)烯丙基改性6-氨基嘌呤制备
称取溴乙醇,在冰水浴条件下溶解在二氯甲烷中,加入三乙胺和甲基丙烯酰氯,在室温条件下搅拌反应12-14h,反应完成后分离滤液,所述滤液依次经冰水洗涤、无水硫酸镁干燥后蒸去二氯甲烷,得到酯化产物;称取6-氨基嘌呤和氢化钠并溶解在无水DMF中,充分搅拌均匀后,加入所述酯化产物,在室温条件下搅拌反应12-14h,反应完成后分离滤液,并用丙酮沉淀数次,真空干燥;其中,
所述溴乙醇与所述二氯甲烷、所述三乙胺、所述甲基丙烯酰氯的质量比例为1:(6-7):(0.1-0.15):(0.9-0.95);所述6-氨基嘌呤与所述氢化钠、所述无水DMF、所述酯化产物的质量比例为1:(0.2-0.22):(10-12):(2.4-2.5);
(2)共聚产物制备
称取所述烯丙基改性6-氨基嘌呤和磺基甜菜碱甲基丙烯酸酯并溶解在无水DMSO中,充分溶解后加入引发剂,经氮吹除氧后封闭反应体系,将反应体系置于70-75℃下恒温反应3-4d,反应完成后冷却,分离滤液,并用丙酮沉淀数次,真空干燥制得共聚产物;其中,
所述烯丙基改性6-氨基嘌呤与所述磺基甜菜碱甲基丙烯酸酯、所述无水DMSO、所述引发剂的质量比例为1:(1.0-1.1):(50-100):(0.07-0.09);
(3)包覆产物制备
称取所述共聚产物并溶解在热的蒸馏水中,加入经乙醇和蒸馏水洗涤后制得的沉淀,充分分散后边搅拌边滴加与所述蒸馏水等体积的丙酮,过滤,滤饼真空干燥后制得,其中,
所述共聚产物与所述沉淀、所述蒸馏水的质量比例为1:1:1000。
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