CN113475717A - 一种益生菌组合物的制备方法 - Google Patents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23P10/00—Shaping or working of foodstuffs characterised by the products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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Abstract
本发明涉及益生菌技术领域,具体涉及一种益生菌组合物的制备方法,包括抗性淀粉的制备、益生菌的活化、混合双菌的制备和微胶囊化等步骤,本发明对添加了抗性淀粉和魔芋多糖的嗜酸乳杆菌和长双岐杆菌的混合双菌进行微胶囊化处理,大大提高混合双菌的耐酸性,大大减少人体胃酸对益生菌的伤害,提高益生菌在体内存活率,并且能够在肠道内快速完成崩解,释放出益生菌,使益生菌真正发挥益生作用。
Description
技术领域
本发明涉及益生菌技术领域,具体涉及一种益生菌组合物的制备方法。
背景技术
益生菌对人体有着多种益生功能,能够调节肠道环境,维持肠道内的菌群平衡,抑制有害菌的生长,并能够代谢出一些有利于人体健康的代谢产物。正常情况下,人体的消化道内生存着各种各样的微生物,这些微生物对人体内的微生态平衡和身体健康起着不可替代的作用,但如果肠道内的微生态被打破,有害菌会大量繁殖,影响人体的健康,益生菌可以在消化道内代谢产生一些有机酸、过氧化氢、细菌素等物质,有效抑制有害菌的生长;当益生菌在肠道内的数量足够多时,代谢产生的有机酸类对人体的肠壁神经起到刺激作用,加快肠道蠕动速度,同时还能提高肠道内的渗透压,使水分更多的进入肠道,达到润滑通便的作用。因此,益生菌越来越广泛的被应用于食品、药品等行业。
益生菌分布广泛,种类繁多,益生作用也千差万别。目前主要分为三大类:一是乳杆菌属益生菌,在生长发育繁殖过程中可以产生大量乳酸,所以也被称为乳酸菌,在人体肠道中有着非常重要的作用,如干酪乳杆菌、嗜酸乳杆菌;二是双岐杆菌属益生菌,能适应肠道内的无氧环境,可以在小肠的中后部分和大肠内生长繁殖,保护身体不受致病菌的侵害,从而提高免疫力,还可以分泌一种双岐因子,起到预防便秘、促进肠道蠕动,如长双岐杆菌、青春双歧杆菌;三是革兰氏阳性球菌,对环境的适应力和抵抗力强,如粪链球菌、乳球菌。
但是,要发挥益生菌益生作用,就必须提高益生菌的存活率,使其在肠道内大量存活,这样才能改善肠道微生态和代谢出足够的代谢产物来刺激肠道。但是益生菌的耐酸性差,人体的胃酸和胆汁对益生菌的伤害极大,很少有益生菌能承受这么恶劣的环境,益生菌在体内存活率较低,达不到益生效果。
发明内容
针对现有技术的不足,本发明提供了一种益生菌组合物的制备方法,对添加了抗性淀粉和魔芋多糖的嗜酸乳杆菌和长双岐杆菌的混合双菌进行微胶囊化处理,大大提高混合双菌的耐酸性,大大减少人体胃酸对益生菌的伤害,提高益生菌在体内存活率,并且能够在肠道内快速完成崩解,释放出益生菌,使益生菌真正发挥益生作用。
为实现以上目的,本发明通过以下技术方案予以实现:
一种益生菌组合物的制备方法,包括以下步骤:
1)抗性淀粉的制备:用蒸馏水将高直链玉米淀粉配置成浓度为20%的溶液,121℃高压灭菌锅中糊化20min,冷却至室温,4℃冰箱中老化24h,重复2次后,加入α-淀粉酶,60℃搅拌水解24h后,调节pH至4.5,加入糖化酶,60℃搅拌水解3h,沸水浴灭菌5min,加入95%的乙醇,静置8-12h后离心,去除上清液,95%的乙醇水洗2次,60℃烘干,粉碎,过100目筛;
2)益生菌的活化:将嗜酸乳杆菌和长双岐杆菌分别以1mL/100mL的接种量接种至MRS液体培养基中,在37℃恒温培养下,培养20-24h,得到活化的嗜酸乳杆菌菌种和长双岐杆菌菌种;
3)混合:将活化的嗜酸乳杆菌菌种以1mL/100mL的接种量接种至含有抗性淀粉的一号混合培养液中,将活化的长双岐杆菌菌种以1mL/100mL的接种量接种至含有魔芋多糖的二号混合培养液中,分别在37℃恒温培养下,培养20-24h后,取出混匀,4℃、4000r/min离心15-20min,得到菌泥;
4)微胶囊化:将步骤3)制得的菌泥与灭菌后的海藻酸钠溶液、碳酸钙溶液混合均匀,再加入含有吐温-80的大豆油,4℃搅拌15-20min,进行乳化,再加入含有冰乙酸的大豆油,4℃搅拌25-30min后,加入醋酸盐缓冲液,静置2-3h,4℃、4000r/min离心10-12min,弃去上层液体,生理盐水洗涤三次,收集得到微胶囊,4℃冷藏保存。
进一步的,所述MRS液体培养基的配方为10g牛肉浸膏、20g葡萄糖、10g蛋白胨、5g酵母浸膏、0.05g四水硫酸锰、1.0mL吐温-80、2g磷酸氢二钾、5g三水乙酸钠、0.58g七水硫酸镁、0.25g硫酸锰、2g柠檬酸二铵和1000mL蒸馏水。
进一步的,所述步骤3)的一号混合培养液为含有0.50%抗性淀粉的MRS液体培养基,所述步骤3)的二号混合培养液为含有0.95%魔芋多糖的MRS液体培养基。
进一步的,所述步骤4)中乳化的搅拌速度是600r/min,所述乳化的时间为15min,所述含有吐温-80的大豆油为每10mL大豆油中含有150μL吐温-80,所述含有冰乙酸的大豆油为每10mL大豆油中含有50μL冰乙酸。
进一步的,所述步骤4)中醋酸盐缓冲液的pH值为5.5。
进一步的,所述步骤4)中菌泥、海藻酸钠、碳酸钙的质量比为2:1:6。
由于采用上述的技术方案,本发明的有益效果如下:
1)本发明对嗜酸乳杆菌和长双岐杆菌的混合双菌进行微胶囊化处理,通过模拟人工胃酸环境,微胶囊化的嗜酸乳杆菌和长双岐杆菌的混合双菌在胃酸中2h后还能保持很高的存活率(78.9%),大大提高混合双菌的耐酸性,大大减少人体胃酸对益生菌的伤害,提高益生菌在体内存活率,并且能够在肠道内快速完成崩解,释放出益生菌,使益生菌真正发挥益生作用;
2)本发明的嗜酸乳杆菌和长双岐杆菌混合双菌中还添加抗性淀粉和魔芋多糖,既有效促进嗜酸乳杆菌和长双岐杆菌的生长繁殖,对益生菌混合双菌的生长具有积极的刺激作用,还能对菌体死亡有抑制作用,提高混合双菌在酸性环境下的存活率;
3)本发明对添加了抗性淀粉和魔芋多糖的嗜酸乳杆菌和长双岐杆菌的混合双菌进行微胶囊化处理,增加了在肠道的缚水能力,抑制小肠对水分的吸收利用,保证肠壁水分充足,增加粪便容量,改善肠道内环境,使肠内厌氧微生物数量增加,促进肠道蠕动,加速体内有毒物质的排泄,同时还能促进肠道内益生菌群生长繁殖,调节肠道微生态环境,并被肠道内微生物发酵产生短链脂肪酸如能够降低肿瘤细胞的扩增率的丁酸。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例,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作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
将活化的嗜酸乳杆菌和长双岐杆菌(菌种由中国普通微生物菌种保藏管理中心提供)分别以1mL/100mL的接种量接种至MRS液体培养基中,在37℃恒温培养下,培养24h。MRS液体培养基中不添加抗性淀粉或魔芋多糖。
实施例2
将活化的嗜酸乳杆菌菌种以1mL/100mL的接种量接种至含有0.50%抗性淀粉的MRS液体培养基中,将活化的长双岐杆菌菌种以1mL/100mL的接种量接种至含有0.95%魔芋多糖的MRS液体培养基中,分别在37℃恒温培养下,培养24h后,取出混匀。
实施例3
将活化的嗜酸乳杆菌和长双岐杆菌分别以1mL/100mL的接种量接种至MRS液体培养基中,在37℃恒温培养下,培养24h后,取出混匀,4℃、4000r/min离心15min,得到菌泥;将菌泥与灭菌后的海藻酸钠溶液、碳酸钙溶液混合均匀,菌泥、海藻酸钠、碳酸钙的质量比为2:1:6,再加入含有600μL吐温-80的大豆油40mL,4℃、600r/min搅拌15min,进行乳化,再加入含有100μL冰乙酸的大豆油20mL,4℃搅拌30min后,加入60mL、pH为5.5醋酸盐缓冲液,静置2h,4℃、4000r/min离心10min,弃去上层液体,生理盐水洗涤三次,收集得到微胶囊,4℃冷藏保存。
实施例4
用蒸馏水将10g高直链玉米淀粉配置成浓度为20%的溶液,121℃高压灭菌锅中糊化20min,冷却至室温,4℃冰箱中老化24h,重复2次后(糊化-冷却循环),加入1mLα-淀粉酶(1%,2700U/mL),60℃搅拌水解24h后,调节pH至4.5,加入1mL糖化酶(1%,1600U/mL),60℃搅拌水解3h,沸水浴灭菌5min,加入沉淀4倍体积的95%的乙醇,静置12h后离心,去除上清液,95%的乙醇水洗2次,60℃烘干,粉碎,过100目筛,制得抗性淀粉;
将活化的嗜酸乳杆菌菌种以1mL/100mL的接种量接种至含有0.50%抗性淀粉的MRS液体培养基中,将活化的长双岐杆菌菌种以1mL/100mL的接种量接种至含有0.95%魔芋多糖的MRS液体培养基中,分别在37℃恒温培养下,培养24h后,取出混匀,4℃、4000r/min离心15min,得到菌泥;
将上述制得的菌泥与灭菌后的海藻酸钠溶液、碳酸钙溶液混合均匀,菌泥、海藻酸钠、碳酸钙的质量比为2:1:6,再加入含有600μL吐温-80的大豆油40mL,4℃、600r/min搅拌15min,进行乳化,再加入含有100μL冰乙酸的大豆油20mL,4℃搅拌30min后,加入60mL、pH为5.5醋酸盐缓冲液,静置2h,4℃、4000r/min离心10min,弃去上层液体,生理盐水洗涤三次,收集得到微胶囊,4℃冷藏保存。
实施例1-4使用的MRS液体培养基的配方为:10g牛肉浸膏、20g葡萄糖、10g蛋白胨、5g酵母浸膏、0.05g四水硫酸锰、1.0mL吐温-80、2g磷酸氢二钾、5g三水乙酸钠、0.58g七水硫酸镁、0.25g硫酸锰、2g柠檬酸二铵和1000mL蒸馏水。
以实施例1-3为对照组(实施例1为嗜酸乳杆菌和长双岐杆菌未经包埋的混合裸菌,实施例2为加入抗性淀粉、魔芋多糖的嗜酸乳杆菌和长双岐杆菌未经包埋的混合裸菌、实施例3为为加入抗性淀粉、魔芋多糖的嗜酸乳杆菌和长双岐杆菌混合微胶囊),以胃蛋白酶制备人工胃液,以胰蛋白酶制备人工肠液,以平板菌落计数法测定实施例1-4的活菌数,结果如表1-2所示:
表1本发明实施例1-4耐胃酸实验存活率(%)
实施例1 | 实施例2 | 实施例3 | 实施例4 | |
30min | 50.0 | 50.5 | 93.0 | 94.8 |
60min | 10.0 | 10.2 | 88.5 | 92.0 |
120min | <5.0 | <5.0 | 75.5 | 78.9 |
表2本发明实施例1-4肠溶性实验的活菌数(cfu/mL)
实施例1 | 实施例2 | 实施例3 | 实施例4 | |
0 | 6.1 | 6.2 | 6.2 | 6.2 |
20min | 6.1 | 6.2 | 6.8 | 7.1 |
30min | 6.1 | 6.1 | 7.8 | 8.0 |
60min | 6.1 | 6.1 | 7.9 | 8.2 |
从表1可以看出,在人工胃酸环境下,未经包埋的实施例1、2中益生菌的存活率下降,实施例1、2的混合双菌裸菌在2h后基本全部死亡,而经过包埋的实施例3、4虽然有所下降,但在胃酸中2h后存活率还能保持在75%以上,添加了抗性淀粉、魔芋多糖的实施例4的益生菌存活率要高于未添加抗性淀粉、魔芋多糖的实施例3,微胶囊能对益生菌混合双菌起到保护作用,帮助益生菌混合双菌抵抗酸性环境的侵蚀,并且抗性淀粉、魔芋多糖对益生菌混合双菌的生长具有积极的刺激作用,能提高其在酸性环境下的存活率。
益生菌组合物微胶囊想要在人体内发挥益生作用,必须在肠道内尽快崩解,释放出益生菌。从表2可以看出,实施例3、4的微胶囊在30min就已经完成崩解,活菌数量比实施例1、2未经包埋的裸菌有大幅度的提高,并且在人工肠液中持续培养,益生菌的肠溶出率也在一直增加。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (6)
1.一种益生菌组合物的制备方法,其特征在于,包括以下步骤:
1)抗性淀粉的制备:用蒸馏水将高直链玉米淀粉配置成浓度为20%的溶液,121℃高压灭菌锅中糊化20min,冷却至室温,4℃冰箱中老化24h,重复2次后,加入α-淀粉酶,60℃搅拌水解24h后,调节pH至4.5,加入糖化酶,60℃搅拌水解3h,沸水浴灭菌5min,加入95%的乙醇,静置8-12h后离心,去除上清液,95%的乙醇水洗2次,60℃烘干,粉碎,过100目筛;
2)益生菌的活化:将嗜酸乳杆菌和长双岐杆菌分别以1mL/100mL的接种量接种至MRS液体培养基中,在37℃恒温培养下,培养20-24h,得到活化的嗜酸乳杆菌菌种和长双岐杆菌菌种;
3)混合:将活化的嗜酸乳杆菌菌种以1mL/100mL的接种量接种至含有抗性淀粉的一号混合培养液中,将活化的长双岐杆菌菌种以1mL/100mL的接种量接种至含有魔芋多糖的二号混合培养液中,分别在37℃恒温培养下,培养20-24h后,取出混匀,4℃、4000r/min离心15-20min,得到菌泥;
4)微胶囊化:将步骤3)制得的菌泥与灭菌后的海藻酸钠溶液、碳酸钙溶液混合均匀,再加入含有吐温-80的大豆油,4℃搅拌15-20min,进行乳化,再加入含有冰乙酸的大豆油,4℃搅拌25-30min后,加入醋酸盐缓冲液,静置2-3h,4℃、4000r/min离心10-12min,弃去上层液体,生理盐水洗涤三次,收集得到微胶囊,4℃冷藏保存。
2.根据权利要求1所述的益生菌组合物的制备方法,其特征在于,所述MRS液体培养基的配方为10g牛肉浸膏、20g葡萄糖、10g蛋白胨、5g酵母浸膏、0.05g四水硫酸锰、1.0mL吐温-80、2g磷酸氢二钾、5g三水乙酸钠、0.58g七水硫酸镁、0.25g硫酸锰、2g柠檬酸二铵和1000mL蒸馏水。
3.根据权利要求1所述的益生菌组合物的制备方法,其特征在于,所述步骤3)的一号混合培养液为含有0.50%抗性淀粉的MRS液体培养基,所述步骤3)的二号混合培养液为含有0.95%魔芋多糖的MRS液体培养基。
4.根据权利要求1所述的益生菌组合物的制备方法,其特征在于,所述步骤4)中乳化的搅拌速度是600r/min,所述乳化的时间为15min,所述含有吐温-80的大豆油为每10mL大豆油中含有150μL吐温-80,所述含有冰乙酸的大豆油为每10mL大豆油中含有50μL冰乙酸。
5.根据权利要求1所述的益生菌组合物的制备方法,其特征在于,所述步骤4)中醋酸盐缓冲液的pH值为5.5。
6.根据权利要求1所述的益生菌组合物的制备方法,其特征在于,所述步骤4)中菌泥、海藻酸钠、碳酸钙的质量比为2:1:6。
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