CN111713685A - 一种微胶囊壁材及其在制备益生菌微胶囊中的应用 - Google Patents
一种微胶囊壁材及其在制备益生菌微胶囊中的应用 Download PDFInfo
- Publication number
- CN111713685A CN111713685A CN202010622001.5A CN202010622001A CN111713685A CN 111713685 A CN111713685 A CN 111713685A CN 202010622001 A CN202010622001 A CN 202010622001A CN 111713685 A CN111713685 A CN 111713685A
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- Prior art keywords
- wall material
- microcapsule wall
- probiotic
- freeze
- probiotics
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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Abstract
本发明公开了一种微胶囊壁材及其在制备益生菌微胶囊中的应用,属于微生物技术领域。本发明提供了一种可显著提高益生菌微胶囊中益生菌存活率的微胶囊壁材,此微胶囊壁材的成分包含酪蛋白酸钠、阴离子多糖以及冻干保护剂;使用此微胶囊壁材制备益生菌微胶囊时,益生菌的包埋率高达79.23%,将使用此微胶囊壁材制备得到的益生菌微胶囊进行冷冻干燥后,益生菌的冻干存活率高达18.21%,并且,将使用此微胶囊壁材制备得到的益生菌微胶囊用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达35.24%。
Description
技术领域
本发明涉及一种微胶囊壁材及其在制备益生菌微胶囊中的应用,属于微生物技术领域。
背景技术
益生菌是活的、对人体健康有益的一类微生物。当适量摄入时,可以抑制消化道致病菌繁殖,维持肠道菌群平衡,促进营养物质的吸收,从而提高机体免疫力。通常认为益生菌要发挥其益生功效,必须在产品与宿主中都保持活性和代谢稳定,一般要求每g或每mL产品中活菌数不少于106CFU。然而,在益生菌产品的生产、运输、销售、贮藏等环节,益生菌活性极易受pH、温度、氧以及宿主消化系统(人体胃部的pH值大约是1.8~3.0)的影响,其活菌数将大幅下降,使最终定植于肠道中的活菌数低于理论值。
微胶囊包埋技术是一种能够对益生菌提供有效保护效果的技术,可以在菌体与外界环境之间形成一层物理屏障,从而延缓不良环境对菌体的损伤。目前,常见的微胶囊包埋技术主要有挤压法、乳化法与喷雾干燥法三种。其中,挤压法通常是先将微胶囊壁材与益生菌菌悬液混合,得到混合液,然后将混合液通过针孔或喷嘴挤出,以液滴形式滴入固化剂,得到益生菌微胶囊。这种微胶囊包埋方法包埋效率低且制得的包埋产物粒径过大,不适用于大规模工业化生产。
乳化法主要是先将益生菌菌悬液与微胶囊壁材混合,得到混合液,然后将混合液注入油相并添加相应的乳化剂,搅拌形成乳液,最后在乳液中添加相应的固化剂,得到益生菌微胶囊。与挤压法相比,乳化法包埋效率更高且包埋产物颗粒更小,但是,与挤压法相比,乳化法制得的包埋产物含水量高、不便贮藏。
除此以外,挤压法和乳化法由于工序较多,其包埋过程中益生菌暴露于有氧环境过长,易引起菌体失活。
喷雾干燥法主要是先将益生菌菌悬液与微胶囊壁材混合,得到混合液,然后将混合液进行喷雾干燥,得到益生菌微胶囊。与乳化法相比,喷雾干燥法解决了包埋产物的贮藏问题,并且,喷雾干燥法的工艺更简单,但是,在喷雾干燥的过程中,益生菌会受到高温影响,将会产生极大的活性损失。
可见,使用现有的微胶囊包埋技术制备益生菌微胶囊均存在制备得到的益生菌微胶囊中益生菌存活率低的问题。因此,急需找到一种提高益生菌微胶囊中益生菌存活率的方法。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种提高益生菌微胶囊中益生菌存活率的方法。
[技术方案]
为解决本发明的技术问题,本发明提供了一种微胶囊壁材,所述微胶囊壁材的成分包含酪蛋白酸钠、阴离子多糖以及冻干保护剂。
在本发明的一种实施方式中,所述阴离子多糖为羧甲基纤维素钠、海藻酸钠、黄原胶或阿拉伯胶中的一种或一种以上。
在本发明的一种实施方式中,所述冻干保护剂为水苏糖、低聚果糖、低聚木糖、低聚半乳糖或低聚异麦芽糖中的一种或一种以上。
在本发明的一种实施方式中,所述微胶囊壁材的成分包含酪蛋白酸钠10g/L~90g/L、阴离子多糖5g/L~20g/L以及冻干保护剂100g/L~200g/L。
在本发明的一种实施方式中,所述微胶囊壁材的成分包含酪蛋白酸钠90g/L、阴离子多糖10g/L以及冻干保护剂100g/L。
本发明还提供了一种制备益生菌微胶囊的方法,所述方法为使用上述微胶囊壁材制备益生菌微胶囊。
在本发明的一种实施方式中,所述方法为将益生菌培养液离心,收集菌泥;将菌泥重悬于生理盐水或缓冲液中,得到菌悬液;将菌悬液与上述微胶囊壁材混合,得到混合液A;在混合液A中添加油相进行搅拌,得到混合液B;在混合液B中添加有机酸进行搅拌,得到混合液C;在混合液C中添加生理盐水进行搅拌,得到混合液D;将混合液D冰浴静置后进行离心,收集沉淀;将沉淀用生理盐水或缓冲液清洗后,得到益生菌微胶囊。
在本发明的一种实施方式中,所述菌泥与生理盐水或缓冲液的质量比为1~2:1~2。
在本发明的一种实施方式中,所述菌泥与生理盐水或缓冲液的质量比为1:1。
在本发明的一种实施方式中,所述菌悬液的pH为6.0~6.5。
在本发明的一种实施方式中,所述菌悬液的pH为6.5。
在本发明的一种实施方式中,所述菌悬液与微胶囊壁材的体积比为1~2:3~9。
在本发明的一种实施方式中,所述菌悬液与微胶囊壁材的体积比为2:5。
在本发明的一种实施方式中,所述混合液A与油相的体积比为1~2:3~9。
在本发明的一种实施方式中,所述混合液A与油相的体积比为1:3。
在本发明的一种实施方式中,所述油相为大豆油。
在本发明的一种实施方式中,在混合液B中添加有机酸使得混合液B的pH为4.4~4.8。
在本发明的一种实施方式中,在混合液B中添加有机酸使得混合液B的pH为4.6。
在本发明的一种实施方式中,所述有机酸为冰醋酸。
在本发明的一种实施方式中,所述混合液C与生理盐水的体积比为1~2:1~3。
在本发明的一种实施方式中,所述混合液C与生理盐水的体积比为1:1。
在本发明的一种实施方式中,所述生理盐水的温度为4℃
在本发明的一种实施方式中,所述冰浴静置的时间为30min。
在本发明的一种实施方式中,所述益生菌包括罗伊氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、约氏乳杆菌、格氏乳杆菌、干酪乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌、卷曲乳杆菌、唾液乳杆菌、清酒乳杆菌、德氏乳杆菌和/或瑞士乳杆菌。
本发明还提供了一种益生菌微胶,所述益生菌微胶是使用上述方法制备得到的。
本发明还提供了上述微胶囊壁材或上述方法在制备益生菌微胶中的应用。
[有益效果]
(1)本发明提供了一种可显著提高益生菌微胶囊中益生菌存活率的微胶囊壁材,此微胶囊壁材的成分包含酪蛋白酸钠、阴离子多糖以及冻干保护剂;使用此微胶囊壁材制备益生菌微胶囊时,益生菌的包埋率高达79.23%,将使用此微胶囊壁材制备得到的益生菌微胶囊进行冷冻干燥后,益生菌的冻干存活率高达18.21%,并且,将使用此微胶囊壁材制备得到的益生菌微胶囊用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达35.24%。
(2)本发明提供了一种可显著提高益生菌微胶囊中益生菌存活率的制备益生菌微胶囊的方法,此方法为使用成分包含酪蛋白酸钠、阴离子多糖以及冻干保护剂的微胶囊壁材制备益生菌微胶囊;使用此方法制备益生菌微胶囊时,益生菌的包埋率高达79.23%,将使用此方法制备得到的益生菌微胶囊进行冷冻干燥后,益生菌的冻干存活率高达18.21%,并且,将使用此方法制备得到的益生菌微胶囊用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达35.24%。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的水苏糖、酪蛋白酸钠、羧甲基纤维素钠、海藻酸钠、黄原胶、阿拉伯胶、冰醋酸、生理盐水购自国药集团化学试剂公司;下述实施例中涉及的大豆油购自欧尚超市;下述实施例中涉及的益生菌为植物乳杆菌,植物乳杆菌记载于公开号为CN102827796A的专利申请文本中,保藏编号为CGMCC No.6077。
下述实施例中涉及的培养基如下:
MRS固体培养基:酵母粉5g/L,无水乙酸钠5g/L,牛肉膏10g/L,无水葡萄糖20g/L,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,琼脂20g/L,吐温-801g/L。
MRS液体培养基:酵母粉5g/L,无水乙酸钠5g/L,牛肉膏10g/L,无水葡萄糖20g/L,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。
模拟胃液:胃蛋白酶3.2g/L、氯化钠2g/L,pH 2.5。
下述实施例中涉及的检测方法如下:
益生菌活菌数的检测方法:采用国标《GB 4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。
益生菌微胶囊包埋率的检测方法:测定微胶囊悬浮液中的总活菌数和2mL菌悬液中的总活菌数,并计算益生菌的包埋率;
其中,益生菌微胶囊包埋率的计算公式如下:
益生菌微胶囊冻干存活率的检测方法:在冷冻真空干燥前测定微胶囊悬浮液中益生菌的活菌数作为微胶囊冻干前活菌浓度,在真空冷冻干燥后,将冻干菌粉用无菌水复溶至真空冷冻干燥前体积,得到复溶液,测定复溶液中益生菌活菌数作为微胶囊冻干后活菌浓度,根据测定结果计算益生菌微胶囊冻干存活率;
其中,益生菌微胶囊冻干存活率的计算公式如下:
益生菌经pH为2.5的模拟胃液处理2h后的存活率的检测方法:称取冻干菌粉0.01g,加入1mL无菌生理盐水复溶,得到复溶液,测定复溶液中双歧杆菌活菌数作为0.01g冻干菌粉酸处理前总活菌数;称取双歧杆菌冻干粉0.01g加入10mL模拟胃液中,37℃培养2h后取样,测定样本中双歧杆菌活菌数作为0.01g冻干菌粉酸处理后总活菌数,根据测定结果计算双歧杆菌经pH为2.5的模拟胃液处理2h后的存活率;
其中,益生菌经pH为2.5的模拟胃液处理2h后的存活率的计算公式如下:
实施例1:益生菌微胶囊和冻干菌粉的制备
具体步骤如下:
配置成分为酪蛋白酸钠90g/L、羧甲基纤维素钠10g/L以及水苏糖100g/L的微胶囊壁材;用接种环蘸取保菌管中的益生菌菌液在MRS固体培养基上划线,于37℃恒温培养24h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃恒温培养12h,得到种子液;将种子液按4%(v/v)的接种量接种至新鲜的MRS液体培养基中,37℃恒温培养12h,得到菌液;将菌液8000g下离心20min,收集菌泥;将菌泥重悬于生理盐水中,得到菌悬液;将菌悬液的pH调节至6.5后(通过质量体积分数为3%的NaOH溶液调节pH),以菌悬液为空白对照,将菌悬液与微胶囊壁材混合,得到混合液A;在混合液A中添加大豆油,500rpm搅拌15min,得到混合液B;在混合液B中添加冰醋酸至pH为4.6,500rpm搅拌5min,得到混合液C;在混合液C中加入预冷的生理盐水(4℃),500rpm搅拌3min,得到混合液D;将混合液D冰浴静置30min后进行离心,收集沉淀;将沉淀用生理盐水重悬后,得到酪蛋白酸钠-羧甲基纤维素钠微胶囊悬浮液,记为SC-CMC微胶囊;其中,菌泥与生理盐水的质量比为1:1,菌悬液与微胶囊壁材的体积比为2:5,混合液A与大豆油的体积比为1:3,混合液C与生理盐水的体积比为1:1。
对SC-CMC微胶囊进行冷冻干燥,得到冻干菌粉;其中冷冻干燥在冷冻干燥机(购自西班牙泰事达公司)内完成,包括预冻、一次干燥和二次干燥,预冻为控制层板1h内降温至-50℃,保持4h,一次干燥为控制层板1.3h升温至-30℃,保持30h,二次干燥为控制层板1h升温至25℃,保持20h。
检测SC-CMC微胶囊中益生菌的包埋率以及冻干菌粉中益生菌的冻干存活率(检测结果见表1)。检测经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率(检测结果见表1)。
由表1可知,使用成分为酪蛋白酸钠90g/L、阴离子多糖10g/L以及冻干保护剂100g/L的微胶囊壁材微胶囊壁材制备得到的SC-CMC微胶囊中,益生菌的包埋率高达79.23±2.34%,使用此微胶囊壁材制备得到的冻干菌粉中,益生菌的冻干存活率高达18.21±0.86%,并且,将使用此微胶囊壁材制备得到的冻干菌粉用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达35.24±1.14%。
表1 SC-CMC微胶囊中益生菌的包埋率、冻干菌粉中益生菌的冻干存活率以及经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率
实施例2:益生菌微胶囊和冻干菌粉的制备
具体步骤如下:
在实施例1的基础上,将羧甲基纤维素钠替换为海藻酸钠(SA),得到酪蛋白酸钠-海藻酸钠微胶囊(SC-SA微胶囊)和冻干菌粉。
检测SC-SA微胶囊中益生菌的包埋率以及冻干菌粉中益生菌的冻干存活率(检测结果见表2)。检测经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率(检测结果见表2)。
由表2可知,使用成分为酪蛋白酸钠90g/L、阴离子多糖10g/L以及冻干保护剂100g/L的微胶囊壁材微胶囊壁材制备得到的SC-SA微胶囊中,益生菌的包埋率高达75.26±3.56%,使用此微胶囊壁材制备得到的冻干菌粉中,益生菌的冻干存活率高达15.33±0.54%,并且,将使用此微胶囊壁材制备得到的冻干菌粉用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达36.78±2.36%。
表2 SC-SA微胶囊中益生菌的包埋率、冻干菌粉中益生菌的冻干存活率以及经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率
实施例3:益生菌微胶囊和冻干菌粉的制备
具体步骤如下:
在实施例1的基础上,将酪蛋白酸钠的浓度调整为70g/L,将羧甲基纤维素钠替换为黄原胶(Xan),并且,将黄原胶(Xan)的浓度调整为5g/L,得到酪蛋白酸钠-黄原胶微胶囊(SC-Xan微胶囊)和冻干菌粉。
检测SC-Xan微胶囊中益生菌的包埋率以及冻干菌粉中益生菌的冻干存活率(检测结果见表3)。检测经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率(检测结果见表3)。
由表3可知,使用成分为酪蛋白酸钠90g/L、阴离子多糖10g/L以及冻干保护剂100g/L的微胶囊壁材微胶囊壁材制备得到的SC-Xan微胶囊中,益生菌的包埋率高达76.84±2.28%,使用此微胶囊壁材制备得到的冻干菌粉中,益生菌的冻干存活率高达12.58±1.37%,并且,将使用此微胶囊壁材制备得到的冻干菌粉用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达35.65±2.42%。
表3 SC-Xan微胶囊中益生菌的包埋率、冻干菌粉中益生菌的冻干存活率以及经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率
实施例4:益生菌微胶囊和冻干菌粉的制备
具体步骤如下:
在实施例1的基础上,将酪蛋白酸钠的浓度调整为50g/L,将羧甲基纤维素钠替换为阿拉伯胶(Arab),并且,将阿拉伯胶(Arab)的浓度调整为6g/L,得到酪蛋白酸钠-阿拉伯胶微胶囊(SC-Arab微胶囊)和冻干菌粉。
检测SC-Arab微胶囊中益生菌的包埋率以及冻干菌粉中益生菌的冻干存活率(检测结果见表4)。检测经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率(检测结果见表4)。
由表4可知,使用成分为酪蛋白酸钠90g/L、阴离子多糖10g/L以及冻干保护剂100g/L的微胶囊壁材微胶囊壁材制备得到的SC-Arab微胶囊中,益生菌的包埋率高达74.34±2.22%,使用此微胶囊壁材制备得到的冻干菌粉中,益生菌的冻干存活率高达14.38±0.36%,并且,将使用此微胶囊壁材制备得到的冻干菌粉用pH为2.5的模拟胃液处理2h后,益生菌的存活率高达32.44±1.08%。
表4 SC-Arab微胶囊中益生菌的包埋率、冻干菌粉中益生菌的冻干存活率以及经pH为2.5的模拟胃液处理2h后的冻干菌粉中益生菌的存活率
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。
Claims (10)
1.一种微胶囊壁材,其特征在于,所述微胶囊壁材的成分包含酪蛋白酸钠、阴离子多糖以及冻干保护剂。
2.如权利要求1所述的一种微胶囊壁材,其特征在于,所述阴离子多糖为羧甲基纤维素钠、海藻酸钠、黄原胶或阿拉伯胶中的一种或一种以上。
3.如权利要求1或2所述的一种微胶囊壁材,其特征在于,所述冻干保护剂为水苏糖、低聚果糖、低聚木糖、低聚半乳糖或低聚异麦芽糖中的一种或一种以上。
4.如权利要求1所述的一种微胶囊壁材,其特征在于,所述微胶囊壁材的成分包含酪蛋白酸钠10g/L~90g/L、阴离子多糖5g/L~20g/L以及冻干保护剂100g/L~200g/L。
5.一种制备益生菌微胶囊的方法,其特征在于,所述方法为使用权利要求1~4任一项所述的微胶囊壁材制备益生菌微胶囊。
6.如权利要求5所述的一种制备益生菌微胶囊的方法,其特征在于,所述方法为将益生菌培养液离心,收集菌泥;将菌泥重悬于生理盐水或缓冲液中,得到菌悬液;将菌悬液与权利要求1~4任一项所述的微胶囊壁材混合,得到混合液A;在混合液A中添加油相进行搅拌,得到混合液B;在混合液B中添加有机酸进行搅拌,得到混合液C;在混合液C中添加生理盐水进行搅拌,得到混合液D;将混合液D冰浴静置后进行离心,收集沉淀;将沉淀用生理盐水或缓冲液清洗后,得到益生菌微胶囊。
7.如权利要求6所述的一种制备益生菌微胶囊的方法,其特征在于,所述菌泥与生理盐水或缓冲液的质量比为1~2:1~2。
8.如权利要求6或7所述的一种制备益生菌微胶囊的方法,其特征在于,所述菌悬液与微胶囊壁材的体积比为1~2:3~9。
9.一种益生菌微胶,其特征在于,所述益生菌微胶是使用权利要求5-8任一项所述的方法制备得到的。
10.权利要求1-4任一项所述的微胶囊壁材或权利要求5-8任一项所述的方法在制备益生菌微胶中的应用。
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