WO2020107580A1 - 以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊及制法 - Google Patents

以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊及制法 Download PDF

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WO2020107580A1
WO2020107580A1 PCT/CN2018/122643 CN2018122643W WO2020107580A1 WO 2020107580 A1 WO2020107580 A1 WO 2020107580A1 CN 2018122643 W CN2018122643 W CN 2018122643W WO 2020107580 A1 WO2020107580 A1 WO 2020107580A1
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arabinoxylan
probiotic
sodium alginate
preparation
mixed solution
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PCT/CN2018/122643
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English (en)
French (fr)
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汪惠丽
陶晗
廖巧明
徐毅
谷小珍
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合肥工业大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals

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  • the application relates to a preparation method of probiotic microcapsules, in particular to a probiotic microcapsule using arabinoxylan-sodium alginate composite gel as a wall material and a preparation method thereof, belonging to the field of food biotechnology.
  • Probiotics refer to a group of living microorganisms that promote the health of the host. Exogenous oral probiotics can significantly improve the host’s intestinal microecological balance, thereby preventing diarrhea, alleviating lactose intolerance, enhancing immunity, and reducing serum Cholesterol and various physiological effects such as inhibiting the formation of tumor cell precursors.
  • probiotics are a type of anaerobic or facultative anaerobic microorganisms. They do not form spores during growth and are less resistant. They also produce some metabolites such as lactic acid and acetic acid that threaten their own growth and reproduction, and survive under extreme conditions. The rate is very low. Encapsulation of probiotics is one of the most effective and practical ways to protect its activity.
  • Sodium alginate is one of the most commonly used wall materials for embedding probiotics. It has a wide range of sources, low price, mild gelation conditions, simple gelation operation, and has the characteristics of pH sensitivity and high biocompatibility, but it is used alone When probiotics are embedded, the protective effect of probiotics is not ideal. For example, the surface pore size of pure sodium alginate microcapsules is not small enough to completely prevent gastric juice from infiltrating. Under low pH conditions, sodium alginate is not stable enough, and it is prone to burst release and Early release phenomenon.
  • the main purpose of the present application is to provide a probiotic microcapsule using arabinoxylan-sodium alginate composite gel as a wall material and a preparation method thereof to overcome the shortcomings in the prior art.
  • the embodiments of the present application provide a preparation method of probiotic microcapsules using arabinoxylan-sodium alginate as a wall material, including:
  • the sterilized mixed solution, probiotic bacterial solution, and laccase are mixed uniformly and subjected to a composite gel reaction, and then solidified to obtain probiotic microcapsules using arabinoxylan-sodium alginate as a wall material.
  • the preparation method specifically includes:
  • step (3) Mix and shake the probiotic liquid obtained in step (1), the sterilized mixed solution obtained in step (2), and laccase to perform a composite gel reaction;
  • step (3) The mixed solution obtained in step (3) is cured in a curing agent at room temperature for 0.5 to 1 hour, and left to stand until the gel beads are formed, that is, probiotic microcapsules using arabinoxylan-sodium alginate as a wall material.
  • the probiotic is preferably Lactobacillus rhamnosus (GR-1), but is not limited thereto.
  • the examples of the present application also provide probiotic microcapsules prepared by the foregoing method using arabinoxylan-sodium alginate as a wall material, and the embedding rate of Lactobacillus rhamnosus is above 82.25%, and the shape is spherical.
  • the average particle size is 1 to 5 mm, the amount of viable bacteria at room temperature is above 1.14 ⁇ 10 10 CFU/mL, and the survival rate after storage at 4°C for 21 days is above 50%.
  • the probiotic microcapsules obtained by using the arabinoxylan-sodium alginate mixed gel as the wall material have good acid resistance and embedding rate, and the single unit can be improved by adding arabinoxylan Sodium alginate-embedded probiotics are unstable and easily decomposed at low pH, which improves the resistance of the core material in a simulated environment, improves the resistance of the strain to gastric acid, and gradually releases the strain in the intestinal juice environment.
  • the arabinoxylan used in this application is a polysaccharide in rice bran edible fiber. The extraction process is simple. It is the only polysaccharide with antioxidant properties in dietary fiber.
  • the microcapsule product has a uniform particle size distribution, a high embedding rate, and has a good protective effect on Lactobacillus rhamnosus, and the microcapsule product also has good storage.
  • FIG. 1 is a graph showing the change in survival rate of GR-1 microbial capsules and GR-1 microcapsules using arabinoxylan and sodium alginate as composite wall materials with time in Example 1 of the present application.
  • FIG. 2 is a graph comparing the embedding rate of Example 1 of the present application with the embedding rates of Comparative Example 1 and Comparative Example 2.
  • FIG. 2 is a graph comparing the embedding rate of Example 1 of the present application with the embedding rates of Comparative Example 1 and Comparative Example 2.
  • FIG. 3 is a preparation flow chart of a GR-1 microcapsule using arabinoxylan-sodium alginate as a wall material in a typical implementation case of this application.
  • Arabinoxylan is a dietary fiber extracted from wheat bran. It is a polysaccharide from rice bran edible fiber. The extraction process is simple. It is the only polysaccharide with antioxidant properties in dietary fiber. To promote the growth of beneficial bacteria such as Bifidobacterium colon in humans and animals, the sources of corn and wheat are rich resources in China. It has physical and chemical properties such as high molecular weight, high viscosity, and oxidized gel. It also has anti-tumor, immune-enhancing, anti-lipid, anti-oxidation and other functions. In addition, arabinoxylan can form a covalently cross-linked gel, which can be decomposed by microorganisms in the intestine. The product is arabinoxylan oligosaccharide, which is a prebiotic. It is known that arabinoxylan is an ideal probiotic Wall material.
  • arabinoxylan in this application can improve the problem of single sodium alginate-embedded probiotics unstable and easily decomposed at low pH, improve the resistance of the core material in a simulated environment, and increase the resistance of the strain to gastric acid And gradually release strains in the environment of intestinal juice, increase the colonization rate in the intestine, and achieve probiotic effect.
  • a preparation method of probiotic microcapsules using arabinoxylan-sodium alginate as a wall material which includes:
  • the sterilized mixed solution, probiotic bacterial solution, and laccase are mixed uniformly and subjected to a composite gel reaction, and then solidified to obtain probiotic microcapsules using arabinoxylan-sodium alginate as a wall material.
  • the preparation method specifically includes:
  • step (3) Mix and shake the probiotic liquid obtained in step (1), the sterilized mixed solution obtained in step (2), and laccase to perform a composite gel reaction;
  • step (3) The mixed solution obtained in step (3) is cured in a curing agent at room temperature for 0.5 to 1 hour, and left to stand until the gel beads are formed, that is, probiotic microcapsules using arabinoxylan-sodium alginate as a wall material.
  • the probiotic is preferably Lactobacillus rhamnosus (hereinafter may be referred to as GR-1 for short), but is not limited thereto.
  • arabinoxylan is alkali-extracted arabinoxylan, but it is not limited thereto.
  • the final concentrations of arabinoxylan and sodium alginate in the sterilized mixed solution in step (2) are both 2 to 4 wt%.
  • step (3) the volume of the probiotic bacterial solution and the sterilized mixed solution is 1:10 to 1:15.
  • the mass volume ratio of the laccase to the probiotic bacterial solution is 0.015 g to 0.02 g: 1 mL, that is, the amount of the laccase added is 0.015 g/mL to 0.02 g/mL probiotic bacterial solution.
  • probiotic bacterial solution is GR-1 cultured to the end of logarithm.
  • the temperature of the shaking in step (3) is 35-37° C., and the time is 20-30 min.
  • step (4) includes: squeezing the mixed liquid obtained in step (3) into a curing agent for curing.
  • the curing agent is a calcium chloride solution with a concentration of 2 to 4 mol/L, but it is not limited thereto.
  • FIG. 3 is arabinoxylan solution ⁇ seaweed Sodium solution ⁇ Arabinoxylan-sodium alginate mixed solution ⁇ mixed solution (GR-1 bacterial solution and arabinoxylan-sodium alginate mixed solution) ⁇ microcapsule curing ⁇ sterile water washing ⁇ GR-1 microcapsule product.
  • the preparation method mainly includes: first disposing the arabinoxylan solution and the sodium alginate solution, mixing the two so that the final concentration is 2 to 4 wt% and sterilizing, and then mixing with GR-1 bacterial solution and an appropriate amount of lacquer The enzymes are mixed and stirred to form a uniform mixed solution. After the mixed solution is squeezed into the curing solution with a syringe to solidify the microcapsules, it is washed with sterile water to finally obtain the GR-1 microcapsule product.
  • the preparation method includes the following steps:
  • MRS broth culture medium is placed, divided into glass test tubes, sealed and placed under 115-121°C sterilization for 20-30 minutes. After the liquid culture medium is cooled to room temperature, draw 200 ⁇ L of the frozen and preserved original strains, insert it into the MRS liquid culture medium, and culture at 35 to 38°C for 20 to 24 hours, and then pass it 1 to 2 times.
  • the activated GR-1 bacterial solution was inoculated into MRS liquid medium, and cultured to the end of logarithm at 35-38°C.
  • Another aspect of the embodiments of the present application also provides probiotic microcapsules prepared by the foregoing method using arabinoxylan-sodium alginate as a wall material, which can improve resistance to gastric acid and improve colonization in human intestines It also has good storage stability and embedding rate. Its embedding rate for Lactobacillus rhamnosus is more than 82.25%.
  • the shape is spherical and the particle size distribution is uniform. The average particle size is 1 to 5 mm, preferably It is 2 ⁇ 3mm, the amount of viable bacteria at room temperature is above 1.14 ⁇ 10 10 CFU/mL, and the survival rate is kept above 50% after storage at 4°C for 21 days.
  • the probiotic microcapsules obtained in the present application can improve the resistance to gastric acid, increase the colonization rate in the human intestine, and also have good storage stability and embedding rate.
  • This application solves the problem of depolymerization of sodium alginate at low pH and makes microcapsules unstable by adding arabinoxylan, improves the resistance of the strain to gastric acid, increases the colonization rate in the intestine, and has good storage .
  • MRS broth culture medium is placed, sterilized at 121°C for 20 min after blocking. After the liquid culture medium was cooled to room temperature, 200 ⁇ L of the frozen and preserved original strains were sucked into the MRS liquid culture medium, and cultured at 37° C. for 24 hours, and then subcultured once.
  • the activated GR-1 bacterial solution was inoculated into MRS liquid medium, and cultured to the end of logarithm at 37°C.
  • Step (3) Preparation of GR-1 microcapsules: Mix the bacterial solution cultured in step (1) with the mixed solution in step (2) at a ratio of 1:10, add laccase at a ratio of 0.015g:1mL and Stir it evenly, put it in a constant temperature shaker and shake it at 37°C for 20min.
  • the embedding rate of GR-1 microcapsules obtained in this example to Lactobacillus rhamnosus GR-1 was 88.17% ⁇ 1.03%, and the average particle size of the microcapsule particles was 2.0 mm.
  • the preparation method of this example is basically the same as that of Example 1, except that the curing time of the microcapsules is 30 minutes.
  • the embedding rate of GR-1 microcapsules obtained in this example to Lactobacillus rhamnosus GR-1 was 90.99% ⁇ 0.81%, and the average particle size of the microcapsule particles was 2.2 mm.
  • the preparation method of this example is basically the same as that of Example 1, except that the curing time of the microcapsules is 45 minutes.
  • the embedding rate of GR-1 microcapsules obtained in this example to Lactobacillus rhamnosus GR-1 was 89.28% ⁇ 0.48%, and the average particle size of the microcapsule particles was 2.0 mm.
  • MRS broth culture medium is placed, sterilized at 115°C for 30 min after blocking. After the liquid culture medium was cooled to room temperature, 200 ⁇ L of the frozen and preserved original strains were sucked into the MRS liquid culture medium and cultured at 38° C. for 20 hours, and then subcultured twice. The activated GR-1 bacterial solution was inoculated into the MRS liquid medium, and cultured to the end of logarithm at 38°C.
  • Step (3) Preparation of GR-1 microcapsules: Mix the bacterial solution cultured in step (1) with the mixed solution in step (2) at a ratio of 1:15, add laccase at a ratio of 0.02g:1mL and Stir it evenly, put it in a constant temperature shaker and shake it at 35°C for 30min.
  • MRS broth culture medium is placed, sterilized at 118°C for 25 min after blocking. After the liquid culture medium was cooled to room temperature, 200 ⁇ L of the frozen and preserved original strains were sucked into the MRS liquid culture medium, and cultured at 35°C for 22 hours, and then subcultured twice.
  • the activated GR-1 bacterial solution was inoculated into MRS liquid medium, and cultured to the end of logarithm at 35°C.
  • Step (3) Preparation of GR-1 microcapsules: Mix the bacterial solution cultured in step (1) and the mixed solution in step (2) at a ratio of 1:12, add laccase at a ratio of 0.018g:1mL and Stir it evenly, put it in a constant temperature shaker and shake it at 36°C for 25min.
  • the preparation method of this example is basically the same as that of Example 1, except that no arabinoxylan solution is added.
  • the embedding rate of GR-1 microcapsules obtained in this example to L. rhamnosus GR-1 was 27.61% ⁇ 1.05%, and the average particle size of the microcapsule particles was 2.5 mm.
  • the preparation method of this example is basically the same as that of Example 1, except that no laccase is added.
  • the embedding rate of GR-1 microcapsules obtained in this example to Lactobacillus rhamnosus GR-1 was 47.07% ⁇ 0.78%, and the average particle size of the microcapsule particles was 2.1 mm.
  • Lactobacillus rhamnosus GR-1 microcapsule product (hereinafter referred to as the composite microcapsule of Example 1) prepared by using arabinoxylan-sodium alginate as the wall material prepared in Example 1 as an example, the storage resistance Determination.
  • the GR-1 bacterial solution and the composite microcapsules of Example 1 were stored at 4°C, and samples were taken every 7 days within 21 days to detect the survival of the bacterial cells.
  • the results are shown in FIG. 1 and it was found that the storage properties of the naked bacterial cells were poor, and after 21 days of storage, the survival rate of the naked bacterial strains dropped to about 15% (see the GR-1 curve in FIG. 1).
  • the storage period of the strain was extended. After 21 days of storage, GR- 1 Survival rate can reach 50%, with good storage.
  • FIG. 2 shows a comparison diagram of the embedding rate of Example 1 of the present application and the embedding rates of Comparative Example 1 and Comparative Example 2.
  • FIG. 2 shows a comparison diagram of the embedding rate of Example 1 of the present application and the embedding rates of Comparative Example 1 and Comparative Example 2.
  • this application solves the problem of destabilization of sodium alginate at low pH and makes microcapsules unstable by adding arabinoxylan, and improves the resistance of the strain to gastric acid , Increases the colonization rate in the intestine, and has good storage.

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Abstract

一种以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊及其制备方法，所述制备方法包括：提供培养至对数末期的益生菌菌液，提供包含有阿拉伯木聚糖和海藻酸钠的混合溶液，并灭菌；将经灭菌的混合溶液、益生菌菌液、漆酶混合均匀并进行复合凝胶反应，之后固化，获得以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。所制备的益生菌微胶囊能够提高对胃酸的抵抗力，提高在人体肠道中的定植率，同时还具有良好的储藏稳定性和的包埋率。

Description

以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊及制法 技术领域
本申请涉及一种益生菌微胶囊的制备方法，特别涉及一种以阿拉伯木聚糖-海藻酸钠复合凝胶为壁材的益生菌微胶囊及其制备方法，属于食品生物技术领域。
背景技术
益生菌指一类具有促进宿主健康的活的微生物，外源口服一定量的益生菌可显著改善宿主肠道微生态平衡，从而起到预防腹泻、缓解乳糖不耐症、增强免疫力、降低血清胆固醇以及抑制肿瘤细胞前体物质形成等多种生理功效。然而，益生菌是一类厌氧或兼性厌氧的微生物，生长过程中不形成芽孢，抗性较差，还会产生一些危及自身生长繁殖的乳酸和乙酸等代谢产物，且极端条件下存活率非常低。将益生菌胶囊化是保护其活性最为有效和实用的方法之一。海藻酸钠是包埋益生菌最常用的壁材之一，具有来源广泛，价格便宜，胶凝条件温和，胶凝操作简单，且具有pH敏感性且生物相容性高的特点，但单独用于益生菌包埋时对益生菌的保护作用不够理想，比如纯海藻酸钠微胶囊表面孔径不够小，不能完全防止胃液渗入，在低pH条件下海藻酸钠不够稳定，且容易出现突释和早释的现象。
发明内容
本申请的主要目的在于提供一种以阿拉伯木聚糖-海藻酸钠复合凝胶为壁材的益生菌微胶囊及其制备方法，以克服现有技术中的不足。
为实现上述发明目的，本申请采用了如下技术方案：
本申请实施例提供了一种以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊的制备方法，其包括：
提供包含有阿拉伯木聚糖和海藻酸钠的混合溶液，并灭菌；
将经灭菌的混合溶液、益生菌菌液、漆酶混合均匀并进行复合凝胶反应，之后固化，获得以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
在一些优选实施例中，所述制备方法具体包括：
(1)取冷冻保藏的益生菌菌种，接入液体培养基中，置于35～38℃下培养20～24小时，之后传代1～2次，将活化后的益生菌菌液接种到液体培养基中，在35～38℃下培养至对数末期；
(2)将阿拉伯木聚糖溶液和海藻酸钠溶液混合均匀后在115～121℃下灭菌20～30min，得到经灭菌的混合溶液；
(3)将步骤(1)所获益生菌菌液、步骤(2)所获经灭菌的混合溶液、漆酶混合并振荡均匀，进行复合凝胶反应；
(4)将步骤(3)所获混合液于固化剂中于常温固化0.5～1h，静置待胶珠形成，即为以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
进一步地，所述益生菌优选为鼠李糖乳杆菌(GR-1)，但不限于此。
本申请实施例还提供了由前述方法制备的以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊，其对鼠李糖乳杆菌的包埋率在82.25％以上，形状呈球形，平均粒径为1～5mm，常温活菌量在1.14×10 10CFU/mL以上，在4℃储存21天后存活率在50％以上。
较之现有技术，本申请所获以阿拉伯木聚糖-海藻酸钠混合凝胶为壁材的益生菌微胶囊具有很好的耐酸性和包埋率，通过加入阿拉伯木聚糖可改善单一海藻酸钠包埋益生菌的在低pH下不稳定易分解的问题，提高了芯材在模拟环境中的抵抗力，提高了菌株对胃酸的抵抗力，并在肠液环境中逐步释放菌株，增加了在肠道内的定植率，达到益生的作用。本申请中采用的阿拉伯木聚糖是一种米糠食用纤维中的多糖，提取工艺简单，是膳食纤维类中唯一一种具有抗氧化性的多糖，可选择性地促进人和动物结肠双歧杆菌等有益菌的生长，其来源玉米和小麦是我国丰富资源。该微胶囊产品粒径分布均一，包埋率高，对鼠李糖乳杆菌有很好的保护作用，同时该微胶囊产品还有良好的储藏性。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是GR-1菌液与本申请实施例1之中以阿拉伯木聚糖和海藻酸钠为复合壁材的GR-1微胶囊的存活率随时间的变化图。
图2是本申请实施例1的包埋率与对照例1、对照例2的包埋率的对比图。
图3为本申请一典型实施案例之中一种以阿拉伯木聚糖-海藻酸钠为壁材的GR-1微胶囊的制备流程图。
具体实施方式
针对现有技术的诸多缺陷，本案发明人经长期研究和大量实践，得以提出本申请的技术方案。为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。此外，下面所描述的本申请各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
阿拉伯木聚糖是从小麦麸皮中提取的一种膳食纤维，是一种米糠食用纤维中的多糖，提取工艺简单，是膳食纤维类中唯一一种具有抗氧化性的多糖，可选择性地促进人和动物结肠双歧杆菌等有益菌的生长，其来源玉米和小麦是我国丰富资源。它具有高分子量、高粘度性和氧化凝胶等理化特性，同时还具有抗肿瘤、增强免疫力、抗血脂、抗氧化等功能。此外，阿拉伯木聚糖能形成共价交联的凝胶，可被肠道内的微生物分解，产物为阿拉伯木寡糖，是一种益生元，可知阿拉伯木聚糖是一种较理想的益生菌壁材。
鉴于此，本申请通过加入阿拉伯木聚糖可改善单一海藻酸钠包埋益生菌的在低pH下不稳定易分解的问题，提高芯材在模拟环境中的抵抗力，提高菌株对胃酸的抵抗力，并在肠液环境中逐步释放菌株，增加在肠道内的定植率，达到益生的作用。
作为本申请技术方案的一个方面，其所涉及的系一种以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊的制备方法，其包括：
提供包含有阿拉伯木聚糖和海藻酸钠的混合溶液，并灭菌；
将经灭菌的混合溶液、益生菌菌液、漆酶混合均匀并进行复合凝胶反应，之后固化，获得以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
在一些优选实施例中，所述制备方法具体包括：
(1)取冷冻保藏的益生菌菌种，接入液体培养基中，置于35～38℃下培养20～24小时，之后传代1～2次，将活化后的益生菌菌液接种到液体培养基中，在35～38℃下培养至对数末期；
(2)将阿拉伯木聚糖溶液和海藻酸钠溶液混合均匀后在115～121℃下灭菌20～30min，得 到经灭菌的混合溶液；
(3)将步骤(1)所获益生菌菌液、步骤(2)所获经灭菌的混合溶液、漆酶混合并振荡均匀，进行复合凝胶反应；
(4)将步骤(3)所获混合液于固化剂中于常温固化0.5～1h，静置待胶珠形成，即为以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
进一步地，所述益生菌优选为鼠李糖乳杆菌(以下可简称为GR-1)，但不限于此。
进一步地，所述阿拉伯木聚糖为碱提阿拉伯木聚糖，但不限于此。
进一步地，步骤(2)中所述经灭菌的混合溶液中阿拉伯木聚糖和海藻酸钠的终浓度均为2～4wt％。
进一步地，步骤(3)中所述益生菌菌液与经灭菌的混合溶液的体积为1：10～1：15。
进一步地，所述漆酶与益生菌菌液的质量体积比为0.015g～0.02g：1mL，亦即，所述漆酶的添加量为0.015g/mL～0.02g/mL益生菌菌液。
进一步地，所述益生菌菌液为培养至对数末期的GR-1。
进一步地，步骤(3)中所述振荡的温度为35～37℃，时间为20～30min。
进一步地，步骤(4)包括：将步骤(3)所获混合液挤压进入固化剂中进行固化。
更进一步地，所述固化剂为浓度为2～4mol/L的氯化钙溶液，但不限于此。
其中，在一些更为具体的实施案例之中，所述以阿拉伯木聚糖-海藻酸钠为壁材的GR-1微胶囊的制备方法如图3所示，为阿拉伯木聚糖溶液→海藻酸钠溶液→阿拉伯木聚糖-海藻酸钠混合溶液→混合溶液(GR-1菌液和阿拉伯木聚糖-海藻酸钠混合溶液)→微胶囊固化→无菌水洗涤→GR-1微胶囊产品。
进一步地，该制备方法主要包括：首先配置阿拉伯木聚糖溶液和海藻酸钠溶液，将二者混合使终浓度均为2～4wt％并灭菌，然后与GR-1菌液和适量的漆酶混合并搅拌均匀，形成均一的混合液，用注射器将混合溶液挤入固化液中对微胶囊进行固化后，用无菌水洗涤最终得到GR-1微胶囊产品。
具体的，该制备方法包括以下步骤：
(1)菌种的活化及培养：配置MRS肉汤培养基，分装至玻璃试管中，封塞后置于115～121℃条件下灭菌20～30min。待液体培养基冷却至室温后，吸取200μL冻保藏的原菌种，接入MRS液体培养基中，置于35～38℃条件下培养20～24小时，之后传代1～2次。将活化后的GR-1菌液接种到MRS液体培养基中，在35～38℃条件下培养至对数末期。
(2)阿拉伯木聚糖-海藻酸钠混合溶液的配置：向无菌水中添加阿拉伯木聚糖，配置成一定浓度的阿拉伯木聚糖溶液。同法配置成一定浓度的海藻酸钠溶液。将二者混匀使得混合后阿拉伯木聚糖和海藻酸钠终浓度均为2～4wt％，搅拌混合均匀后在115～121℃条件下灭菌20～30min，取出冷却至室温后置于4℃条件下备用。
(3)GR-1微胶囊的制备：将步骤(1)中的菌液与步骤(2)中的混合溶液按照1：10～1：15的比例混合均匀，添加适量漆酶并搅拌均匀，放入恒温振荡器中进行振荡均匀。
(4)GR-1微胶囊的固化：将上述制备的混合液用50mL注射器挤入氯化钙溶液中。静置待胶珠形成，即为所得微胶囊，将固化后的微胶囊用无菌水进行洗涤，过滤后进行冷冻干燥，收集得到GR-1微胶囊产品。
本申请实施例的另一个方面还提供了由前述方法制备的以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊，其能够提高对胃酸的抵抗力，提高在人体肠道中的定植率，同时还具有良好的储藏稳定性和的包埋率，其对鼠李糖乳杆菌的包埋率在82.25％以上，形状呈球形，粒径分布均匀，平均粒径为1～5mm，优选为2～3mm，常温活菌量在1.14×10 10CFU/mL以上，在4℃储存21天后存活率都保持在50％以上。
藉由上述技术方案，本申请所获益生菌微胶囊能够提高对胃酸的抵抗力，提高在人体肠道中的定植率，同时还具有良好的储藏稳定性和的包埋率。本申请通过加入阿拉伯木聚糖解决海藻酸钠在低pH下解聚使得微胶囊不稳定的问题，提高了菌株对胃酸的抵抗力，增加了在肠道内的定植率，并且具有良好的储藏性。
下面结合若干优选实施例及附图对本申请的技术方案做进一步详细说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本申请保护的范围。
实施例1
(1)菌种的活化及培养：配置MRS肉汤培养基，封塞后置于121℃条件下灭菌20min。待液体培养基冷却至室温后，吸取200μL冷冻保藏的原菌种，接入MRS液体培养基中，置于37℃条件下培养24小时，之后传代1次。将活化后的GR-1菌液接种到MRS液体培养基中，在37℃条件下培养至对数末期。
(2)阿拉伯木聚糖-海藻酸钠混合溶液的配置：配置混合后终浓度为2wt％阿拉伯木聚糖溶液和2wt％海藻酸钠溶液，将二者混合均匀后在121℃条件下灭菌20min，取出冷却至室温 后置于4℃条件下备用。
(3)GR-1微胶囊的制备：将步骤(1)中培养的菌液与步骤(2)中的混合溶液按照1：10的比例混合均匀，按照0.015g:1mL的比例添加漆酶并搅拌均匀，放入恒温振荡器中于37℃振荡20min均匀。
(4)GR-1微胶囊的固化：将上述制备的混合液用50mL注射器挤入浓度为3mol/L的氯化钙溶液中。静置60min，待胶珠形成，即为所得微胶囊，将固化后的微胶囊用无菌水进行洗涤，过滤后进行冷冻干燥，收集得到GR-1微胶囊产品。
本实施例所得GR-1微胶囊对鼠李糖乳杆菌GR-1的包埋率达88.17％±1.03％，微胶囊颗粒平均粒径为2.0mm。
实施例2
本实施例的制备方法与实施例1基本一致，不同之处在于：微胶囊固化时间为30min。
本实施例所得GR-1微胶囊对鼠李糖乳杆菌GR-1的包埋率达90.99％±0.81％，微胶囊颗粒平均粒径为2.2mm。
实施例3
本实施例的制备方法与实施例1基本一致，不同之处在于：微胶囊固化时间为45min。
本实施例所得GR-1微胶囊对鼠李糖乳杆菌GR-1的包埋率达89.28％±0.48％，微胶囊颗粒平均粒径为2.0mm。
实施例4
(1)菌种的活化及培养：配置MRS肉汤培养基，封塞后置于115℃条件下灭菌30min。待液体培养基冷却至室温后，吸取200μL冷冻保藏的原菌种，接入MRS液体培养基中，置于38℃条件下培养20小时，之后传代2次。将活化后的GR-1菌液接种到MRS液体培养基中，在38℃条件下培养至对数末期。
(2)阿拉伯木聚糖-海藻酸钠混合溶液的配置：配置混合后终浓度为3wt％阿拉伯木聚糖溶液和3wt％海藻酸钠溶液，将二者混合均匀后在115℃条件下灭菌30min，取出冷却至室温后置于4℃条件下备用。
(3)GR-1微胶囊的制备：将步骤(1)中培养的菌液与步骤(2)中的混合溶液按照1：15的比例混合均匀，按照0.02g:1mL的比例添加漆酶并搅拌均匀，放入恒温振荡器中于35℃振荡30min均匀。
(4)GR-1微胶囊的固化：将上述制备的混合液用50mL注射器挤入浓度为2mol/L的氯 化钙溶液中。静置60min，待胶珠形成，即为所得微胶囊，将固化后的微胶囊用无菌水进行洗涤，过滤后进行冷冻干燥，收集得到GR-1微胶囊产品。
实施例5
(1)菌种的活化及培养：配置MRS肉汤培养基，封塞后置于118℃条件下灭菌25min。待液体培养基冷却至室温后，吸取200μL冷冻保藏的原菌种，接入MRS液体培养基中，置于35℃条件下培养22小时，之后传代2次。将活化后的GR-1菌液接种到MRS液体培养基中，在35℃条件下培养至对数末期。
(2)阿拉伯木聚糖-海藻酸钠混合溶液的配置：配置混合后终浓度为4wt％阿拉伯木聚糖溶液和4wt％海藻酸钠溶液，将二者混合均匀后在118℃条件下灭菌25min，取出冷却至室温后置于4℃条件下备用。
(3)GR-1微胶囊的制备：将步骤(1)中培养的菌液与步骤(2)中的混合溶液按照1：12的比例混合均匀，按照0.018g:1mL的比例添加漆酶并搅拌均匀，放入恒温振荡器中于36℃振荡25min均匀。
(4)GR-1微胶囊的固化：将上述制备的混合液用50mL注射器挤入浓度为4mol/L的氯化钙溶液中。静置30min，待胶珠形成，即为所得微胶囊，将固化后的微胶囊用无菌水进行洗涤，过滤后进行冷冻干燥，收集得到GR-1微胶囊产品。
对照例1
本实施例的制备方法与实施例1基本一致，不同之处在于：未添加阿拉伯木聚糖溶液。
本实施例所得GR-1微胶囊对鼠李糖乳杆菌GR-1的包埋率达27.61％±1.05％，微胶囊颗粒平均粒径为2.5mm。
对照例2
本实施例的制备方法与实施例1基本一致，不同之处在于：未添加漆酶。
本实施例所得GR-1微胶囊对鼠李糖乳杆菌GR-1的包埋率达47.07％±0.78％，微胶囊颗粒平均粒径为2.1mm。
测试例
以实施例1所制备的以阿拉伯木聚糖-海藻酸钠为壁材的鼠李糖乳杆菌GR-1微胶囊产品(以下简称为实施例1的复合微胶囊)为例，进行耐储藏性测定。
将GR-1菌液和实施例1的复合微胶囊在4℃条件下贮存，在21天内每隔7天取样检测菌体的存活情况。结果如图1所示，结果发现，裸露菌体的储藏性较差，在存储21天以后， 裸露菌株的存活率下降到15％左右(参见图1中的GR-1曲线)。而对于以阿拉伯木聚糖-海藻酸钠为壁材的GR-1复合微胶囊(参见图1中的AX-SA-GR-1曲线)，延长了菌株的贮藏期，储藏21天后，GR-1存活率能达到50％，具有良好的储藏性。
图2示出了本申请实施例1的包埋率与对照例1、对照例2的包埋率的对比图。
综上所述，藉由实施例1-5的技术方案，本申请通过加入阿拉伯木聚糖解决海藻酸钠在低pH下解聚使得微胶囊不稳定的问题，提高了菌株对胃酸的抵抗力，增加了在肠道内的定植率，并且具有良好的储藏性。
此外，本案发明人还参照实施例1-5的方式，以本说明书中列出的其它原料和条件等进行了试验，并同样成功制得了具有相同性能的以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
需要说明的是，在本文中，在一般情况下，由语句“包括……”限定的要素，并不排除在包括所述要素的步骤、过程、方法或者实验设备中还存在另外的相同要素。
应当理解，以上较佳实施例仅用于说明本申请的内容，除此之外，本申请还有其他实施方式，但凡本领域技术人员因本申请所涉及之技术启示，而采用等同替换或等效变形方式形成的技术方案均落在本申请的保护范围内。

Claims (10)

  1. 一种以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊的制备方法，其特征在于包括：
    提供包含有阿拉伯木聚糖和海藻酸钠的混合溶液，并灭菌；
    将经灭菌的混合溶液、益生菌菌液、漆酶混合均匀并进行复合凝胶反应，之后固化，获得以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
  2. 根据权利要求1所述的制备方法，其特征在于具体包括：
    (1)取冷冻保藏的益生菌菌种，接入液体培养基中，置于35～38℃下培养20～24小时，之后传代1～2次，将活化后的益生菌菌液接种到液体培养基中，在35～38℃下培养至对数末期；
    (2)将阿拉伯木聚糖溶液和海藻酸钠溶液混合均匀后在115～121℃下灭菌20～30min，得到经灭菌的混合溶液；
    (3)将步骤(1)所获益生菌菌液、步骤(2)所获经灭菌的混合溶液、漆酶混合并振荡均匀，进行复合凝胶反应；
    (4)将步骤(3)所获混合液于固化剂中于常温固化0.5～1h，静置待胶珠形成，即为以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊。
  3. 根据权利要求1或2所述的制备方法，其特征在于：所述益生菌包括鼠李糖乳杆菌。
  4. 根据权利要求1所述的制备方法，其特征在于：所述阿拉伯木聚糖为碱提阿拉伯木聚糖。
  5. 根据权利要求2所述的制备方法，其特征在于，步骤(2)中所述经灭菌的混合溶液中阿拉伯木聚糖和海藻酸钠的终浓度均为2～4wt％。
  6. 根据权利要求2所述的制备方法，其特征在于：步骤(3)中所述益生菌菌液与经灭菌的混合溶液的体积为1：10～1：15；和/或，所述漆酶与益生菌菌液的质量体积比为0.015g～0.02g：1mL。
  7. 根据权利要求2所述的制备方法，其特征在于：步骤(3)中所述振荡的温度为35～37℃，时间为20～30min。
  8. 根据权利要求2所述的制备方法，其特征在于，步骤(4)包括：将步骤(3)所获混合液挤压进入固化剂中进行固化；优选的，所述固化剂包括氯化钙溶液；优选的，所述氯化钙溶液的浓度为2～4mol/L。
  9. 根据权利要求2所述的制备方法，其特征在于还包括：以无菌水对所述益生菌微胶囊进行洗涤，过滤后进行冷冻干燥，并收集。
  10. 由权利要求1-9中任一项所述方法制备的以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊，其对鼠李糖乳杆菌的包埋率在82.25％以上，形状呈球形，平均粒径为1～5mm，常温活菌量在1.14×10 10CFU/mL以上，在4℃储存21天后存活率在50％以上。
PCT/CN2018/122643 2018-11-26 2018-12-21 以阿拉伯木聚糖-海藻酸钠为壁材的益生菌微胶囊及制法 WO2020107580A1 (zh)

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