技术领域Technical Field
本发明属于食品生物技术和医药技术领域，具体涉及一种基于异质蛋白复合凝聚的益生菌微胶囊制备方法及其应用。The invention belongs to the field of food biotechnology and pharmaceutical technology, and specifically relates to a method for preparing probiotic microcapsules based on heterogeneous protein complex coagulation and an application thereof.
背景技术Background Art
益生菌是一类可以在宿主肠道内发挥有益健康功效的活的微生物。研究表明，益生菌在人体中不仅能够促进食物的消化，还能够与肠道中的有害细菌进行氧气竞争，维持肠道菌群平衡。然而，益生菌在加工、储运和消化过程中容易受到环境因素的影响造成菌活力的下降或失活，使益生菌功效无法达到预期效果，而微囊化常被作为改善这一问题的重要手段。Probiotics are a type of live microorganisms that can exert beneficial health effects in the host's intestines. Studies have shown that probiotics can not only promote food digestion in the human body, but also compete with harmful bacteria in the intestines for oxygen and maintain the balance of intestinal flora. However, probiotics are easily affected by environmental factors during processing, storage, transportation and digestion, causing the activity of the bacteria to decrease or become inactivated, making the efficacy of probiotics unable to achieve the expected effect. Microencapsulation is often used as an important means to improve this problem.
目前，国内外已经发展了多种微囊化技术应用于益生菌，例如喷雾干燥法、流化床法、挤压法、乳化法等。然而，这些方法均存在着诸多的短板，如喷雾干燥法由于在干燥过程中存在失水造成的渗透胁迫，再加上热空气对细胞的热损伤，会对微生物菌体造成严重伤害；挤压法虽操作简单，成本低廉，但制得的微胶囊尺寸较大，应用范围受限；乳化法由于涉及乳化中剧烈搅拌的过程，会对微生物菌体造成伤害，同时，由于油脂成分的引入，会导致氧化稳定性不佳等问题。与之相比，复合凝聚法具有操作条件温和，粒径尺寸较小，不引入不稳定成分等优点，因此成为微囊化益生菌的良好选择。At present, a variety of microencapsulation technologies have been developed and applied to probiotics at home and abroad, such as spray drying, fluidized bed method, extrusion method, emulsification method, etc. However, these methods all have many shortcomings. For example, the spray drying method will cause serious damage to the microbial cells due to the osmotic stress caused by water loss during the drying process, coupled with the thermal damage of hot air to the cells; although the extrusion method is simple to operate and low in cost, the size of the microcapsules produced is large and the scope of application is limited; the emulsification method will cause damage to the microbial cells due to the violent stirring process involved in the emulsification, and at the same time, due to the introduction of oil components, it will lead to problems such as poor oxidative stability. In comparison, the complex coacervation method has the advantages of mild operating conditions, small particle size, and no introduction of unstable components, so it has become a good choice for microencapsulated probiotics.
复合凝聚是由带相反电荷的聚电解质间发生静电相互作用引起的液-液相分离现象，形成聚电解质含量差别很大的富集相和稀释相。异质蛋白复合凝聚是复合凝聚的一种特殊情况，其中液体浓缩相由至少两种异源蛋白质构成。目前，异质蛋白复合凝聚已被用于脂溶性物质和水溶性物质的微囊化，可以有效地保护和输送一系列敏感(热不稳定、pH敏感或易氧化)的生物活性物质。与传统的多糖-蛋白复合凝聚微胶囊相比，异质蛋白复合凝聚微胶囊具有高负载、高包封、可控释放、抗极端环境等优点，拥有高表面疏水性及快速成膜能力，分子间空隙小且在胃消化过程中保留率高，是益生菌活性保护递送载体构建的优质来源。然而，异质蛋白复合凝聚作为一种新兴的包埋方法，其在益生菌微囊化领域的应用鲜有报道。Complex coacervation is a liquid-liquid phase separation phenomenon caused by electrostatic interactions between oppositely charged polyelectrolytes, forming enriched and diluted phases with greatly different polyelectrolyte contents. Heterogeneous protein complex coacervation is a special case of complex coacervation, in which the liquid concentrated phase is composed of at least two heterologous proteins. At present, heterogeneous protein complex coacervation has been used for the microencapsulation of fat-soluble and water-soluble substances, and can effectively protect and deliver a series of sensitive (heat-labile, pH-sensitive or easily oxidized) bioactive substances. Compared with traditional polysaccharide-protein complex coacervation microcapsules, heterogeneous protein complex coacervation microcapsules have the advantages of high loading, high encapsulation, controlled release, and resistance to extreme environments. They have high surface hydrophobicity and rapid film-forming ability, small intermolecular gaps, and high retention rate during gastric digestion. They are a high-quality source for the construction of probiotic active protection delivery carriers. However, as an emerging encapsulation method, heterogeneous protein complex coacervation has rarely been reported in the field of probiotic microencapsulation.
本专利与《一种基于全水相复合凝聚的益生菌微胶囊及其制备方法》(公开号：CN109700032A)、《一种含益生菌的冻干皮克林乳剂及其制备方法》(公开号：CN114698847A)以及《一种基于复合凝聚法制备益生菌微胶囊的方法与应用》(公开号：CN115381101A)中的所申请的权利保护方向有着本质不同。中国专利CN109700032A公开了一种基于全水相复合凝聚的益生菌微胶囊制备方法，该方法通过将菌泥、A型明胶溶液、酪蛋白酸钠溶液混合后，调节pH，诱导复合凝聚反应，制备微胶囊。该专利中复合凝聚法制得的益生菌微胶囊稳定性和对益生菌的保护能力均相对欠佳，特别是缺乏耐胃酸的能力，不适宜应用于将益生菌送入人体肠道发挥益生作用。中国专利CN114698847A公开了一种含益生菌的冻干皮克林乳剂制备方法，该方法将益生菌菌粉加入到油相中，将油菌混合物加入到水相中，剪切分散得到皮克林乳液进一步干燥得到含益生菌的冻干皮克林乳剂。该专利中复合凝聚法制备微胶囊的过程中含有乳化步骤，且其包埋是通过油/水或水/油/水体系来包埋活性成分，制备工艺复杂，包埋率低，不适用益生菌等不宜长时间暴露于空气中的敏感芯材。中国专利CN115381101A公开了一种基于复合凝聚法制备益生菌微胶囊的方法，该方法通过以改性大豆蛋白和植物或微生物来源的多糖为壁材，以益生菌为芯材，并使用非酶方式的双重固化剂对壁材进行固化处理制备益生菌微胶囊。该专利中利用了最常见的多糖-蛋白质复合凝聚法作为微胶囊壁材保护益生菌提升存活率，然而其在胃肠道中不易降解，难以控制生物活性物质在肠道中释放，不适宜用作益生菌类活性物质保护递送载体。This patent is essentially different from the rights protection direction applied for in "A probiotic microcapsule based on full-water phase composite coagulation and its preparation method" (publication number: CN109700032A), "A freeze-dried Pickering emulsion containing probiotics and its preparation method" (publication number: CN114698847A) and "A method and application of preparing probiotic microcapsules based on composite coagulation" (publication number: CN115381101A). Chinese patent CN109700032A discloses a method for preparing probiotic microcapsules based on full-water phase composite coagulation. The method prepares microcapsules by mixing bacterial mud, type A gelatin solution, and sodium caseinate solution, adjusting the pH, and inducing composite coagulation reaction. The stability and protection ability of the probiotic microcapsules prepared by the composite coagulation method in this patent are relatively poor, especially the lack of gastric acid resistance, and are not suitable for delivering probiotics into the human intestine to exert probiotic effects. Chinese patent CN114698847A discloses a method for preparing a freeze-dried Pickering emulsion containing probiotics, in which the probiotic powder is added to the oil phase, the oil-bacteria mixture is added to the water phase, the Pickering emulsion is obtained by shear dispersion, and the Pickering emulsion is further dried to obtain a freeze-dried Pickering emulsion containing probiotics. The process of preparing microcapsules by the composite coacervation method in this patent contains an emulsification step, and the embedding is to embed the active ingredients through an oil/water or water/oil/water system. The preparation process is complicated and the embedding rate is low. It is not suitable for sensitive core materials such as probiotics that are not suitable for long-term exposure to the air. Chinese patent CN115381101A discloses a method for preparing probiotic microcapsules based on the composite coacervation method. The method uses modified soy protein and polysaccharides from plants or microorganisms as wall materials, probiotics as core materials, and uses a non-enzymatic dual curing agent to cure the wall materials to prepare probiotic microcapsules. This patent uses the most common polysaccharide-protein complex coagulation method as the microcapsule wall material to protect probiotics and improve survival rate. However, it is not easily degraded in the gastrointestinal tract, and it is difficult to control the release of bioactive substances in the intestine. It is not suitable for use as a protective delivery carrier for probiotic active substances.
上述3项专利并不影响本发明专利的创造性与创新性的体现，而恰恰是进一步改善益生菌微胶囊的耐胃酸能力差、不稳定、操作繁琐、难以实现可控释放等不够理想的技术问题，因此开发一种适宜益生菌的异质蛋白复合凝聚法来制备益生菌微胶囊成为亟待解决的问题。这将为益生菌类产品开发提供重要的科学依据，对推动大健康产业延链增值具有重要意义。The above three patents do not affect the creativity and innovation of the present invention, but are precisely to further improve the poor gastric acid resistance, instability, cumbersome operation, and difficulty in achieving controlled release of probiotic microcapsules. Therefore, the development of a heterogeneous protein complex coagulation method suitable for probiotics to prepare probiotic microcapsules has become an urgent problem to be solved. This will provide an important scientific basis for the development of probiotic products and is of great significance to promoting the extension and value-added of the health industry.
发明内容Summary of the invention
本发明的目的在于克服现有技术之缺陷，提供了一种基于异质蛋白复合凝聚的益生菌微胶囊制备方法，该方法是基于卵白蛋白/溶菌酶体系的复合凝聚，优点是操作条件温和、成本低、不含有机溶剂、耐热性和耐湿性好、高负载、高包封、可控释放、抗极端环境、成膜性能佳且分子间空隙小等，对芯材起到良好的保护和缓释作用。The purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing probiotic microcapsules based on heterogeneous protein complex coagulation. The method is based on the complex coagulation of ovalbumin/lysozyme system, and has the advantages of mild operating conditions, low cost, no organic solvent, good heat resistance and moisture resistance, high load, high encapsulation, controlled release, resistance to extreme environment, good film-forming performance and small intermolecular gaps, etc., which provides good protection and sustained release for the core material.
为实现上述目的，本发明通过下述技术方案来实现：To achieve the above object, the present invention is implemented by the following technical solutions:
1.基于异质蛋白复合凝聚的益生菌微胶囊的制备方法，其特征在于，所述方法包括如下步骤：1. A method for preparing probiotic microcapsules based on heterogeneous protein complex coagulation, characterized in that the method comprises the following steps:
步骤一、培养冷冻保藏的益生菌，离心，弃上清，获得菌泥，重悬于无菌生理盐水中，备用；Step 1: Cultivate the frozen probiotics, centrifuge, discard the supernatant, obtain the bacterial slurry, and resuspend it in sterile saline for later use;
步骤二、称取一定质量的卵白蛋白粉末溶解于超纯水中，搅拌混匀，制备成质量浓度为1～5％的卵白蛋白溶液，于4℃下冷藏备用；Step 2: Weigh a certain amount of egg white protein powder and dissolve it in ultrapure water, stir and mix to prepare an egg white protein solution with a mass concentration of 1 to 5%, and refrigerate at 4°C for later use;
步骤三、称取一定质量的溶菌酶粉末溶解于超纯水中，搅拌混匀，制备成质量浓度为1～5％的溶菌酶溶液，于4℃下冷藏备用；Step 3, weigh a certain amount of lysozyme powder and dissolve it in ultrapure water, stir and mix to prepare a lysozyme solution with a mass concentration of 1 to 5%, and refrigerate at 4°C for later use;
步骤四、称取一定质量的多酚粉末，于室温下放置备用；Step 4, weigh a certain amount of polyphenol powder and place it at room temperature for later use;
步骤五、在室温下，将所述菌悬液、多酚、卵白蛋白溶液、溶菌酶溶液混合，400rpm下混匀30min，得到多酚-异质蛋白-益生菌混合溶液；所述菌泥添加量为：每1g卵白蛋白添加108～1012cfu的菌泥，所述卵白蛋白与溶菌酶的质量比为1:4～4:1，所述多酚添加量为0.1％～1w/w％；Step 5: at room temperature, the bacterial suspension, polyphenols, ovalbumin solution and lysozyme solution are mixed, and mixed at 400 rpm for 30 min to obtain a polyphenol-heterogeneous protein-probiotic mixed solution; the bacterial sludge is added in an amount of 108 to 1012 cfu of bacterial sludge per 1 g of ovalbumin, the mass ratio of ovalbumin to lysozyme is 1:4 to 4:1, and the polyphenol is added in an amount of 0.1% to 1 w/w%;
步骤六、调节步骤五制得的多酚-异质蛋白-益生菌混合溶液pH至6.5～8.5，400rpm下持续搅拌30min，诱导复合凝聚反应完全，静置，弃上清，制备湿态微胶囊；Step 6: adjusting the pH of the polyphenol-heterogeneous protein-probiotic mixed solution obtained in step 5 to 6.5-8.5, stirring continuously at 400 rpm for 30 min to induce the complex coacervation reaction to be complete, standing, discarding the supernatant, and preparing wet microcapsules;
步骤七、将步骤六得到的湿态微胶囊冷冻干燥，得到益生菌微胶囊干粉。Step 7: freeze-dry the wet microcapsules obtained in step 6 to obtain probiotic microcapsule dry powder.
2.所述益生菌包括罗伊氏乳杆菌、植物乳杆菌、干酪乳杆菌中的一种或多种；所述多酚包括儿茶素、表儿茶素和表没食子儿茶素中的一种或多种。2. The probiotics include one or more of Lactobacillus reuteri, Lactobacillus plantarum, and Lactobacillus casei; and the polyphenols include one or more of catechin, epicatechin, and epigallocatechin.
3.所述卵白蛋白与溶菌酶的质量比为1：1。3. The mass ratio of ovalbumin to lysozyme is 1:1.
4.所述步骤(5)中多酚、卵白蛋白溶液、溶菌酶溶液与菌泥混合后，添加终浓度为0～5w/w％的小分子糖的粉末，搅拌均匀。4. After the polyphenols, egg white protein solution, lysozyme solution and bacterial mud are mixed in step (5), small molecule sugar powder with a final concentration of 0 to 5 w/w% is added and stirred evenly.
5.所述小分子糖包括葡萄糖、蔗糖、果糖、海藻糖、麦芽糖中的一种或多种。5. The small molecule sugar includes one or more of glucose, sucrose, fructose, trehalose and maltose.
6.所述步骤(6)中pH为7.0。6. In the step (6), the pH is 7.0.
7.所述冷冻干燥的条件为：控制温度为-20℃～-80℃，冷冻时间为24～48h。7. The freeze-drying conditions are: controlling the temperature to be -20°C to -80°C and the freezing time to be 24 to 48 hours.
8.基于异质蛋白复合凝聚的益生菌微胶囊，通过上述的方法制备得到。8. Probiotic microcapsules based on heterogeneous protein complex coagulation are prepared by the above method.
9.所述的益生菌微胶囊在制备营养强化酸奶中的应用。9. Use of the probiotic microcapsules in preparing nutritionally fortified yogurt.
与现有技术相比，本发明具有如下优点和效果：Compared with the prior art, the present invention has the following advantages and effects:
1、本发明提供的基于异质蛋白复合凝聚的益生菌微胶囊制备方法，采用卵白蛋白/溶菌酶复合凝聚体系，在中性pH范围内静电复合、微相分离形成液滴结构，干燥得到微胶囊粉末。第一、不需要乳化步骤，简化了方法，节约了材料，减短了微胶囊制备时间，易于工业化生产；第二、有效提高了益生菌的包埋率，具有良好的环境和消化稳定性；第三，能够得到微米尺寸的益生菌微胶囊，易于在多种产品形态中添加，应用范围广泛。1. The method for preparing probiotic microcapsules based on heterogeneous protein complex coagulation provided by the present invention adopts an ovalbumin/lysozyme complex coagulation system, forms a droplet structure through electrostatic compounding and microphase separation in a neutral pH range, and obtains microcapsule powder by drying. First, no emulsification step is required, which simplifies the method, saves materials, shortens the preparation time of microcapsules, and is easy for industrial production; second, the embedding rate of probiotics is effectively improved, and it has good environmental and digestive stability; third, micron-sized probiotic microcapsules can be obtained, which are easy to add to a variety of product forms and have a wide range of applications.
2、本发明通过合理使用多酚(儿茶素、表儿茶素、表没食子儿茶素)固化剂，其耐高温和耐酸碱性良好，在pH 2.0～7.0范围内稳定，且活性成分不仅具有强大的抗氧化能力，还具有调节肠道微生物群落的作用，可以增强益生菌的保健作用。此外，多酚使得产品结构稳定性良好，能够有效保护益生菌在贮藏期间的活性，且能够实现在胃液环境中对所包埋益生菌的保护，可以将益生菌递送到肠道中并迅速释放，以发挥益生菌的益生作用。2. The present invention uses polyphenol (catechin, epicatechin, epigallocatechin) curing agent in a reasonable manner, which has good high temperature resistance and acid and alkali resistance, is stable in the range of pH 2.0 to 7.0, and the active ingredients not only have strong antioxidant capacity, but also have the function of regulating intestinal microbial communities, which can enhance the health care effect of probiotics. In addition, polyphenols make the product structure stable, can effectively protect the activity of probiotics during storage, and can achieve protection of the embedded probiotics in the gastric fluid environment, can deliver the probiotics to the intestines and release them quickly to exert the probiotic effect of the probiotics.
3、本发明以益生菌细胞对冷、热、储存条件下耐受性作为复合凝聚微胶囊环境稳定性评价指标，以益生菌细胞在体外模拟胃肠道消化过程中的存活率作为消化稳定性评价指标：3. The present invention uses the tolerance of probiotic cells to cold, hot and storage conditions as the environmental stability evaluation index of the composite coacervate microcapsules, and uses the survival rate of probiotic cells in the in vitro simulated gastrointestinal digestion process as the digestion stability evaluation index:
(1)本专利采用加热条件下益生菌耐受性作为复合凝聚益生菌微胶囊环境稳定性的评价指标，经测定，益生菌存活率仅下降了15～20％。(1) This patent uses the tolerance of probiotics under heating conditions as an evaluation index for the environmental stability of composite coacervate probiotic microcapsules. It has been determined that the survival rate of probiotics only decreased by 15-20%.
(2)本专利采用冷冻储藏条件下益生菌耐受性作为复合凝聚益生菌微胶囊环境稳定性的评价指标，经测定，益生菌存活率仅下降了5～10％。(2) This patent uses the tolerance of probiotics under frozen storage conditions as an evaluation index for the environmental stability of composite coacervate probiotic microcapsules. It has been determined that the survival rate of probiotics only decreased by 5-10%.
(3)本专利采用体外模拟胃肠道消化条件下益生菌存活率作为复合凝聚益生菌微胶囊消化稳定性的评价指标，经测定，与游离菌相比，微胶囊中益生菌活力提高40～50％。(3) This patent uses the survival rate of probiotics under in vitro simulated gastrointestinal digestion conditions as an evaluation index for the digestion stability of composite coacervate probiotic microcapsules. It has been determined that the activity of probiotics in microcapsules is increased by 40-50% compared with free bacteria.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为不同pH的异质蛋白复合凝聚物的光学形态图；FIG1 is an optical morphology diagram of heterogeneous protein complex coacervates at different pH values;
图2为复合凝聚益生菌微胶囊冻干粉宏观图片；Figure 2 is a macroscopic picture of the composite coacervated probiotic microcapsule freeze-dried powder;
图3为复合凝聚益生菌微胶囊冻干粉微观图片；Figure 3 is a microscopic picture of the composite coacervated probiotic microcapsule freeze-dried powder;
图4为复合凝聚益生菌微胶囊的环境稳定性结果；FIG4 is the environmental stability results of the complex coacervated probiotic microcapsules;
图5为添加复合凝聚益生菌微胶囊的酸奶图片。Figure 5 is a picture of yogurt with added complex coacervate probiotic microcapsules.
具体实施方式DETAILED DESCRIPTION
为了令本发明的目的、特征、优点更加明显易懂，下面结合附图中涉及的具体实施方式对本发明的实施例进行清楚、完整地描述。显然，所描述的实施例仅为本发明的一部分实施例，而不是全部实施例。基于本发明的实施例，本领域技术人员在未进行创造性劳动前提下获得的所有其它实施例，如只改变用途而不改变权利要求涉及基本原理的实施例，都属于本发明保护的范围。In order to make the purpose, features and advantages of the present invention more obvious and understandable, the embodiments of the present invention are clearly and completely described below in conjunction with the specific implementation methods involved in the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work, such as embodiments that only change the purpose without changing the basic principles involved in the claims, belong to the scope of protection of the present invention.
实施例1：Embodiment 1:
培养冷冻保藏的益生菌，离心，弃上清，获得1×1010CFU/mL菌泥，重悬于无菌生理盐水中，备用。称取卵白蛋白粉末8g溶解于400mL超纯水中，搅拌混匀，制备成质量浓度为2％的卵白蛋白溶液，于4℃下冷藏备用。称取溶菌酶粉末8g溶解于400mL超纯水中，搅拌混匀，制备成质量浓度为2％的溶菌酶溶液，于4℃下冷藏备用。称取多酚(儿茶素、表儿茶素、表没食子儿茶素)粉末400mg，于室温下放置备用。将2mL菌悬液、400mg多酚、100mL卵白蛋白溶液、100mL溶菌酶溶液、4g蔗糖(终浓度为2w/w％)混合，400rpm下混匀30min，得到多酚-异质蛋白-益生菌混合溶液。调节混合溶液pH至7.0，400rpm下持续搅拌30min，诱导复合凝聚反应完全，静置，弃上清，制备湿态微胶囊，再在-80℃条件下冷冻干燥48h，得到复合凝聚益生菌微胶囊。对益生菌微胶囊环境稳定性进行测定，在55℃水浴加热3小时后，复合凝聚益生菌微胶囊存活率仅下降了15％；在-20℃冷冻储藏120天后，复合凝聚益生菌微胶囊存活率仅下降了5％。Cultivate the frozen probiotics, centrifuge, discard the supernatant, obtain 1×1010 CFU/mL bacterial mud, resuspend in sterile physiological saline for standby use. Weigh 8g of ovalbumin powder and dissolve it in 400mL ultrapure water, stir and mix, prepare an ovalbumin solution with a mass concentration of 2%, and refrigerate at 4°C for standby use. Weigh 8g of lysozyme powder and dissolve it in 400mL ultrapure water, stir and mix, prepare a lysozyme solution with a mass concentration of 2%, and refrigerate at 4°C for standby use. Weigh 400mg of polyphenol (catechin, epicatechin, epigallocatechin) powder and place it at room temperature for standby use. Mix 2mL of bacterial suspension, 400mg of polyphenol, 100mL of ovalbumin solution, 100mL of lysozyme solution, and 4g of sucrose (final concentration of 2w/w%), mix at 400rpm for 30min, and obtain a polyphenol-heterogeneous protein-probiotic mixed solution. The pH of the mixed solution was adjusted to 7.0, and the mixture was stirred at 400 rpm for 30 minutes to induce the complex coacervation reaction to be complete. The mixture was allowed to stand and the supernatant was discarded to prepare wet microcapsules, which were then freeze-dried at -80°C for 48 hours to obtain complex coacervation probiotic microcapsules. The environmental stability of the probiotic microcapsules was determined. After heating in a 55°C water bath for 3 hours, the survival rate of the complex coacervation probiotic microcapsules decreased by only 15%; after being frozen and stored at -20°C for 120 days, the survival rate of the complex coacervation probiotic microcapsules decreased by only 5%.
实施例2：Embodiment 2:
培养冷冻保藏的益生菌，离心，弃上清，获得1×1010CFU/mL菌泥，重悬于无菌生理盐水中，备用。称取卵白蛋白粉末10g溶解于250mL超纯水中，搅拌混匀，制备成质量浓度为4％的卵白蛋白溶液，于4℃下冷藏备用。称取溶菌酶粉末10g溶解于250mL超纯水中，搅拌混匀，制备成质量浓度为4％的溶菌酶溶液，于4℃下冷藏备用。称取多酚(儿茶素、表儿茶素、表没食子儿茶素)粉末800mg，于室温下放置备用。将2mL菌悬液、800mg多酚、100mL卵白蛋白溶液、100mL溶菌酶溶液、4g果糖(终浓度为2w/w％)混合，400rpm下混匀30min，得到多酚-异质蛋白-益生菌混合溶液。调节混合溶液pH至7.25，400rpm下持续搅拌30min，诱导复合凝聚反应完全，静置，弃上清，制备湿态微胶囊，再在-80℃条件下冷冻干燥48h，得到复合凝聚益生菌微胶囊。对益生菌微胶囊环境稳定性进行测定，在55℃水浴加热3小时后，复合凝聚益生菌微胶囊存活率仅下降了17％；在-20℃冷冻储藏120天后，复合凝聚益生菌微胶囊存活率仅下降了6％。Cultivate the frozen probiotics, centrifuge, discard the supernatant, obtain 1×1010 CFU/mL bacterial mud, resuspend in sterile physiological saline for standby use. Weigh 10g of ovalbumin powder and dissolve it in 250mL ultrapure water, stir and mix, prepare an ovalbumin solution with a mass concentration of 4%, and refrigerate at 4°C for standby use. Weigh 10g of lysozyme powder and dissolve it in 250mL ultrapure water, stir and mix, prepare a lysozyme solution with a mass concentration of 4%, and refrigerate at 4°C for standby use. Weigh 800mg of polyphenol (catechin, epicatechin, epigallocatechin) powder and place it at room temperature for standby use. Mix 2mL of bacterial suspension, 800mg of polyphenol, 100mL of ovalbumin solution, 100mL of lysozyme solution, and 4g of fructose (final concentration of 2w/w%), mix at 400rpm for 30min, and obtain a polyphenol-heterogeneous protein-probiotic mixed solution. The pH of the mixed solution was adjusted to 7.25, and the mixture was stirred at 400 rpm for 30 minutes to induce the complex coacervation reaction to be complete. The mixture was allowed to stand and the supernatant was discarded to prepare wet microcapsules, which were then freeze-dried at -80°C for 48 hours to obtain complex coacervation probiotic microcapsules. The environmental stability of the probiotic microcapsules was determined. After heating in a 55°C water bath for 3 hours, the survival rate of the complex coacervation probiotic microcapsules decreased by only 17%; after being frozen and stored at -20°C for 120 days, the survival rate of the complex coacervation probiotic microcapsules decreased by only 6%.
实施例3：Embodiment 3:
培养冷冻保藏的益生菌，离心，弃上清，获得1×1010CFU/mL菌泥，重悬于无菌生理盐水中，备用。称取卵白蛋白粉末8g溶解于400mL超纯水中，搅拌混匀，制备成质量浓度为2％的卵白蛋白溶液，于4℃下冷藏备用。称取溶菌酶粉末8g溶解于400mL超纯水中，搅拌混匀，制备成质量浓度为2％的溶菌酶溶液，于4℃下冷藏备用。称取多酚(儿茶素、表儿茶素、表没食子儿茶素)粉末400mg，于室温下放置备用。将2mL菌悬液、400mg多酚、100mL卵白蛋白溶液、100mL溶菌酶溶液、4g蔗糖(终浓度为2w/w％)混合，400rpm下混匀30min，得到多酚-异质蛋白-益生菌混合溶液。调节混合溶液pH至7.0，400rpm下持续搅拌30min，诱导复合凝聚反应完全，静置，弃上清，制备湿态微胶囊，再在-80℃条件下冷冻干燥48h，得到复合凝聚益生菌微胶囊。准备500mL常温纯牛奶，倒入1g益生菌微胶囊菌粉搅拌均匀，43℃恒温发酵8小时制作含复合凝聚益生菌微胶囊的酸奶。Cultivate the frozen probiotics, centrifuge, discard the supernatant, obtain 1×1010 CFU/mL bacterial mud, resuspend in sterile physiological saline for standby use. Weigh 8g of ovalbumin powder and dissolve it in 400mL ultrapure water, stir and mix, prepare an ovalbumin solution with a mass concentration of 2%, and refrigerate at 4°C for standby use. Weigh 8g of lysozyme powder and dissolve it in 400mL ultrapure water, stir and mix, prepare a lysozyme solution with a mass concentration of 2%, and refrigerate at 4°C for standby use. Weigh 400mg of polyphenol (catechin, epicatechin, epigallocatechin) powder and place it at room temperature for standby use. Mix 2mL of bacterial suspension, 400mg of polyphenol, 100mL of ovalbumin solution, 100mL of lysozyme solution, and 4g of sucrose (final concentration of 2w/w%), mix at 400rpm for 30min, and obtain a polyphenol-heterogeneous protein-probiotic mixed solution. The pH of the mixed solution was adjusted to 7.0, and the mixture was stirred continuously at 400 rpm for 30 min to induce the complex coacervation reaction to be complete. The mixture was allowed to stand, and the supernatant was discarded to prepare wet microcapsules, which were then freeze-dried at -80 °C for 48 h to obtain complex coacervation probiotic microcapsules. 500 mL of room temperature pure milk was prepared, 1 g of probiotic microcapsule powder was poured into the mixture and stirred evenly, and the yogurt containing complex coacervation probiotic microcapsules was prepared at a constant temperature of 43 °C for 8 hours.
实施例4：Embodiment 4:
培养冷冻保藏的益生菌，离心，弃上清，获得1×1010CFU/mL菌泥，重悬于无菌生理盐水中，备用。称取卵白蛋白粉末10g溶解于250mL超纯水中，搅拌混匀，制备成质量浓度为4％的卵白蛋白溶液，于4℃下冷藏备用。称取溶菌酶粉末10g溶解于250mL超纯水中，搅拌混匀，制备成质量浓度为4％的溶菌酶溶液，于4℃下冷藏备用。称取多酚(儿茶素、表儿茶素、表没食子儿茶素)粉末800mg，于室温下放置备用。将2mL菌悬液、800mg多酚、100mL卵白蛋白溶液、100mL溶菌酶溶液、4g果糖(终浓度为2w/w％)混合，400rpm下混匀30min，得到多酚-异质蛋白-益生菌混合溶液。调节混合溶液pH至7.25，400rpm下持续搅拌30min，诱导复合凝聚反应完全，静置，弃上清，制备湿态微胶囊，再在-80℃条件下冷冻干燥48h，得到复合凝聚益生菌微胶囊。准备1000mL常温纯牛奶，倒入1g益生菌微胶囊菌粉搅拌均匀，43℃恒温发酵10小时制作含复合凝聚益生菌微胶囊的酸奶。Cultivate the frozen probiotics, centrifuge, discard the supernatant, obtain 1×1010 CFU/mL bacterial mud, resuspend in sterile physiological saline for standby use. Weigh 10g of ovalbumin powder and dissolve it in 250mL ultrapure water, stir and mix, prepare an ovalbumin solution with a mass concentration of 4%, and refrigerate at 4°C for standby use. Weigh 10g of lysozyme powder and dissolve it in 250mL ultrapure water, stir and mix, prepare a lysozyme solution with a mass concentration of 4%, and refrigerate at 4°C for standby use. Weigh 800mg of polyphenol (catechin, epicatechin, epigallocatechin) powder and place it at room temperature for standby use. Mix 2mL of bacterial suspension, 800mg of polyphenol, 100mL of ovalbumin solution, 100mL of lysozyme solution, and 4g of fructose (final concentration of 2w/w%), mix at 400rpm for 30min, and obtain a polyphenol-heterogeneous protein-probiotic mixed solution. The pH of the mixed solution was adjusted to 7.25, and the mixture was stirred continuously at 400 rpm for 30 min to induce the complex coacervation reaction to be complete. The mixture was allowed to stand, and the supernatant was discarded to prepare wet microcapsules, which were then freeze-dried at -80 °C for 48 h to obtain complex coacervation probiotic microcapsules. 1000 mL of room temperature pure milk was prepared, 1 g of probiotic microcapsule powder was poured into the mixture and stirred evenly, and the yogurt containing complex coacervation probiotic microcapsules was prepared at a constant temperature of 43 °C for 10 hours.
说明书未详细描述的部件组合特征属于公知技术轻易想到或者实施本发明时容易确定且无异议的内容。上述方案，仅为本申请较佳的几个实施方式的描述，但本申请的保护范围不仅限于此，任何熟悉该技术的人能在本申请描述的范围内轻易实现，而不改变权利要求涉及基本原理的变化或替换，都应涵盖在本申请的保护范围之内，即本申请保护范围应以权利要求保护范围为准。The component combination features not described in detail in the specification belong to the contents that are easily thought of by the known technology or easily determined and undisputed when implementing the present invention. The above scheme is only a description of several preferred implementation methods of the present application, but the protection scope of the present application is not limited to this. Any person familiar with the technology can easily implement within the scope of the description of the present application without changing the changes or substitutions involved in the basic principles of the claims, which should be covered within the protection scope of the present application, that is, the protection scope of the present application should be based on the protection scope of the claims.
| Application Number | Priority Date | Filing Date | Title |
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| CN202410775078.4ACN118697051A (en) | 2024-06-17 | 2024-06-17 | Preparation method and application of probiotic microcapsules based on heterogeneous protein complex coacervation |
| Application Number | Priority Date | Filing Date | Title |
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| CN202410775078.4ACN118697051A (en) | 2024-06-17 | 2024-06-17 | Preparation method and application of probiotic microcapsules based on heterogeneous protein complex coacervation |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202410775078.4APendingCN118697051A (en) | 2024-06-17 | 2024-06-17 | Preparation method and application of probiotic microcapsules based on heterogeneous protein complex coacervation |
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| CN (1) | CN118697051A (en) |
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