CN112335884A - 一种新型益生菌微球及其制备方法 - Google Patents
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- CN112335884A CN112335884A CN202011219590.9A CN202011219590A CN112335884A CN 112335884 A CN112335884 A CN 112335884A CN 202011219590 A CN202011219590 A CN 202011219590A CN 112335884 A CN112335884 A CN 112335884A
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Abstract
本发明的目的在于提供一种益生菌微球的制备方法。本发明的新型益生菌微球包括含益生菌和保护剂的芯材、含益生元的海藻酸盐的囊材和含壳聚糖的被膜共同构成的三层微球结构。该微球具有制备工艺简单,成本低,口服抗酸性好,稳定性强,菌体生存率高等特点。可用于一种或多种益生菌的包裹、定植和增殖,具有良好的应用前景。
Description
技术领域
本发明涉及一种新型益生菌微球及其制备的方法,属于生物保健品技术领域。
背景技术
随着肠道菌群研究的逐渐深入,益生菌在人体肠道内发挥的做的作用越来越明显。益生菌是指健康人体肠道内的对人体健康有益的一类菌群。已有报道的益生菌种类繁多,其中可用于食品的益生菌分为六类。第一类是双歧杆菌属,包括两歧双歧杆菌、婴儿双歧杆菌、长双歧杆菌、短双歧杆菌以及青春双歧杆菌;第二类是乳杆菌属,包括德氏乳杆菌保加利亚种、嗜酸乳杆菌、干酪乳杆菌干酪亚种以及罗伊氏乳杆菌在内的14种菌;第三类是链球菌属中的嗜热链球菌;第四类是眀串球菌数中的肠膜明串珠菌肠膜亚种;第五类是葡萄球菌属中的小牛葡萄球菌、木糖葡萄球菌和肉葡萄球菌;第六类是芽孢杆菌属中的凝结芽孢杆菌。现有的研究已经表明益生菌制剂在调控维生素吸收代谢,促进肠蠕动以及肠道免疫调节方面发挥重要作用。
海藻酸钠是一类天然高分子材料,能与多价阳离子物质发生交联反应形成凝胶。海藻酸钠因其具有优异的生物相容性和安全性,广泛应用于食品、药品以及医疗器械等领域。海藻酸钠是一种无毒、无刺激性、生物相容性良好的生物材料,可用于制备基于海藻酸钠的微球,包裹诸如细菌在内的微生物,在人体内发挥治疗作用。壳聚糖是一种价格便宜并且性质优良的天然高分子材料,其具有良好的生物相容性、可降解性和成膜性,这些性质使其成为制备微胶囊较好的壁材,同时壳聚糖作为壁材可以起到控制芯材释放的作用。
微球包裹技术是指利用高分子材料将均匀分散的液体甚至是固体混合物进行包裹,形成具有一定空间结构的微球。通过相关的技术处理可是微球具有一定的抗酸性、一定抗机械挤压以及良好的运输储存能力。常用制备微球的高分子材料有明胶、卡拉胶、黄原胶以及海藻酸盐等。壳聚糖和海藻酸钠为壁材所制备的海藻酸钠-壳聚糖微胶囊具有较好的耐酸性、肠溶性和贮存稳定性等特点。益生菌作为一种功能性食品,具有良好的益生功效。但如何使增强益生菌产品的口服抗酸性、在肠道的稳定快速增值性是此类产品共同面临的技术壁垒。
发明内容
本发明内容包括提供了一种新型益生菌微球的制备方法。所述微球具有制备工艺简单,成本低,口服抗酸性好,稳定性强,菌体生存率高等特点。可用于一种或多种益生菌的包裹、定植和增殖,具有良好的应用前景。
本发明内容包括提供了一种新型益生菌微球的制备方法。具体包括以下几个步骤:
(1)益生菌在培养基中发酵培养,经离心得到菌泥,将菌泥与益生元和保护剂混匀得到芯材。
(2)海藻酸盐溶液混合得到微球的芯材载体。再将芯材载体与菌泥混合,充分搅拌混匀得到微球囊壁。
(3)微球囊壁材料滴加入含有氯化钙和壳聚糖的溶液中,通过静电离子交换反应形成微球。
优选的,步骤(1)所述益生菌包括:两歧双歧杆菌、婴儿双歧杆菌、长双歧杆菌、短双歧杆菌、青春双歧杆菌、德氏乳杆菌保加利亚种、嗜酸乳杆菌、干酪乳杆菌干酪亚种、罗伊氏乳杆菌、嗜热链球菌、肠膜明串珠菌肠膜亚种、小牛葡萄球菌、木糖葡萄球菌、肉葡萄球菌和凝结芽孢杆菌中的一种或几种组合。
优选的,步骤(1)中所述培养基为MRS培养基,包括:牛肉膏10.0 g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0 g,柠檬酸二氨2.0g,MgSO4·7 H2O 0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0 mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
优选的,步骤(1)所述保护剂为白蛋白、明胶、可溶性淀粉、糊精,肉汁、果胶、阿拉伯胶、羟甲基纤维素、藻类等,以及天然混合物如脱脂牛奶、血清等中的一种或他们的混合物。
优选的,步骤(2)中所述益生元为菊粉、低聚果糖、抗性淀粉、改性淀粉、普鲁兰多糖、甘露聚糖和发明人已授权专利(授权号:CN 108079001 B)中木聚糖改性产物中的一种或者他们的混合物,尤其是优选发明人已授权专利(授权号:CN 108079001 B)中木聚糖改性产物。
优选的,步骤(2)中所述海藻酸盐不是唯一可使用的天然高分子材料,可选的材料还有:明胶、酪蛋白、玉米朊、果胶或它的衍生物、海藻酸或它的盐、琼脂、结冷胶、卡拉胶、红藻胶等。
优选的,步骤(3)所述氯化钙和壳聚糖溶液为混合溶液,具有一定的离子强度。
优选的,所述步骤(4)中,氯化钙溶液质量分数为1.5%-2.5%,壳聚糖溶液质量分数为0.3%-0.5%,并将调节pH 值至5-6。
优选的,所述微球的直径为4-8 mm,所述微球的包封率为70%-90%。
优选的,所述步骤(3)中,选用直径为0.5 mm软管为材料,通过恒流泵流速0.3 mL/min-1 mL/min的流速滴加进入氯化钙/壳聚糖溶液中进行成膜交联反应,形成稳定凝胶。微球采用真空干燥,温度30℃-40℃,干燥时间15-30 h。
所述益生菌微球具有以下特点:
优选的,所述益生菌微球室温存储崩解后存活率为:73%-85%。
优选的,所述益生菌微球具有良好的抗胃酸能力,在人工模拟胃液搅拌4 h后人能维持良好的微球形态,在模拟肠液中崩解时间为2 h,菌体存活率为60%-80%。
优选的,添加发明人已授权专利(授权号:CN 108079001 B)中木聚糖改性产物能够明显促进益生菌的增殖,优选添加的量为0.1%-2%(质量分数)
具体实施方式
以下对本发明的具体实施方式进行详细说明,应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
本发明的新型益生菌微球的制备包括微球芯材载体、芯材和微球壁组成。所述芯材为白蛋白、明胶、可溶性淀粉、糊精,肉汁、果胶、阿拉伯胶、羟甲基纤维素、藻类等,以及天然混合物如脱脂牛奶、血清等中的一种或他们的混合物。
所述芯材载体为菊粉、低聚果糖、抗性淀粉、改性淀粉、普鲁兰多糖、甘露聚糖和发明人已授权专利(授权号:CN 108079001 B)中木聚糖改性产物中的一种或者他们的混合物,尤其是优选发明人已授权专利(授权号:CN 108079001 B)中木聚糖改性产物。
所述微球壁为明胶、酪蛋白、玉米朊、果胶或它的衍生物、海藻酸或它的盐、琼脂、结冷胶、卡拉胶、红藻胶等。尤其优选的,氯化钙和壳聚糖溶液为混合溶液,具有一定的离子强度。优选质量分数为1.5%-2.5%,壳聚糖溶液质量分数为0.3%-0.5%,并将调节pH 值至5-6。
所述益生菌种类包括:两歧双歧杆菌、婴儿双歧杆菌、长双歧杆菌、短双歧杆菌、青春双歧杆菌、德氏乳杆菌保加利亚种、嗜酸乳杆菌、干酪乳杆菌干酪亚种、罗伊氏乳杆菌、嗜热链球菌、肠膜明串珠菌肠膜亚种、小牛葡萄球菌、木糖葡萄球菌、肉葡萄球菌和凝结芽孢杆菌中的一种或几种组合。
本发明的新型的制备方法包括:
(1)制备活化益生菌菌种:将一种或多种益生菌菌粉接种至已灭菌冷却的MRS液体培养基中,在 37℃恒温培养进入稳定期后,4000 r/min离心并用生理盐水洗涤,收集浓缩菌液;MRS液体培养基为:牛肉膏10.0 g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0 g,柠檬酸二氨2.0 g,MgSO4·7 H2O 0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0 mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
(2)微球芯材的配制:取20%-30%(质量分数)白蛋白、明胶、可溶性淀粉、糊精,肉汁、果胶、阿拉伯胶、羟甲基纤维素、藻类等,以及天然混合物如脱脂牛奶、血清等中的一种或他们的混合物,加入40%-50%(质量分数)的步骤(1)中所得菌泥,充分搅拌混匀。
(3)芯材载体溶液的配制:取2%-5%(质量分数)的发明人已获得专利授权的合成化合物木聚糖丁酸脂或木聚糖乙酸酯,与相同质量的菊粉和低聚果糖,加入步骤(3)中的芯材中,充分搅拌混匀。
(4)向步骤(3)溶液中加入到2%-5%(质量分数)海藻酸钠,混合均匀,取10-20mL芯材混合液备用。
(5)壳聚糖氯化钙溶液配制: 配制1%冰乙酸溶液,随后加入氯化钙,使其终浓度为1.5%-2.5%,备用;取40-60 mL、质量分数为0.3%-0.5%的壳聚糖溶液、调节其pH 值至5-6,将壳聚糖溶液倒入上述稀释后的氯化钙溶液中进行固化0.5-1小时,得到壳聚糖氯化钙溶液。
(6)微球的制备:将步骤(4)中的芯材混合液装入容器,用恒流泵以0.3 mL/min-1mL/min的流速加入到步骤(5)的壳聚糖氯化钙溶液中,经离子交换形成凝胶小球,并持续搅拌1 h。最后置于低温喷雾干燥机中干燥得微球。
实施例1
(1)制备活化益生菌菌种:将长双歧杆菌菌粉接种至已灭菌冷却的MRS液体培养基中,在 37℃恒温培养进入稳定期后,4000 r/min离心并用生理盐水洗涤,收集浓缩菌液;MRS液体培养基为:牛肉膏10.0 g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0g,柠檬酸二氨2.0 g,MgSO4·7 H2O 0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
(2)微球芯材的配制:取20%(质量分数)脱脂牛奶加入40%(质量分数)的步骤(1)中所得菌泥,充分搅拌混匀。
(3)芯材载体溶液的配制:取2%(质量分数)的发明人已获得专利授权的合成化合物木聚糖乙酸酯,与相同质量的菊粉和低聚果糖,加入步骤(2)中的芯材中,充分搅拌混匀。
(4)向步骤(3)溶液中加入到2.5%(质量分数)海藻酸钠,混合均匀,取20 mL芯材混合液备用。
(5)壳聚糖氯化钙溶液配制: 配制1%冰乙酸溶液,随后加入氯化钙,使其终浓度为2.5%,备用;取60 mL、质量分数为0.35%的壳聚糖溶液、调节其pH 值至5.5,将壳聚糖溶液倒入上述稀释后的氯化钙溶液中进行固化1 h,得到壳聚糖氯化钙溶液。
(6)微球的制备:将步骤(5)中的芯材混合液装入容器,用恒流泵以0.5 mL/min的流速加入到步骤(5)的壳聚糖氯化钙溶液中,经离子交换形成凝胶小球,并持续搅拌1 h。最后置于低温喷雾干燥机中干燥得微球。
实施例2
(1)制备活化益生菌菌种:将鼠李糖乳杆菌和长双歧杆菌菌粉分别接种至已灭菌冷却的MRS液体培养基中,在 37℃恒温培养进入稳定期后,4000 r/min离心并用生理盐水洗涤,收集浓缩菌液,将三种浓缩菌液以等质量混合;MRS液体培养基为:牛肉膏10.0 g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0 g,柠檬酸二氨2.0 g,MgSO4·7 H2O0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0 mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
(2)微球芯材的配制:取25%(质量分数)脱脂牛奶加入45%(质量分数)的步骤(1)中所得菌泥,充分搅拌混匀。
(3)芯材载体溶液的配制:取2%(质量分数)的发明人已获得专利授权的合成化合物木聚糖乙酸酯,与相同质量的菊粉和低聚果糖,加入步骤(3)中的芯材中,充分搅拌混匀。
(4)向步骤(4)溶液中加入到2.5%(质量分数)海藻酸钠,混合均匀,取20 mL芯材混合液备用。
(5)壳聚糖氯化钙溶液配制: 配制1%冰乙酸溶液,随后加入氯化钙,使其终浓度为2.5%,备用;取60 mL、质量分数为0.35%的壳聚糖溶液、调节其pH 值至5.5,将壳聚糖溶液倒入上述稀释后的氯化钙溶液中进行固化1 h,得到壳聚糖氯化钙溶液。
(6)微球的制备:将步骤(5)中的芯材混合液装入容器,用恒流泵以0.5 mL/min的流速加入到步骤(5)的壳聚糖氯化钙溶液中,经离子交换形成凝胶小球,并持续搅拌2 h。最后置于低温喷雾干燥机中干燥得微球。
实施例3
(1)制备活化益生菌菌种:将鼠李糖乳杆菌、长双歧杆菌和嗜酸乳杆菌菌粉分别接种至已灭菌冷却的MRS液体培养基中,在 37℃恒温培养进入稳定期后,4000 r/min离心并用生理盐水洗涤,收集浓缩菌液,将三种浓缩菌液以等质量混合;MRS液体培养基为:牛肉膏10.0g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0 g,柠檬酸二氨2.0 g,MgSO4·7 H2O 0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0 mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
(2)微球芯材的配制:取25%(质量分数)脱脂牛奶加入50%(质量分数)的步骤(1)中所得菌泥,充分搅拌混匀。
(3)芯材载体溶液的配制:取5%(质量分数)的发明人已获得专利授权的合成化合物木聚糖乙酸酯,与相同质量的菊粉和低聚果糖,加入步骤(3)中的芯材中,充分搅拌混匀。
(4)向步骤(4)溶液中加入到5%(质量分数)海藻酸钠,混合均匀,取20 mL芯材混合液备用。
(5)壳聚糖氯化钙溶液配制: 配制1%冰乙酸溶液,随后加入氯化钙,使其终浓度为3%,备用;取60 mL、质量分数为0.35%的壳聚糖溶液、调节其pH 值至5.5,将壳聚糖溶液倒入上述稀释后的氯化钙溶液中进行固化1 h,得到壳聚糖氯化钙溶液。
(6)微球的制备:将步骤(5)中的芯材混合液装入容器,用恒流泵以0.5 mL/min的流速加入到步骤(5)的壳聚糖氯化钙溶液中,经离子交换形成凝胶小球,并持续搅拌1 h。最后置于低温喷雾干燥机中干燥得微球。
将上述实施例获得的微胶囊进行应用性测试:
1. 人工胃液耐受试验:取微球2-5 g,加入人工胃液50 mL,37℃搅拌4 h后取出微球,将微球置入0.1 mol/L的柠檬酸钠溶液中进行崩解,后离心得到菌体,通过稀释涂布的方法计算菌体的存活率,结果见表1。
所述人工胃液的配制方法为:精密称取胃蛋白酶3.2 g,NaCl 2.0 g,于烧杯中,加适量去离子水充分溶解,HCl调pH 值至1.2,转入1 L容量瓶中加去离子水定容至刻度线,摇匀备用。
2. 肠液溶解性试验:将经过人工胃液处理3h后未溶解的微球转移至人工肠液中37℃处理2 h,每隔20 min取样,后离心得到菌体,通过稀释涂布的方法计算菌体释放率,见过见表2。测定菌体在人工肠液中的释放情况,结果发现微胶囊在人工肠液中处理120min后基本崩解完全。
所述人工肠液的配制方法为:精密称取KH2PO4 6.8 g,加去离子水250 mL溶解,加0.2 mol/L NaOH溶液77 mL和500 mL去离子水,再加胰酶10.0 g溶解后调pH值至6.8±0.1即可。
附表1 不同实施案例中微球在人工胃液中的存活率(%)
实施方式 | 实施例1 | 实施例2 | 实施例3 |
存活率(%) | 98 | 99. | 98 |
附表2 不同实施案例中微球在人工肠液中菌体的释放率(%)
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
Claims (7)
1.一种益生菌微球,其特征在于,
包括微球被膜、微球壁和微球芯材,
所述微球被膜由壳聚糖和氯化钙制备而成,
所述微球芯材中的益生元包含符合发明人授权专利(授权号: CN 108079001 B))中的益生元、菊粉和低聚果糖制备而成,
所述微球芯材由益生菌和白蛋白、明胶、可溶性淀粉、糊精,肉汁、果胶、阿拉伯胶等,以及天然混合物如脱脂牛奶、血清等中的一种或他们的混合物制备而成。
2.根据权利要求1所述的益生菌微球,其特征在于,
所述微球包括如下重量组份:海藻酸钠2-5份,壳聚糖2-4份,氯化钙1-2份、木聚糖乙酸酯或/和木聚糖丁酸酯、菊粉和低聚果糖一种或混合物15-25份、益生菌20-30份、脱脂牛奶10-20份。
3.根据权利要求1所述的益生菌微球,其特征在于,
所述微球的直径为4-8 mm,所述微球的包封率为73%-85%。
4.根据权利要求1所述的益生菌微球,其特征在于,
所述益生菌包含长双歧杆菌、鼠李糖乳杆菌和嗜酸乳杆菌中的一种或其任意混合物。
5.根据权利要求1-4中任一项所述的益生菌微球的制备方法,其特征在于,
所述方法包括:
(1)制备活化益生菌菌种:将菌种接种至MRS液体培养基中,37℃恒温培养进入稳定期后,离心并用生理盐水洗涤,收集浓缩菌液,
(3)微球芯材的配制:取20-30份明胶、可溶性淀粉、糊精、果胶、阿拉伯胶、脱脂牛奶中的一种或他们的混合物,加入40-50份的步骤(1)中所得菌泥,充分搅拌混匀,
(4)芯材载体溶液的配制:取2-5份的发明人已获得专利授权的合成化合物,与相同质量的菊粉和低聚果糖,加入步骤(3)中的芯材中,充分搅拌混匀,
(5)向步骤(4)溶液中加入到2-5份(质量分数)海藻酸钠,混合均匀,取10-20 mL芯材混合液备用,
(6)壳聚糖氯化钙溶液配制: 配制1%冰乙酸溶液,随后加入氯化钙,使其终浓度为1.5%-2.5%,备用;取40-60 mL、质量分数为1-2份的壳聚糖溶液、调节其pH 值至5-6,将壳聚糖溶液倒入上述稀释后的氯化钙溶液中进行固化0.5-1小时,
(7)微球的制备:将步骤(5)中的芯材混合液装入容器,用恒流泵以0.3 mL/min-1 mL/min的流速加入到步骤(6)的壳聚糖氯化钙溶液中,经离子交换形成凝胶小球,并持续搅拌1h,最后置于低温喷雾干燥机中干燥得微球。
6.根据权利要求5所述的益生菌微球的制备方法,其特征在于,
所述步骤(1)中,MRS液体培养基为:牛肉膏10.0 g,蛋白胨10.0 g,酵母膏5.0 g,三水合乙酸钠5.0 g,葡萄糖20.0 g,柠檬酸二氨2.0 g,MgSO4·7 H2O 0.58 g,K2HPO4 2.0 g,MnSO4·H2O 0.2 g,吐温80 1.0 mL,蒸馏水1000 mL,pH 6.5,121℃条件下灭菌20 min。
7.根据权利要求6所述的益生菌微球的制备方法,其特征在于,
所述步骤(4)中,氯化钙溶液质量分数为1.5%-2.5%,壳聚糖溶液质量分数为0.5%-1%,并将调节pH 值至5-6。
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CN113499325A (zh) * | 2021-07-08 | 2021-10-15 | 成都邦家乐君生物科技有限公司 | 一种用于益生菌活性保护的生物质基包封材料及包封方法 |
CN114223795A (zh) * | 2021-12-21 | 2022-03-25 | 西南科技大学 | 一种仔猪用益生菌饲料的制备方法 |
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CN113499325A (zh) * | 2021-07-08 | 2021-10-15 | 成都邦家乐君生物科技有限公司 | 一种用于益生菌活性保护的生物质基包封材料及包封方法 |
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