CN111150068A - 一种以复合材料为壁材的益生菌微胶囊及其制备方法 - Google Patents
一种以复合材料为壁材的益生菌微胶囊及其制备方法 Download PDFInfo
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- CN111150068A CN111150068A CN201911043477.7A CN201911043477A CN111150068A CN 111150068 A CN111150068 A CN 111150068A CN 201911043477 A CN201911043477 A CN 201911043477A CN 111150068 A CN111150068 A CN 111150068A
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Abstract
本发明涉及一种益生菌微胶囊的壁材的处理方法、益生菌微胶囊及其制备方法。
Description
对相关申请的引用
本申请要求了2018年11月6日提交的中国专利申请号201811314761.9的优先权,将申请的全部内容并入本文作为说明书的一部分。
技术领域
本发明涉及食品生物技术领域。更具体地,涉及一种以复合材料为壁材的益生菌微胶囊及其制备方法。
背景技术
益生菌作为一类对宿主健康有一定促进作用的微生物活体,在治疗保健等方面具有多重生理功能。然而,在实际生产、贮藏、运输等过程中,益生菌受到食品组分(酸、添加剂等)、环境温度及宿主消化系统(胃酸、酶、胆盐等)的影响,导致活菌数大幅下降,最终定植于肠道中的活菌数低于理论上能发挥益生作用的最小值。
为了有效提高益生菌活菌数量,本发明采用微胶囊技术对益生菌进行包埋,通过在益生菌活菌体周围形成一层或多层类似胶囊的保护膜,能显著提高益生菌在不利环境中的存活率,其中,壁材的选择、包埋方式、以及造粒方式对微胶囊保护作用的发挥至关重要。
尽管作为微胶囊的壁材的材料种类繁多,但单一的壁材很难满足制备微胶囊的各方面需要。例如,碳水化合物水溶性好,黏度低,但因结构上不具备亲脂集,乳化性和成膜性较差,大都缺乏达到高微胶囊化效率所需的界面特性,单独使用不能有效地包埋芯材。蛋白质因其具有良好的功能特性而被作为壁材广泛应用于微胶囊领域,它会起到促进乳状液形成,并通过减少界面张力及在油滴周围形成一层保护膜,因此乳化性极佳,成膜性良好,可得到较高的芯材保留率。但是,蛋白质含量、质构、蛋白和非蛋白成分的组成等内在的差异,及外界温度、离子强度、pH值等外界因素都会对其功能性质造成影响。
微胶囊技术的引入对于食品中敏感成分的保护,风味、感观方面的修饰都有着重大的意义,而壁材的组配又对微胶囊产品的功能和性质起着决定性的作用。而且,传统的挤压造粒方式也导致微胶囊成型稳定性较差,且不易进行规模化生产。
因此开发一种能较好地保护益生菌活性的复合壁材和相应的益生菌微胶囊,并且寻找一种使微胶囊成型较好且易实现扩大化生产的优化的造粒方式使得微胶囊技术能够在食品和药品领域得到更广泛的应用是当前亟待解决的课题。
发明内容
本发明的目的之一是提供了一种用于益生菌微胶囊的复合壁材、益生菌微胶囊及其制备方法。以本发明的复合壁材能够优化壁材的性状,相对于单一种类材料的壁材能够形成更为致密的网络结构,具有一定的强度和弹性,增加微胶囊的稳定性并能够更好地保护芯材。使用本发明的复合壁材制备的益生菌微胶囊的应用及保存条件受环境的影响较小,应用更加广泛,可应用到药品、保健品及食品领域。
本发明的另一目的是通过喷雾冷冻法制备采用上述复合壁材的益生菌微胶囊,全程于非热条件下操作,很大程度上避免制造微胶囊过程中益生菌的死亡,保持了其活性。而且,通过喷雾冷冻的造粒方式生产的微胶囊冷冻后滴入固化液中,相对现有的挤压造粒直接滴入固化液中,呈现的颗粒形状更好,能够更好地保护益生菌。同时,本发明的喷雾冷冻技术相对于挤压造粒的方式更易实现微胶囊批量化、规模化生产,具有重要的商业价值。
本发明还涉及一种益生菌微胶囊的制备方法,所述益生菌微胶囊具有一层或多层包埋结构,包括:菌芯颗粒和任选的至少一层包覆所述菌芯颗粒的外壳,其中,所述菌芯颗粒包括芯材和第一层壁材,所述芯材包括一种或多种益生菌粉或菌泥,所述芯材被所述第一层壁材所包覆,以及所述至少一层包覆所述菌芯颗粒的外壳包括两层或更多层壁材,分别为第二层壁材、第三层壁材或者更多层壁材,其中,所述菌芯颗粒通过喷雾冷冻法并采用上述复合壁材制备。
定义
除非另有说明,本文使用的所有技术和科学术语具有与本发明所属领域的技术人员通常理解相同的含义,但如有冲突,则以本说明书中的定义为准。
如说明书和权利要求书中所用,单数形式“一”、“一个”和“该(所述)”包括复数形式,除非上下文另有明确说明。
如无特殊说明,本说明书中的百分比(%)均为重量百分比(重量%)。
在说明书和权利要求书中使用的涉及组分量、工艺条件等的所有数值或表述在所有情形中均应理解被“约”修饰。术语“约”当指数量或数值范围时,意思是所指数量或者数值范围是试验变异性内(或统计学实验误差内)的近似值,因此该数量或者数值范围可以在所述数量或数值范围的例如+5之间变化。
涉及相同组分或性质的所有范围均包括端点,该端点可独立地组合。由于这些范围是连续的,因此它们包括在最小值与最大值之间的每一数值。还应理解的是,本申请引用的任何数值范围预期包括该范围内的所有子范围。
当本发明针对物理性质例如分子量或者针对化学性质范围时,应包括范围的所有组合和亚组合以及其内的具体实施方式。术语“包含”(以及相关术语例如“含有”或“含”或“具有”或“包括”)包括这样一些实施方式,该实施方式为例如,物质、组合物、方法或过程等的任何组合,其“由所描述的特征组成”或者“基本上由所描述的特征组成”。
本发明使用的缩写具有在食品、生物学和化学领域的通常含义。
益生菌:本发明的益生菌微胶囊内的益生菌类型,使用国家批准的任何益生菌作为微胶囊包埋芯材的保护对象。包括:青春双歧杆菌、动物双歧杆菌(乳双歧杆菌)、两歧双歧杆菌、短双歧杆菌、婴儿双歧杆菌、长双歧杆菌、嗜酸乳杆菌、干酪乳杆菌、卷曲乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳亚种、发酵乳杆菌、格氏乳杆菌、瑞士乳杆菌、约氏乳杆菌、副干酪乳杆菌、植物乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌、唾液乳杆菌、嗜热链球菌、凝结芽孢杆菌等益生菌中一种或多种。具体如附表A-D所示。
A.可用于食品的菌种名单
B.可用于保健食品的益生菌菌种名单
序号 | 名称 | 拉丁学名 | 备注 |
一 | 双歧杆菌属 | Bifidobacterium | |
1 | 两歧双歧杆菌 | Bifidobacterium bifidum | |
2 | 婴儿双歧杆菌 | Bifidobacterium infantis | |
3 | 长双歧杆菌 | Bifidobacterium longum | |
4 | 短双歧杆菌 | Bifidobacterium breve | |
5 | 青春双歧杆菌 | Bifidobacterium adolescentis | |
二 | 乳杆菌属 | Lactobacillus | |
1 | 保加利亚乳杆菌 | Lactobacillus bulgaricus | |
2 | 嗜酸乳杆菌 | Lactobacillus acidophilus | |
3 | 干酪乳杆菌干酪亚种 | Lactobacillus casei subsp.Casei | |
4 | 罗伊氏乳杆菌 | Lactobacillus reuteri | |
5 | 鼠李糖乳杆菌 | Lactobacillus rhamnosus | |
三 | 链球菌属 | Streptococcus | |
1 | 嗜热链球菌 | Streptococcus thermophilus |
C.可用于婴幼儿食品的菌种名单
D.可用于保健食品的真菌菌种名单
序号 | 中文名 | 英文名 |
1 | 酿酒酵母 | Saccharomyces cerevisiae |
2 | 产朊假丝酵母 | Cadida atilis |
3 | 乳酸克鲁维酵母 | Kluyveromyces lactis |
4 | 卡氏酵母 | Saccharomyces carlsbergensis |
5 | 蝠蛾拟青霉 | Paecilomyces hepiali Chen et Dai,sp.Nov |
6 | 蝙蝠蛾被毛孢 | Hirsutella hepiali Chen et Shen |
7 | 灵芝 | Ganoderma lucidum |
8 | 紫芝 | Ganoderma sinensis |
9 | 松杉灵芝 | Ganoderma tsugae |
10 | 红曲霉 | Monacus anka |
11 | 紫红曲霉 | Monacus purpureus |
壁材:用来包覆、保护或控制释放芯材的成囊物质称为微胶囊的壁材、囊壁或囊壳。壁材是构成微胶囊外壳的材料,也可以称为包衣或包材。
在选择壁材时要考虑壁材本身的性能,如渗透性、稳定性、机械强度、溶解性、可聚合性、电性能、吸湿性及成膜性等,对于生物活性物质的芯材,还要着重考虑壁材的毒性,与芯材的相容性。在本发明中选择具有无毒、成膜性或成球性较好、免疫原性低、生物相容性好、可降解且产物无毒副作用的壁材。
在本发明中,优选与活菌具有很好的生物相容性的成膜材料、食品中可选用的肠溶材料、具有阻湿性的成膜材料、具有阻氧性的成膜材料。
优选的,具体的实例包括下列的一种或多种的组合:
(1)可食用蛋白类:包括动物蛋白如乳蛋白、卵蛋白、酪蛋白,植物蛋白如谷类蛋白等;
优选的,本发明的益生菌微胶囊的壁材选自乳清蛋白:
包括浓缩乳清蛋白(WPC)、分离乳清蛋白(WPI)或乳清蛋白肽,尤其优选分离乳清蛋白(WPI);其中,WPI是在浓缩乳清蛋白(WPC)的基础上经过进一步工艺处理得到的高纯度乳清蛋白,纯度可达90%以上,且更易消化吸收,安全无毒无副作用;
乳蛋白:包括酪蛋白或乳清蛋白。乳清蛋白质是指溶解分散在乳清中的蛋白,约占乳蛋白质的18%~20%,可分为热稳定和热不稳定乳清蛋白两部分。
浓缩乳清蛋白(WPC)
将乳清直接烘干后,可得到乳清粉末,其中的乳清蛋白极低,一般为百分之十几,不超过百分之三十。乳清经过澄清、超滤、干燥等过程后得到的产物就是浓缩乳清蛋白。过滤程度的不同可以得到蛋白浓度从34-80%不等的产品。
分离乳清蛋白(WPI)
分离乳清蛋白是在浓缩乳清蛋白的基础上经过进一步的工艺处理得到的高纯度乳清蛋白,纯度可达90%以上。其价格昂贵,是浓缩乳清蛋白的2-3倍,但是它也更容易消化吸收。分离乳清蛋白的真正妙处在于它的营养价值,它拥有高含量的优质蛋白,能为某些特定需要的人群比如婴儿和住院病人提供所需优质蛋白。此外,分离乳清蛋白所含有的生物活性化合物如α-乳白蛋白和β-乳球蛋白、乳铁蛋白以及免疫球蛋白,都为市场注入了新鲜的活力。
乳清蛋白肽
乳清蛋白肽是乳清蛋白的水解产物,是乳清蛋白的精华,它在机体中能更快地参与肌肉合成的过程。
优选的,本发明的益生菌微胶囊的壁材选自下列中的一种或者多种的组合:
植物蛋白:包括油料种子蛋白、豆类蛋白质、谷类蛋白等,
其中,油料种子蛋白包括:花生蛋白、芝麻蛋白、油菜子蛋白、向日葵蛋白、棉子蛋白、红花蛋白、椰子蛋白等;
其中,豆类蛋白包括:大豆蛋白、蚕豆蛋白、豌豆蛋白、绿豆蛋白、红小豆蛋白、芸豆蛋白等;
其中,谷类蛋白包括:稻类(籼稻、粳稻、糯稻)蛋白、麦类(小麦、大麦、燕麦、黑麦)蛋白、玉米蛋白、高粱蛋白、粟蛋白、黍蛋白、黄米蛋白、荞麦蛋白等,以及薯类蛋白:包括甘薯蛋白、马铃薯蛋白、山药蛋白、芋蛋白、木薯蛋白等;
其中,玉米蛋白包括玉米醇溶蛋白、玉米胚蛋白等;
优选的,本发明的益生菌微胶囊的壁材选自玉米蛋白,包括玉米醇溶蛋白、玉米胚蛋白,尤其优选玉米醇溶蛋白。
(2)油脂类:
本发明中,油脂是油和脂肪的统称,是脂肪族羧酸与甘油所形成的酯,在室温下呈液态的称为油,呈固态或半固态的的称为脂肪。天然油脂大都是混合甘油酯。从植物种子中得到的大多为油,来自动物的大多为脂肪。
本发明中,根据脂肪酸的种类,各种天然脂肪酸分子是由不同碳链(4~24C)所组成的直链脂肪酸。除个别例外,碳原子均为双数。此类脂肪酸有两种分类法:一种是根据碳原子数将脂肪酸分为短链(4-6C)、中链(8-12C)及长链(12C以上)脂肪酸。另一种是将脂肪酸分为饱和及不饱和脂肪酸。饱和脂肪酸的一般分子式为CnH2nO2,而不饱和脂肪酸带有1、2、3个以至更多的双键,其一般分子式为CnH2n-2O2、CnH2n-4O2、CnH2n-6O2。其中有两个以上双键的亚油酸、亚麻酸及花生四烯酸称为多不饱和脂肪酸。除直接脂肪酸外,还有环状脂肪酸,大枫子油中的大枫子油酸与亚大枫子油酸。
本发明中的油脂中的脂肪酸大多是含偶数碳原子的饱和的或不饱和的脂肪酸,常见的有肉豆蔻酸(C14)、软脂酸(C16)、硬脂酸(C18)等饱和酸和棕榈油酸(C16,单烯)、油酸(C18,单烯)、亚油酸(C18,二烯)、亚麻酸(C18,三烯)等不饱和酸。某些油脂中含有若干特殊的脂肪酸,如桐油中的桐油酸,菜油中的油菜酸,蓖麻油中的蓖麻酸,椰子油中的橘酸等。
根据来源将脂肪分成动物性脂肪和植物性脂肪。动物性脂肪又有两大类,一类为水产动物脂肪,如鱼类、虾、海豹等另一类是陆生动物脂肪,其中大部分含饱和脂肪酸和较少量的不饱和脂肪酸。植物性脂肪如棉子油、花生油、菜子油、豆油等,其脂肪中主要含不饱和脂肪酸,而且多不饱和脂肪酸(亚油酸)含量很高,占脂肪总量的40~50%。但椰子油中的脂肪酸主要是饱和的脂肪酸。
中链甘油三酯(MCT):其为己酸(C6)到月桂酸(C12)组成的甘油三酯,尤其是指其组成中的脂肪酸为辛酸、癸酸等六碳链到十二碳链的脂肪酸的一种结构脂质,天然的MCT主要来源于椰子油、棕榈油等植物油中。低于己酸构成的甘油三酯为短链脂肪酸甘油三酯(SCT),高于月桂酸组成的甘油三酯称为长链脂肪酸甘油三酯(LCT)。
棕榈油:又称棕油、棕皮油,包括:毛棕榈油(CPO)、棕榈粕(PE)、毛棕榈仁油(CPKO)、棕榈仁粕(PKE)、-精炼棕榈油(RBD PO)、棕榈油色拉油(RBD PKO)、棕榈油酸(PFAD)、棕榈液油(简称OLEAN)、棕榈硬脂(简称STEARINE或ST)等。棕榈油在常温下呈半固态,其稠度和熔点在很大程度上取决于游离脂肪酸的含量。常把低酸值的棕榈油叫做软油,高酸值的油则叫做硬油。
本发明的益生菌微胶囊的壁材优选选自棕榈油,尤其优选33℃棕榈油、40℃棕榈油、44℃棕榈油、52℃棕榈油、58℃棕榈油,更优选熔点为40℃以上的棕榈油,尤其是熔点为40-50℃的棕榈油,例如熔点为40℃的棕榈油。
优选的,本发明的益生菌微胶囊的壁材选自下列中的一种或者多种的组合:
植物油:包括代可可脂、可可脂、菜籽油、大豆油、玉米油、花生油、棉籽油、葵花籽油、棕榈油(固体棕榈油或者液体棕榈油)、棕仁油、椰子油等;或者
动物油:猪油、牛油、鱼油、乳脂、羊脂等。
优选的,本发明的益生菌微胶囊的壁材选自优选熔点为40℃以上的油脂,尤其是熔点为40-50℃的油脂,例如熔点为40℃的棕榈油或MCT。
尤其优选下列中的一种或者多种的组合:中链甘油三酯(MCT)、代可可脂、可可脂、棕榈油、卵磷脂、棕榈油甘油单酯、氢化油脂(如:氢化棕榈油、硬化油、氢化大豆油)等,它们的熔点优选40℃以上,尤其优选熔点为40-50℃。
(3)其他材料:
包括:海藻酸钠、虫胶、CMC-Na、胶凝糖、黄原胶、k-角叉菜胶、醋酸邻苯二甲酸纤维素、麦芽糊精、淀粉、糊精、蔗糖、乳糖、葡聚糖、玉米糖浆、果胶、阿拉伯胶、壳聚糖、乙酰化单双甘油脂肪酸酯、魔芋胶、卡拉胶、蜡质或明胶等的一种或者多种的组合。
复合壁材
本发明的复合壁材是指将两种或者多种前述壁材混合复配以得到更优性能的壁材。本发明的复合壁材具有良好的协同作用,有利于提高壁材中含有这些成分的微胶囊的稳定性和致密性,改善微胶囊产品的品质,进而促进微胶囊技术在各行业的应用。
亲水胶体:通常是指能溶解于水,并在一定条件下充分水化形成黏稠、滑腻或胶冻溶液的大分子物质,在食品、医药、化工及其他许多领域中广泛应用。亲水胶体按来源可分为:植物分泌物,如果胶、瓜尔豆胶、阿拉伯胶等;微生物发酵、代谢产物,如黄原胶、结冷胶等;海藻胶提取物,如卡拉胶、琼脂、海藻酸盐等。
果胶:果胶是从植物细胞壁中提取的天然多糖类高分子化合物,柑橘果皮、苹果糊、甜菜浆等是提取果胶最常见的原料。根据果胶分子中酯化的半乳糖醛酸基的比例可将其分为高甲氧基果胶(包括高甲氧基水溶性番茄果胶与高甲氧基柑橘果胶等)和低甲氧基果胶(酯化度50%为区分值)。
固化剂:
本发明中的固化剂包含至少一种下列化合物的水溶液:乙酸钠、冰乙酸、柠檬酸、柠檬酸钠、钙盐、表面活性剂。
固化剂优选乙酸钠、冰乙酸和表面活性剂混合液,这样可以使蛋白在非共价键和共价键(如二硫键)的可共同作用下形成不可逆的化学凝胶,并结合表面活性剂的作用,提高蛋白颗粒凝胶强度和在分散相中的稳定性。
优选的,固化剂浓度为约0.5mol/L,pH为约4.5至约5.3,并含有占固化液溶液体积约0.01%~0.1%的表面活性剂。
具体实施方案
本发明涉及一种复合壁材,其包括两种或者多种壁材。
本发明涉及一种复合壁材,其包括蛋白质和亲水胶体,或者由蛋白质和亲水胶体组成。
更优选的,本发明的复合壁材包括蛋白质和亲水胶体,或者由蛋白质和亲水胶体组成,其中:
所述蛋白质选自上述可食用蛋白,例如动物蛋白如乳蛋白、卵蛋白、酪蛋白,植物蛋白如谷类蛋白等;所述亲水胶体选自上述植物分泌物、微生物发酵或代谢来源的胶体、海藻胶提取物等。
尤其优选的,本发明的复合壁材包括蛋白质和亲水胶体,或者由蛋白质和亲水胶体组成,其中:
所述蛋白质选自下列的一种或多种:乳蛋白(酪蛋白或乳清蛋白,如分离乳清蛋白、浓缩乳清蛋白、乳清蛋白肽)、卵蛋白、酪蛋白、花生蛋白、芝麻蛋白、油菜子蛋白、向日葵蛋白、棉子蛋白、红花蛋白、椰子蛋白、大豆蛋白、蚕豆蛋白、豌豆蛋白、绿豆蛋白、红小豆蛋白、芸豆蛋白、稻类(籼稻、粳稻、糯稻)蛋白、麦类(小麦、大麦、燕麦、黑麦)蛋白、玉米蛋白(玉米醇溶蛋白、玉米胚蛋白等)、高粱蛋白、粟蛋白、黍蛋白、黄米蛋白、荞麦蛋白、甘薯蛋白、马铃薯蛋白、山药蛋白、芋蛋白、木薯蛋白等;
所述亲水胶体选自下列的一种或多种:果胶(如桃胶)、阿拉伯胶、k-角叉菜胶、刺槐豆胶、卡拉胶、黄原胶、瓜尔豆胶、罗望子胶、亚麻籽胶、皂荚豆胶、黄蓍胶、印度树胶、刺梧桐胶、魔芋胶、印度芦荟提取液、菊糖、仙草多糖、亚麻籽胶、凝结多糖、普鲁兰糖、结冷胶、海藻酸丙二醇酯等。
在一个实施方案中,本发明的复合壁材包括一种或多种乳蛋白和一种或多种亲水胶体,其中:
优选的,乳蛋白选自酪蛋白或乳清蛋白,如分离乳清蛋白、浓缩乳清蛋白、乳清蛋白肽;
优选的,亲水胶体选自果胶、卡拉胶、黄原胶、瓜尔豆胶。
在一个实施方案中,上述壁材中,蛋白质的浓度为9~13%(重量)。
在一个实施方案中,上述壁材中,亲水胶体和蛋白质的重量比为约1:500~约1:25,优选的,亲水胶体和蛋白质的重量比为约1:450、约1:400、约1:350、约1:300、约1:250、约1:200、约1:150、约1:100、约1:50或其间的任意范围。
在一个实施方案中,上述用于益生菌微胶囊的复合壁材经下列步骤进行预处理:
(1)配制蛋白质和亲水胶体的混合溶液,即将蛋白质、亲水胶体与水混合、搅拌,
(2)充分溶解后对该混合溶液进行加热处理,使蛋白质变性,
(3)冷却后可形成含有亲水胶体的稳定的蛋白变性溶液,即为“壁材溶液”。
优选的,在上述用于益生菌微胶囊的复合壁材的预处理步骤中:
(i)所述的蛋白质和亲水胶体的混合溶液是将乳清蛋白按照约9~13%(重量),分散于去离子水中,按照亲水胶体和蛋白质重量比1:500~1:25(优选的,亲水胶体和蛋白质的重量比为约1:450、约1:400、约1:350、约1:300、约1:250、约1:200、约1:150、约1:100、约1:50或其间的任意范围)添加果胶,于约2~8℃恒速搅拌4~16小时,转速170RPM至240RPM;
(ii)所述的壁材溶液是将蛋白质和亲水胶体的混合溶液置于约75~96℃(优选约75℃、约76℃、约77℃、约78℃、约79℃、约80℃、约81℃、约82℃、约83℃、约84℃、约85℃、约86℃、约87℃、约88℃、约89℃、约90℃、约91℃、约92℃、约93℃、约94℃、约95℃、约96℃,或其间的任意范围),约30至约180分钟的热处理(优选约35、约40、约45、约50、约55、约60、约65、约70、约75、约80、约85、约90、约95、约100、约105、约110、约115、约120、约125、约130、约135、约140、约145、约150、约155、约160、约165、约170、约175分钟,或其间的任意范围),转速85至115rpm/每分钟;之后
(iii)迅速置于冰浴中冷却,冷却温度约-20℃~约4℃;
(iv)于冰箱中4℃条件下存放约10小时~约60小时后使用(优选约10、约11、12、约13、约14、约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约26、约27、约28、约29、约30、约31、约32、约33、约34、约35、约36、约37、约38、约39、约40、约41、约42、约43、约44、约45、约46、约47、约48、约49、约50、约51、约52、约53、约54、约55、约56、约57、约58、约59、约60小时,或其间的任意范围)。
在一个实施方案中,本发明的提供一种益生菌微胶囊的制备方法,其包括使用上述制备的用于益生菌微胶囊的复合壁材包埋益生菌的步骤,具体为:
(1)将本发明中的益生菌的菌粉或菌泥加入到上述制备的复合壁材溶液中,菌粉或菌泥与壁材溶液的比例(重量)为:约1:1~约1:19,优选为:约1:1、约1:2、约1:3、约1:4、约1:5、约1:6、约1:7、约1:8、约1:9、约1:10、约1:11、约1:12、约1:13、约1:14、约1:15、约1:16、约1:17、约1:18、约1:19或其间的任意范围,尤其优选为约1:3~4:6,
并置于30℃恒温水浴锅中,同时低强度搅拌,优选转速约85至115RPM(更优选约95RPM),待温度均匀且二者混合均匀后,优选200目过滤待用;
(2)将上述混合均匀的益生菌的菌粉和壁材溶液以喷雾方式造粒;
(3)进行冷冻冻结和固化,其中,
优选的,在室温下,使用喷雾装置将上述含有益生菌的壁材混悬液通过优选约50~约300μm喷嘴将混悬液喷出,更优选约约50、约60、约70、约80、约90、约100、约110、约120、约130、约140、约150、约160、约170、约180、约190、约200、约210、约220、约230、约240、约250、约260、约270、约280、约290、约300μm或其间的任意范围,尤其优选约100μm;
优选的,频率为约400~约5000Hz,更优选约400、约1000、约1500、约2000、约2500、约3000、约4000、约5000Hz或其间的任意范围,尤其优选1000Hz;
优选的,喷头供料压力为约100~约3000mbar,更优选约100、约200、约300、约400、约500、约600、约700、约800、约900、约1000、约1200、约1500、约1800、约2000、约2500、约3000mbar或其间的任意范围,尤其优选约800mbar;
颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为约-40~0℃,更优选约-40℃、约-35℃、约-30℃、约-25℃、约-20℃、约-15℃、约-10℃、约-5℃、约0℃,尤其优选约-40℃和约-20℃;
落入固化液中,固化时间至少约10分钟,
优选的,固化液为浓度约为0.5mol/L,pH约4.5至5.3的含有占固化液溶液体积约0.01%~0.1%的吐温-20的乙酸-乙酸钠缓冲液;
最终在固化液中固化成球型的微胶囊;
优选的,以50目~500目过滤去除固化液,收集湿粒;
(4)任选将湿粒在无水乙醇中浸泡约10~30分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
(5)将上述(4)所得的微胶囊湿粒进行干燥,直至完全干燥后收集所得微胶囊,
其中,优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度约-60℃~约0℃,更优选约-50℃、-40℃、约-35℃、约-30℃、约-25℃、约-20℃、约-15℃、约-10℃、约-5℃、约0℃,尤其优选约-50℃。
在一个实施方案中,本发明还涉及一种益生菌微胶囊的制备方法,所述益生菌微胶囊具有一层或多层包埋结构,包括:菌芯颗粒和任选的至少一层包覆所述菌芯颗粒的外壳,其中,所述菌芯颗粒包括芯材和第一层壁材,所述芯材包括一种或多种益生菌粉或菌泥,所述芯材被所述第一层壁材所包覆,以及所述至少一层包覆所述菌芯颗粒的外壳包括两层或更多层壁材,分别为第二层壁材、第三层壁材或者更多层壁材,其中,包括以下步骤:
(1)单层微胶囊(即菌芯颗粒)制备:根据上文描述喷雾冷冻法并采用上述复合壁材制备;和任选的(2)多层微胶囊制备:将上述单层微胶囊干粒作为菌芯颗粒,在菌芯颗粒外均匀涂布一层壁材(优选通过流化床喷雾造粒法),并任选喷涂固化液使胶囊壁固化而不溶于水,将所制微胶囊干燥(优选于流化床中干燥),收集双层微胶囊干粒或者将其继续再次或多次包衣。
本发明还涉及一种益生菌微胶囊的制备方法,所述益生菌微胶囊具有一层或多层包埋结构,包括以下步骤:
(1)第一层微胶囊的包被:根据上文描述喷雾冷冻法并采用上述复合壁材制备;和任选的
(2)第二层微胶囊的包被:将步骤(1)得到的微胶囊与第二壁材混匀,通过流化床喷雾法制备微胶囊,将所制微胶囊干燥并收集双层微胶囊干粒;
(3)任选将步骤(2)得到的微胶囊继续于流化床中进行第三次或更多次包衣。
在一个实施方案中,上述方法制备所得的益生菌微胶囊的第二层、第三层或更多层壁材选自可食用蛋白、油脂或其他材料中的一种或多种组合。
优选的,上述可食用蛋白选自动物蛋白或者植物蛋白中的一种或多种组合:
其中,动物蛋白为适用于本发明的益生菌微胶囊的任意动物蛋白,例如本发明中定义的任意动物蛋白中的一种或多种组合,优选乳蛋白、卵蛋白、酪蛋白等,更优选乳清蛋白,包括浓缩乳清蛋白(WPC)、分离乳清蛋白(WPI)或乳清蛋白肽,尤其优选分离乳清蛋白(WPI);
其中,植物蛋白为适用于本发明的益生菌微胶囊的任意植物蛋白,例如本发明中定义的任意植物蛋白中的一种或多种组合,包括油料种子蛋白、豆类蛋白质、谷类蛋白等,
其中,油料种子蛋白优选:花生蛋白、芝麻蛋白、油菜子蛋白、向日葵蛋白、棉子蛋白、红花蛋白、椰子蛋白等;
其中,豆类蛋白优选:大豆蛋白、蚕豆蛋白、豌豆蛋白、绿豆蛋白、红小豆蛋白、芸豆蛋白等;
其中,谷类蛋白优选:稻类(籼稻、粳稻、糯稻)蛋白、麦类(小麦、大麦、燕麦、黑麦)蛋白、玉米蛋白、高粱蛋白、粟蛋白、黍蛋白、黄米蛋白、荞麦蛋白等,以及薯类蛋白:包括甘薯蛋白、马铃薯蛋白、山药蛋白、芋蛋白、木薯蛋白等;
其中,玉米蛋白优选玉米醇溶蛋白、玉米胚蛋白等。
优选的,上述油脂为适用于本发明的益生菌微胶囊的任意油脂,例如本发明中定义的油脂中的一种或多种组合,优选选自下列中的一种或者多种的组合:
植物油:包括可可脂、菜籽油、大豆油、玉米油、花生油、棉籽油、葵花籽油、棕榈油(固体棕榈油或者液体棕榈油)、棕仁油、椰子油等;或者
动物油:猪油、牛油、鱼油、乳脂、羊脂等;
更优选的,本发明的益生菌微胶囊的壁材选自熔点为40℃以上的油脂,尤其是熔点为40-50℃的油脂。
尤其优选下列中的一种或者多种的组合:中链甘油三酯(MCT)、可可脂、棕榈油、卵磷脂、棕榈油甘油单酯、氢化油脂(如:氢化棕榈油、硬化油、氢化大豆油)等。
优选的,上述其他材料选自海藻酸钠、虫胶、CMC-Na、胶凝糖、黄原胶、k-角叉菜胶、醋酸邻苯二甲酸纤维素、麦芽糊精、淀粉、糊精、蔗糖、乳糖、葡聚糖、玉米糖浆、果胶、阿拉伯胶、壳聚糖、乙酰化单双甘油脂肪酸酯、魔芋胶、卡拉胶、蜡质或明胶等的一种或者多种的组合。
在一个实施方案中,上述方法制备所得的益生菌微胶囊的壁材选自下列中的一种或者多种的组合:
植物蛋白,例如大豆蛋白、大米蛋白、小麦蛋白、玉米蛋白等;优选为玉米蛋白,包括玉米醇溶蛋白、玉米胚蛋白,尤其优选玉米醇溶蛋白,或
动物蛋白,例如乳清蛋白、酪蛋白等;优选浓缩乳清蛋白(WPC)、分离乳清蛋白(WPI)或乳清蛋白肽,尤其优选分离乳清蛋白(WPI);或者
油脂,例如熔点为40℃以上的油脂,优选中链甘油三酯(MCT)、棕榈油、可可脂、氢化油脂(如:氢化棕榈油、硬化油、氢化大豆油)、卵磷脂、棕榈油甘油单酯、椰子油、豆油、花生油、葵花籽油,或者
其他材料,例如海藻酸钠、虫胶、CMC-Na、胶凝糖、黄原胶、k-角叉菜胶、醋酸邻苯二甲酸纤维素、麦芽糊精、淀粉、糊精、蔗糖、乳糖、葡聚糖、玉米糖浆、果胶、阿拉伯胶、壳聚糖、乙酰化单双甘油脂肪酸酯、魔芋胶、卡拉胶、蜡质或明胶。
在一个实施方案中,上述益生菌微胶囊的制备方法中,以上述方法所得的益生菌微胶囊以1000重量份微胶囊干粒为基准,
益生菌菌泥或菌粉为约50至约500重量份,更优选:约50重量份、约100重量份、约150重量份、约200重量份、约250重量份、约300重量份、约350重量份、约400重量份、约450重量份、约500重量份或其间的任意范围,尤其优选约250~约400重量份;
第一层壁材为约150至约950重量份,更优选:约200重量份、约250重量份、约300重量份、约350重量份、约400重量份、约450重量份、约500重量份、约550重量份、约600重量份、约650重量份、约700重量份、约750重量份、约800重量份、约850重量份、约900重量份、约950重量份或其间的任意范围,尤其优选约450~约750重量份;
任选的第二层壁材为约0至约350重量份,更优选:100重量份、150重量份、200重量份、250重量份、300重量份、350重量份或其间的任意范围,尤其优选200-300重量份;
任选的第三层约0至约350重量份,更优选:约100重量份、约150重量份、约200重量份、约250重量份、约260重量份、约300重量份、约350重量份或其间的任意范围,尤其优选约200~约300重量份。
在一个实施方案中,上述益生菌微胶囊的制备方法中,菌粉或菌泥和第一层壁材的比例为:约1:1~约1:19,优选为:约1:1、约1:2、约1:3、约1:4、约1:5、约1:6、约1:7、约1:8、约1:9、约1:10、约1:11、约1:12、约1:13、约1:14、约1:15、约1:16、约1:17、约1:18、约1:19或其间的任意范围,尤其优选为约1:3~4:6。
优选的,上述益生菌微胶囊的制备方法,其中,第(1)第一层微胶囊的包被的步骤中制得的湿颗粒经干燥后粒径为约50-300um,优选约50-150um,更优选:约50um、约100um、约200um、约250um、约300um或其间的任意范围。
在一个实施方案中,本发明的提供一种益生菌微胶囊的制备方法,其包括使用上述制备的用于益生菌微胶囊的复合壁材包埋益生菌的步骤,其中,
所述复合壁材包括一种或多种乳蛋白和一种或多种亲水胶体,
优选的,乳蛋白选自酪蛋白或乳清蛋白,如分离乳清蛋白、浓缩乳清蛋白、乳清蛋白肽;
优选的,亲水胶体选自果胶、卡拉胶、黄原胶、瓜尔豆胶;
更优选的,所述复合壁材包括分离乳清蛋白和果胶;
(1)上述用于益生菌微胶囊的复合壁材经下列步骤进行预处理:
a.配制蛋白质和亲水胶体的混合溶液,蛋白质的浓度(重量)为约9~13%,亲水胶体和蛋白质重量比为约1:200、约1:100、约1:50或其间的任意范围,并于约2~8℃恒速搅拌4~16小时,转速170RPM至240RPM;
b.所述的壁材溶液是将蛋白质和亲水胶体的混合溶液置于约78~80℃,约45分钟的热处理转速85至115rpm/每分钟;之后
c.迅速置于冰浴中冷却,冷却温度约-20℃~约4℃;
d.于冰箱中4℃条件下存放约14小时后使用;
(2)采用喷雾冷冻法制备微胶囊:
e.将益生菌(如,凝结芽孢杆菌)的菌粉或菌泥加入到上述制备的复合壁材溶液中,其中,菌粉或菌泥与壁材溶液的比例为:约1:3~4:6(优选1:3),
优选的,上述益生菌微胶囊以1000重量份微胶囊干粒为基准,益生菌菌泥或菌粉为为约50至约500重量份(优选约250至约400重量份),复合壁材为约150至约950重量份(优选,约300至约650重量份);
f.将上述菌粉或菌泥与壁材溶液置于约30℃恒温水浴锅中,同时低强度搅拌,优选转速85至115RPM,待温度均一且二者混合均匀后待用;
g.将上述混合均匀的益生菌和壁材溶液以喷雾方式造粒,优选约50~约300μm(更优选约100μm)喷嘴将混悬液喷出,频率为约400~约5000Hz(优选约1000Hz),喷头供料压力为约100~约3000mbar(优选约800mbar);
喷出的颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为约-40~0℃,更优选约-40℃或-20℃;
h.在固化液中固化时间至少约10分钟,形成球型的微胶囊,优选的,以50目~500目过滤去除固化液,收集湿粒;
优选的,固化液为浓度约为0.5mol/L,pH约4.5至5.3的含有占固化液溶液体积约0.01%~0.1%的吐温-20的乙酸-乙酸钠缓冲液;
i.任选将上述所得的湿粒在无水乙醇中浸泡约10~30分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
j.将上述i中所得的微胶囊湿粒进行干燥,直至完全干燥后收集所得微胶囊,其中,优选的,经鼓风干燥或更优选的经冷冻干燥,
优选的,冷冻干燥的温度温度约-60℃~约0℃,更优选约-50℃;
(3)双层微胶囊的制备:
k.任选将步骤(2)得到的微胶囊与第二壁材混匀,优选通过流化床喷雾法制备微胶囊,将所制微胶囊干燥并收集双层微胶囊干粒;
优选的,所述第二层壁材为WPI或油脂(优选为中链甘油三酯(MTC))或者其组合物;
优选的,采用流化床法进行第二次包衣,优选采用升温至25至75℃乳清蛋白凝胶液,优选流化床进风温度为60至80℃;
(4)多层微胶囊的制备:
l.任选将步骤(3)得到的微胶囊继续于流化床中进行第三次或更多次包衣,
优选的,第三层壁材为WPI,并将WPI与水混匀制成乳清蛋白溶液或者凝胶液,将该溶液或者凝胶液升温至约25至约75℃(优选为约50℃);所述流化床进风温度为约60至约80℃(优选为约65℃);
优选的,在上述双层油脂颗粒外均匀喷涂WPI溶液,其中第三层蛋白固形物含量为0至约300重量份。
本发明还涉及一种益生菌微胶囊,所述益生菌微胶囊具有一层或多层包埋结构,其根据上述的益生菌微胶囊的制备方法制备得到。
优选的,上述的双层或多层益生菌微胶囊,其中第一层包衣后得到的菌芯颗粒外径为约50~约300μm,双层或多层微胶囊外径为约100~约500μm。
本发明的益生菌微胶囊可运用于食品和保健品行业例如膳食补充剂中。本发明还涉及益生菌微胶囊作为食品添加剂的应用,例如用于热加工食品或冷冻食品中,优选用于发酵风味食品、饮料、巧克力、糖果如口香糖、烘焙食品例如布丁、果蔬汁食品等。
优选的,本发明的益生菌微胶囊为乳制品添加剂,更优选为添加到牛奶、酸奶、奶酪、冰激凌、奶粉、乳制品饮料中。
优选的,含有益生菌的微胶囊的添加量为0.03~0.15%。
实施例
以下实施例中益生菌添加量为1010cfu/mL,(其为凝结芽孢杆菌)。
实施例1~5和相应对比例:
制备复合壁材时,壁材材料使用分离乳清蛋白、果胶,本发明的实施例1~5、对比例1~5的具体操作如下:
实施例1
制备壁材溶液:将分离乳清蛋白分散于无菌去离子水中,配制成质量分数为11%的乳清蛋白溶液,,再按照果胶和分离乳清蛋白干物质重量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40~0℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.01%~0.1%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡10~30分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-60~0℃。
本实施例中所得壁材溶液流动性强,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为2.1×1010cfu·mL-1。
对比例1
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成质量分数为8%的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本对比例中所得壁材溶液流动性强,用此壁材制得的微胶囊湿粒塌陷,呈扁平状。微胶囊干粒内活菌数为7.7×109cfu·mL-1。
实施例2
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:200加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本实施例中所得壁材溶液有一定流动性,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为2.3×1010cfu·mL-1。
对比例2
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,不加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本对比例中所得壁材溶液有一定流动性,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为4.4×109cfu·mL-1。
实施例3
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:50加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本实施例中所得壁材溶液有一定流动性,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为1.3×1010cfu·mL-1。
对比例3
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:20加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本对比例中所得壁材溶液流动性差,用此壁材制备微胶囊是堵塞喷头,无颗粒产生。
实施例4
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,80℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本实施例中所得壁材溶液有一定流动性,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为2.5×1010cfu·mL-1。
对比例4
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,70℃,低强度搅拌95RPM,保持45分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本对比例中所得壁材溶液粘度低,流动性强,用此壁材制得的微胶囊湿粒有拖尾,不规则,呈扁平状。微胶囊干粒内活菌数为6.4×107cfu·mL-1。
实施例5
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持35分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本实施例中所得壁材溶液有一定流动性,用此壁材制得的微胶囊湿粒呈球形,圆滑。微胶囊干粒内活菌数为7.9×109cfu·mL-1。
对比例5
制备壁材溶液,将分离乳清蛋白分散于无菌去离子水中,配制成11%(w/w)的乳清蛋白溶液,自然pH,再按照果胶和分离乳清蛋白干物质质量比1:100加入果胶,于4℃低强度恒速搅拌16小时,转速180RPM,使其充分水合至溶液澄清,无结块。将所得蛋白溶液置于恒温水浴锅中加热,78℃,低强度搅拌95RPM,保持20分钟。随后立即放入冷水槽中冷却,4℃储存14小时备用,所得即为壁材溶液。
造粒方法:在室温下,使用喷雾装置将上述含有益生菌的壁材混悬浮液通过优选100μm喷嘴将混悬液喷出,优选频率1000Hz,优选喷头供料压力800mbar,
随后颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为-40℃;
最终落入浓度为0.5mol/L,pH 4.5至5.3的含有占固化液溶液体积0.04%的吐温-20的乙酸-乙酸钠缓冲液中,固化时间至少10分钟,微胶囊在固化液中固化成球型;
过滤去除固化液,收集湿粒;
再将湿粒在无水乙醇中浸泡20分钟以便在后续步骤中促进颗粒干燥并去除杂菌,收集获得湿粒;
将所得的微胶囊湿粒进行干燥,直至微胶囊完全干燥后收集所得,
优选的,经鼓风干燥或更优选的,经冷冻干燥,优选的温度-50C。
本对比例中所得壁材溶液粘度低,流动性强,用此壁材制得的微胶囊湿粒有拖尾,不规则,呈扁平状。微胶囊干粒内活菌数为6.3×107cfu·mL-1。
实施例6~10和相应的对比例:
以下实施例和对比例中的壁材溶液按照实施例1方法制备。
实施例6
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本实施例制得的微胶囊干粒呈球形,圆滑,有光泽,平均粒径120μm,微胶囊干粒内活菌数为2.1×1010cfu·mL-1。
对比例6
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头40μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本对比例中制得的微胶囊为碎片状,平均粒径50μm,微胶囊干粒内活菌数为3.5×107cfu·mL-1。
实施例7
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率2000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本实施例中制得的微胶囊呈球形,圆滑,有光泽,平均粒径115μm,微胶囊干粒内活菌数为1.9×1010cfu·mL-1。
对比例7
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率3000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本对比例中因频率太高,液体发生粘连,无颗粒产生。
实施例8
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力1000mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本实施例中制得的微胶囊呈球形,圆滑,有光泽,平均粒径100μm,微胶囊干粒内活菌数为2.8×1010cfu·mL-1。
对比例8
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力50mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本对比例中因供料压力太高,液体雾化效果差,无颗粒产生。
实施例9
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-20℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本实施例中制得的微胶囊呈球形,圆滑,有光泽,平均粒径110μm,微胶囊干粒内活菌数为1.8×1010cfu·mL-1。
对比例9
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度5℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-50℃,直至完全干燥后取出即获得微胶囊干粒。
本对比例中制得的微胶囊呈球形,平均粒径130μm,微胶囊干粒内活菌数为1.1×1010cfu·mL-1。
实施例10
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度-30℃,直至完全干燥后取出即获得微胶囊干粒。
本实施例中制得的微胶囊呈球形,圆滑,有光泽,平均粒径110μm,微胶囊干粒内活菌数为4.4×1010cfu·mL-1。
对比例10
制备益生菌与壁材混合悬浮液,将壁材溶液置于30℃恒温水浴中,按照菌粉或菌泥和壁材溶液质量比1:3迅速加入菌粉或菌泥,低强度搅拌95RPM,待温度均匀且混合均匀后,200目过滤后待用。
制备固化液,配制0.5M,pH 4.6的乙酸-乙酸钠缓冲液,室温,加入占固化液溶液体积0.04%的吐温-20,搅拌均匀待用。
喷雾冷冻设备设定,使用喷头100μm,频率1000Hz,喷头供料压力800mbar,将菌和蛋白悬浮混合液保持160RPM搅拌通过喷头喷入有冷空气循环的腔体中,腔体温度-40℃,随后落入制备好的固化液中固化,固化液保持搅拌,搅拌速率300RPM,固化20分钟。
将含有颗粒的固化液经200目过滤、去除固化液,收集湿粒,再将所得湿粒浸泡于无水乙醇中,10分钟后取出,收集湿粒,放入容器中,待干燥。
将湿粒进行冷冻干燥中,温度0℃,直至完全干燥后取出即获得微胶囊干粒。
本对比例中制得的微胶囊呈球形,平均粒径120μm,微胶囊干粒内活菌数为1.3×1010cfu·mL-1。
制备复合壁材时,壁材材料使用分离乳清蛋白、果胶,本发明的实施例1~5、对比例1~2的具体操作如下:
本发明中实施例1-5、对比例1-5结果及评价如下:
在使用冷冻喷雾装置进行喷雾造粒时,本发明的实施例6-10、对比例6-10的具体操作如下:
以下实施例中壁材溶液按照实施例1方法制备。
本发明中实施例6-10、对比例6-10产品结果及评价如下:
Claims (11)
1.一种用于益生菌微胶囊的复合壁材的预处理方法,所述复合壁材包括一种或多种蛋白质和亲水胶体,或者由一种或多种蛋白质和亲水胶体组成,其中,所述复合壁材经下列步骤进行预处理:
(1)配制蛋白质和亲水胶体的混合溶液,即将蛋白质、亲水胶体与水混合、搅拌,
(2)充分溶解后对该混合溶液进行加热处理,使蛋白质变性,
(3)冷却后可形成含有亲水胶体的稳定的蛋白变性溶液,即为“壁材溶液”。
2.根据上述权利要求1中所述的预处理方法,其中,蛋白质的浓度为9~13%重量。
3.根据上述权利要求中任一项所述的预处理方法,其中,亲水胶体和蛋白质重量比1:500~1:25。
4.根据上述权利要求中任一项所述的预处理方法,其中,
(1)将蛋白质和亲水胶体的混合溶液于约2~8℃恒速搅拌4~16小时;
(2)将蛋白质和亲水胶体的混合溶液置于约75~96℃,约30至约180分钟的热处理;之后
(3)迅速冷却,冷却温度约-20℃~约4℃;
(4)于冰箱中4℃条件下存放约10小时~约60小时后使用。
5.根据上述权利要求中任一项所述的预处理方法,其中,所述蛋白质选自上述可食用蛋白,例如动物蛋白如乳蛋白、卵蛋白、酪蛋白,植物蛋白如谷类蛋白等;所述亲水胶体选自上述植物分泌物、微生物发酵或代谢来源的胶体、海藻胶提取物等,
优选的,所述乳蛋白选自酪蛋白或乳清蛋白如分离乳清蛋白、浓缩乳清蛋白;
优选的,所述亲水胶体选自下列的一种或多种:果胶(如桃胶)、阿拉伯胶、k-角叉菜胶、刺槐豆胶、卡拉胶、黄原胶、瓜尔豆胶、罗望子胶、亚麻籽胶、皂荚豆胶、黄蓍胶、印度树胶、刺梧桐胶、魔芋胶、印度芦荟提取液、菊糖、仙草多糖、亚麻籽胶、凝结多糖、普鲁兰糖、结冷胶、海藻酸丙二醇酯。
6.一种用于益生菌微胶囊的复合壁材,所述复合壁材包括一种或多种蛋白质和亲水胶体,或者由一种或多种蛋白质和亲水胶体组成,其根据权利要求1~5任一项的预处理方法制备得到。
7.一种益生菌微胶囊的制备方法,其包括使用权利要求1~5中任一项的预处理方法制备复合壁材的步骤,具体为:
(1)将益生菌的菌粉或菌泥加入到上述制备的复合壁材溶液中,并于约30℃低强度搅拌,待混合均匀后,优选过滤待用;
(2)将上述混合均匀的益生菌的菌粉和壁材溶液以喷雾冷冻方式造粒;
(3)进行固化得到微胶囊湿颗粒;
(4)将上述(3)所得的微胶囊湿颗粒进行干燥,收集得到微胶囊干颗粒。
8.根据权利要求7的益生菌微胶囊的制备方法,其中:
喷雾装置的喷嘴优选约50~约300μm;
优选的,频率为约400~约5000Hz;
优选的,喷头供料压力为约100~约3000mbar;
优选的,颗粒在下落过程中在有冷空气循环的腔体中冷冻冻结,腔体温度优选为约-40~0℃。
9.根据权利要求7~8的任一项的益生菌微胶囊的制备方法,其中,一种益生菌微胶囊的制备方法,其中,还包括制备多层微胶囊的步骤:将采用复合壁材包衣得到的上述微胶囊干粒作为菌芯颗粒,在菌芯颗粒外均匀涂布第二层壁材,并任选喷涂固化液使胶囊壁固化而不溶于水,将所制的双层微胶囊干燥,收集所述双层微胶囊干粒或者将其继续第三次或多次包衣得到多层微胶囊。
10.一种益生菌微胶囊,所述益生菌微胶囊具有一层或多层包埋结构,其根据权利要求1~9任一项的益生菌微胶囊的制备方法制备得到。
11.前述权利要求10中所述的益生菌微胶囊益生菌微胶囊作为食品和保健品行业例如膳食补充剂的应用,例如用于热加工食品或冷冻食品中,优选用于发酵风味食品、饮料、巧克力、糖果如口香糖、烘焙食品例如布丁、果蔬汁食品更优选的,本发明的益生菌微胶囊为乳制品添加剂,如添加到牛奶、酸奶、奶酪、冰激凌、奶粉、乳制品饮料中。
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