CN112544978B - 一种可在肠道定点释放的微胶囊包埋的益生菌及其制备方法 - Google Patents
一种可在肠道定点释放的微胶囊包埋的益生菌及其制备方法 Download PDFInfo
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- CN112544978B CN112544978B CN202011389984.9A CN202011389984A CN112544978B CN 112544978 B CN112544978 B CN 112544978B CN 202011389984 A CN202011389984 A CN 202011389984A CN 112544978 B CN112544978 B CN 112544978B
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Abstract
本发明公开了一种可在肠道定点释放的微胶囊包埋的益生菌及其制备方法。该制备方法包括将扩增培养后的益生菌制备成W/O型芯材,然后包覆乳清蛋白和海藻酸钠构成的壁材,再依次经过Ca2+一次固化、纳晶纤维素涂层和Ca2+二次固化。本发明的包埋益生菌的微胶囊不但肠溶性好,而且在模拟胃肠消化后,益生菌活性仍能达到106CFU/g左右。
Description
技术领域
本发明属于益生菌技术领域,更具体地,本发明涉及一种可在肠道定点释放的微胶囊包埋益生菌及其制备方法。
背景技术
益生菌指活的微生物制剂,其通过调节宿主黏膜与系统免疫功能和改善宿主的肠道微生态平衡,达到促进营养吸收、防治肠道相关疾病、增强机体免疫力和延缓衰老等作用。据日本发酵乳与乳酸菌饮料协会规定益生菌含量必须至少为106CFU/g才有足够的活菌发挥保健功能。然而益生菌对外界环境的抵抗能力很弱,较多研究已证实酸奶和发酵乳中的益生菌的存活率很低,发酵乳制品在~18℃的条件下贮存8~12周后,活菌数从109CFU/g下降到104CFU/g以下。
肠道微胶囊包埋益生菌化一直是国内外研究的热点,微胶囊包埋后可降低外界环境对细胞的损伤。微胶囊包埋的益生菌的存活率可提高80~95%。例如,中国专利申请公开号:CN111264870A,公开了一种高环境抗性的微胶囊包埋益生菌及其制备方法;中国专利申请公开号:CN111134334A,公开了一种微胶囊包埋益生菌软颗粒及制备方法;中国专利申请公开号:CN109453207A,公开了一种硒化海藻酸钠与硒化壳聚糖包覆的益生菌双层微胶囊的制备方法。中国专利申请公开号:CN109619593A,公开了一种益生菌双层微胶囊及其制备方法;中国专利申请公开号:CN105533684A,公开了一种以抗性淀粉为壁材制备的肠道微胶囊包埋益生菌的方法;中国专利申请公开号:CN109464425A,公开了一种益生菌包埋颗粒及其制备方法。这些现有技术都不同程度地提高了益生菌的存活率,但是这些微胶囊包埋的益生菌具有不能在肠道定点高效释放、制备成本较高(喷雾干燥或冷冻干燥)、微胶囊粒径分布较广、贮藏时间短等缺陷。
另外,曹晨(中国油脂2019,44(12),143-148)和郭战阳、郑召军(中国油脂2019,44(08),65-71)将益生菌包埋在内水相凝胶化的多重乳液中,尽管水相凝胶化的多重乳液可解决益生菌的抗消化和肠道定植问题,然而贮藏时间短。中国专利申请号201610008369.6公开了一种益生菌发酵前包被微胶囊制剂,其包括由益生菌与海藻酸钠-碳酸钙组成的囊材和由大豆油和Span 80组成的壁材,该微胶囊制剂能有效地防止菌体失活,还能防止胃液的破坏;中国专利申请号201810787547.9公开了一种微胶囊益生菌粉,其以还原性多糖与蛋白的美拉德反应产物为微胶囊壁材包封益生菌芯材,得到的微胶囊益生菌粉抗逆性强,经过模拟胃液模拟肠液连续处理后的存活率高;在常温、冷藏条件下贮存较长时间仍能保持很高的活力。但是,这两个专利申请文件公开的技术方案都不能解决益生菌在肠道内的疏水隔水性能,从而导致益生菌的利用率大大降低。
发明内容
针对现有技术存在的技术问题,本发明的一方面的目的在于提供可在肠道定点释放的微胶囊包埋益生菌及其制备方法,该微胶囊包埋益生菌不但在常规存储和胃液中稳定性好,而且在肠道中定点释放能力强。为实现本发明的目的,本发明采用的技术方案如下:
一种可在肠道定点释放的微胶囊包埋的益生菌,该微胶囊包括W/O型芯材和壁材,益生菌位于W/O型芯材中,壁材由乳清蛋白和海藻酸钠构成,依次经过Ca2+一次固化、纳晶纤维素涂层和Ca2+二次固化,其中Ca2+一次固化所用的水溶液中Ca2+的浓度大于Ca2+二次固化所用的水溶液中Ca2+的浓度。
优选地,上述Ca2+一次固化所用的水溶液中Ca2+的浓度为5.0~10.0%(w/v),Ca2+二次固化所用的水溶液中Ca2+的浓度为1.0~3.0%(w/v)。
优选地,上述可在肠道定点释放的微胶囊包埋的益生菌中W/O乳液芯材、乳清蛋白、海藻酸钠和纳晶纤维素的重量比为1~2:3~5:3~5:0.5~1。
优选地,上述益生菌选自动物双歧杆菌、乳双歧杆菌、短双歧杆菌、鼠李糖乳杆菌、干酪乳杆菌、嗜酸乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌和肠膜明串珠菌中的一种或多种。
优选地,上述W/O型芯材的水相选自甘油、牛血清白蛋白(BSA)、海藻酸钠、肽、胶原蛋白和壳聚糖中的一种或多种。
优选地,上述W/O型芯材的油相选自中链甘油三酯(MCT)、玉米油、海藻油、橄榄油、豆油中的一种或多种。
优选地,W/O乳液芯材中益生菌与水相的重量比为5~10:95~90。
优选地,W/O乳液芯材中水相与油相的重量比为1~2:1~2。
本发明另一方面提供了上述可在肠道定点释放的微胶囊包埋益生菌的制备方法,该制备方法包括以下步骤:
(1)益生菌的培养:将益生菌活化后,加入到培养基中传代培养,将益生菌培养液进行离心,收集益生菌的菌落;
(2)W/O型芯材的制备:将收集的菌落与水相混合形成内水相,再与油相混合搅拌形成W/O型乳液芯材,
(3)微胶囊壁的制备:首先将步骤(2)的芯材先与海藻酸盐水溶液混合,然后在搅拌过程中向其中加入乳清蛋白水溶液,其中所述芯材、海藻酸盐和所述乳清蛋白的重量比例为1~2:3~5:3~5,
(4)一次Ca2+固化:步骤(3)的混合液在搅拌下滴加到浓度为5.0~10.0%(w/v),pH为3.0~5.0的CaCl2水溶液中,室温下,固化20~40min,收集微胶囊,用质量浓度为0.5~1.5%的NaCl水溶液清洗至中性;
(5)纳晶纤维素涂层:将步骤(4)制备的微胶囊搅拌下加入到质量浓度为0.3~2%的纳晶纤维素溶液中,室温下,固化1~10min;
(6)二次Ca2+固化:将步骤(5)制备的混合液在搅拌下分散到浓度为1.0~3.0%(w/v),pH为3.0~5.0的CaCl2水溶液中进行固化,室温下,固化20~40min,收集微胶囊,用质量浓度为0.5~1.5%的NaCl水溶液清洗至中性,冷冻干燥12h,保藏备用。
优选地,步骤(2)中益生菌与水相的重量比为5~10:95~90。
优选地,步骤(2)中水相与油相的重量比为1~2:1~2。
优选地,上述乳清蛋白水溶液制备方法为将乳清蛋白加入水中,持续搅拌1~3h后,4000~8000rpm离心5~15min,上清液即为乳清蛋白水溶液。
优选地,上述乳清蛋白水溶液的质量浓度为10.0~30.0g/L。
优选地,上述海藻酸盐水溶液的质量浓度为10.0~30.0g/L。
优选地,纳晶纤维素与海藻酸盐的重量用量比为3~5:0.5~1。
优选地,Ca2+一次固化步骤中,CaCl2与海藻酸盐的重量用量比为0.1~0.3:5~10。
优选地,Ca2+二次固化步骤中,CaCl2与海藻酸盐的重量用量比为0.01~0.05:5~10。
优选地,步骤(4)中NaCl水溶液的质量浓度为0.85%。
优选地,步骤(6)中NaCl水溶液的质量浓度为0.85%。
本发明再一方面提供了可在肠道定点释放的微胶囊包埋益生菌的用途,该微胶囊包埋益生菌可用于保健产品和食品中的添加剂或作为具有治疗作用的药剂。
本发明的微胶囊包埋益生菌的芯材为W/O型,更适合益生菌存活,壁材由乳清蛋白和海藻酸盐构成,并且利用纳晶纤维素(棒状或带状)封闭微胶囊壁材上的细孔和裂缝,提高微胶囊的隔酸和隔氧效果,保护益生菌所处环境对它的存活影响,极大降低了胃酸的侵蚀,并且使微胶囊包埋益生菌到达人体肠道后,在肠道酶和肠道微生物作用下定点释放益生菌。
与现有技术相比,本发明的方法具有如下有益效果:
(1)本发明的微胶囊包埋益生菌材料安全、益生菌存活率高、稳定性好,并且其制备方法制备方法操作方便、包埋率高。
(2)本发明的微胶囊包埋益生菌通过二次固化包埋技术在海藻酸盐(钙)壁材中加入纳晶纤维素(棒状或带状),使得微胶囊壁材上的细孔和裂缝被封闭,克服了海藻酸盐体系由于多孔性导致的不稳定性,不能完全抵制胃液的渗透作用的缺陷,提高了微胶囊隔酸和隔氧效果,保护益生菌免受胃酸侵蚀,在肠道酶和肠道微生物存在条件下定点释放能力增强。因而,本发明的微胶囊包埋益生菌的存活率更高,在胃环境中不崩解,而在肠液中易破裂,高效释放出芯材。
附图说明
图1是本发明的微胶囊包埋益生菌的制备方法的流程图。
图2A是仅用海藻酸钠作为壁材的微胶囊包埋益生菌的SEM图,图2B、2C和2D分别是实施例1-3制备的微胶囊包埋的益生菌的SEM图。
图3A和图3B分别是仅用海藻酸钠作为壁材的微胶囊包埋LC2W(对照)以及实施例1-3制备的湿微胶囊包埋LC2W(分别用T1、T2和T3表示)的湿产品和冷冻干燥产品的粒径和包埋效率。
图4是在25℃、40℃、50℃和60℃温度条件下,未包埋的LC2W(即,Free LC2W)以及实施例1-3制备的微胶囊包埋LC2W(分别用T1、T2和T3表示)的活性。
图5A和5B分别是在4℃和25℃条件下,在常规条件下的贮藏期内(120天)未包埋的LC2W(即,Free LC2W)、仅用海藻酸钠作为壁材的微胶囊包埋LC2W(对照)、以及实施例1-3制备的微胶囊包埋LC2W(分别用T1、T2和T3表示)的活性。
图6A是在模拟胃液消化0、60、和120min后,未包埋的LC2W(即,Free LC2W)、仅用海藻酸钠作为壁材的微胶囊包埋LC2W(对照)以及实施例1-3制备的微胶囊包埋的LC2W(分别用T1、T2和T3表示)的存活率(活性);图6B显示了模拟胃液消化120min后,再在模拟肠液消化80min和120min(即胃肠道共计分别消化200min和300min)后,未包埋的LC2W、仅用海藻酸钠作为壁材的微胶囊包埋LC2W以及实施例1-3制备微胶囊包埋LC2W的存活率(活性)。
具体实施方式
本发明的描述中,“溶液”没有特殊说明的话,一般指水溶液。
本发明的描述中,“多种”指两种或两种以上。
本发明的描述中,“W/O型芯材”是由内部的水相(亲水性物质)和外部的油相(亲油性物质)构成的油包水型芯材,其中益生菌与内部的水相混合在一起。
本发明的描述中,w/v是指质量体积比,例如“水溶液中Ca2+的浓度为1.0~3.0%(w/v)”是指100mL水溶液中含有1.0~3.0g的Ca2+。
本发明的描述中,益生菌菌粉包括但不局限于动物双歧杆菌、乳双歧杆菌、短双歧杆菌、鼠李糖乳杆菌、干酪乳杆菌、嗜酸乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌和肠膜明串珠菌。
本发明的描述中,水相包括但不局限于甘油、牛血清白蛋白(BSA)、海藻酸钠、肽、胶原蛋白和壳聚糖,油相包括但不局限于中链甘油三酯、、玉米油、海藻油、橄榄油和豆油,并且上述水相和油相可任意组合。
测量微胶囊包埋益生菌的粒径的方法
本发明中测量微胶囊包埋益生菌的粒径的方法按照下面的文献中记载的方法进行:
Li,X.Y.,Chen,X.G.,Sun,Z.W.,Park,H.J.,Cha,D.S.(2011).Preparation ofalginate/chitosan/carboxymethyl chitosan complex microcapsules andapplication in Lactobacillus casei ATCC 393[J].Carbohydrate Polymers,83,1479-1485.
微胶囊包埋益生菌的包埋率测量的方法
本发明中微胶囊包埋益生菌的包埋率测量的方法按照下面的文献中记载的方法进行:
Karimi,N.,Alizadeh,A.,Almasi,H.,Hanifian,S.(2020)Preparation andcharacterization of whey protein isolate/polydextrosebased nanocomposite filmincorporated with cellulose nanofiber and L.plantarum:A new probiotic activepackaging system[J].LWT-Food Science and Technology,121,108978。其中包埋效率(EE)的计算公式为:EE(%)=NN0×100%,其中N为微囊释放的活细胞数,N0为微囊化前浓缩的活细胞数。
微胶囊包埋益生菌的细胞活性测定方法
本发明中微胶囊包埋益生菌的细胞活性测定方法按照下面的文献中记载的方法进行:
Yonekura,L.,Sun,Y.H.,Soukoulis,C.,Fisk,I.(2014).Microencapsulation ofLactobacillus acidophilus NCIMB 701748in matrices containing soluble fibre byspray drying:Technological characterization,storage stability and survivalafter in vitro digestion[J].Journal of Functional Foods,6,205-214.
其中细胞存活率的计算公式为:其中A为储存后微胶囊中活菌数,A0为原微胶囊中活菌数。
胃肠道中微胶囊包埋益生菌的活性测定方法
本发明中胃肠道中微胶囊包埋益生菌的活性测定方法参照下列两篇文献中记载的胃肠道中细菌的活性测定方法进行:
Yasmin,I.,Saeed,M.,Pasha,I.,&Zia,M.A.(2018).Development of wheyprotein concentrate-pectin-alginate based delivery system to improve survivalof b.longum bl-05in simulated gastrointestinal conditions.Probiotics&Antimicrobial Proteins,11(2):413-426.
Ji,R.,Wu,J.H.,Zhang,J.L.,Wang,T.,Zhang,X.D.,Shao,L.,Chen,D.J.,Wang,J.,2019.Extending viability of bifidobacterium longum in chitosan-coatedalginate microcapsules using emulsification and internal gelationencapsulation technology.Frontiers in Microbiology,10,1389.
下面结合具体实施例,进一步阐述本发明。具体实施例是在以本发明技术方案为前提下进行实施的,给出了详细的实施方式和操作过程。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件进行。除非另有说明,比例和百分数按重量计。
下列实施例1~3中,干酪乳酸菌LC2W由上海光明乳业研究院提供。
实施例1
微胶囊包埋干酪乳酸菌LC2W的制备包括以下步骤:
(1)益生菌的培养:干酪乳酸菌LC2W在水浴锅中进行活化30min,水浴锅的温度为37℃,加入10%的菌液到MRS培养基(灭菌后)中传代培养,至菌株活菌数为109CFU/g以上,将1L干酪乳酸菌LC2W的培养液,经6000rpm离心10min后,收集菌体沉淀物;
(2)W/O型芯材的制备:为提高益生菌的存活率和包埋率,将收集好的干酪乳酸菌LC2W菌落(109-10CFU/g)与甘油(保护剂)混合形成内水相(其中,甘油的用量为1g干酪乳酸菌LC2W菌落使用12mL甘油),内水相再与中链甘油三酯(国药化学试剂有限公司)搅拌形成W/O型乳液芯材(其中,中链甘油三酯的用量为1g干酪乳酸菌LC2W菌落使用50m中链甘油三酯),搅拌速度为600rpm;
(3)壁材的制备:配制20g/L的乳清蛋白水溶液,持续搅拌2h后,6000rpm离心10min,保留上清液待用;配制20g/L的海藻酸钠水溶液,加热搅拌至完全溶解,冷却至室温,备用;将步骤(2)中的W/O型乳液芯材先与海藻酸钠水溶液混合,在慢速搅拌过程中再加入乳清蛋白水溶液,搅拌速度为300rpm,搅拌时间为15min,其中W/O型乳液芯材、海藻酸钠和乳清蛋白的重量比为2:3:3;
(4)Ca2+一次固化:配制浓度为8%(w/v),的CaCl2水溶液,并用盐酸调整其pH为4.0,在高压灭菌锅中杀菌,采用1mL 0.3mm孔径的针管吸取步骤(3)的混合液,滴加到上述CaCl2水溶液中(其中步骤(3)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.1~0.3:5~10),固化30min,搅拌速度400rpm,滴加速度为2-3滴/s,然后离心收集微胶囊,再用质量浓度的0.9%的NaCl水溶液清洗至中性、备用;
(5)纳晶纤维素涂层:将步骤(4)制备的微胶囊搅拌(400rpm)下加入到灭菌后的质量浓度为1.2%的纳晶纤维素水溶液中(其中纳晶纤维素的用量为1g海藻酸钠使用10g晶纤维素),搅拌固化5min;
(6)Ca2+二次固化:将步骤(5)制备的混合液再分散到浓度为4%(w/v)的CaCl2水溶液中(其中步骤(5)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.01~0.05:5~10),进行固化30min,搅拌速度400rpm,收集微胶囊,再用质量浓度为0.9%的NaCl水溶液清晰至中性,冷冻干燥3h,保藏备用。
实施例2
微胶囊包埋干酪乳酸菌LC2W的制备包括以下步骤:
(1)益生菌的培养:干酪乳酸菌LC2W在水浴锅中进行活化30min,水浴锅的温度为37℃,加入10%的菌液到MRS培养基(灭菌后)中传代培养,至菌株活菌数为109CFU/g以上,将1L干酪乳酸菌LC2W的培养液,经6000rpm离心10min后,收集菌体沉淀物;
(2)W/O型芯材的制备:为提高益生菌的存活率和包埋率,将收集好的菌落(109- 10CFU/g)与甘油(保护剂)混合形成内水相(其中甘油的用量为1g干酪乳酸菌LC2W菌落使用18mL甘油)混合形成内水相,再与玉米油(其中,玉米油的用量为1g干酪乳酸菌LC2W菌落使用60mL玉米油),搅拌形成W/O型乳液芯材,搅拌速度为800rpm。
(3)壁材的制备:配制20g/L的乳清蛋白水溶液,持续搅拌2h后,6000rpm离心10min,保留上清液待用。配制20g/L的海藻酸钠水溶液,加热搅拌至完全溶解,冷却至室温,备用;将步骤(2)中的W/O型乳液芯材先与海藻酸钠水溶液混合,在慢速搅拌过程中再加入乳清蛋白水溶液,搅拌速度为200rpm,搅拌时间为20min,其中W/O型乳液芯材、海藻酸钠和乳清蛋白的重量比为1.5:4:3。
(4)Ca2+一次固化:配制浓度为5%(w/v)的CaCl2水溶液,并用盐酸调整其pH为4.0,在高压灭菌锅中杀菌,采用1mL 0.3mm孔径的针管吸取步骤(3)的混合液,滴加到上述CaCl2水溶液中(其中步骤(3)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.1~0.3:5~10),固化时间30min,搅拌速度400rpm,滴加速度为2-3滴/s,然后离心收集微胶囊,再用质量浓度为0.9%的NaCl水溶液清洗至中性、备用;
(5)纳晶纤维素涂层:将步骤(4)制备的微胶囊加入到灭菌后的质量浓度为2%的纳晶纤维素溶液中(其中纳晶纤维素的用量为1g海藻酸钠使用10g晶纤维素),搅拌固化2min;
(6)Ca2+二次固化:将步骤(5)制备的混合液再分散到浓度为4%(w/v)的CaCl2水溶液(用盐水调节该CaCl2溶液的pH为4.0)中(其中步骤(5)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.01~0.05:5~10),进行固化40min,搅拌速度400rpm,收集微胶囊,再用质量浓度为0.9%的NaCl水溶液清晰至中性,冷冻干燥3h,保藏备用。
实施例3
微胶囊包埋干酪乳酸菌LC2W的制备包括以下步骤:
(1)益生菌的培养:干酪乳酸菌LC2W在水浴锅中进行活化30min,水浴锅的温度为37℃,加入10%的菌液到MRS培养基(灭菌后)中传代培养,至菌株活菌数为109CFU/g以上;将1L干酪乳酸菌LC2W的培养液,经6000rpm离心10min后,收集菌体沉淀物;
(2)W/O型芯材的制备:为提高益生菌的存活率和包埋率,将收集好的菌落(109- 10CFU/g)与牛血清白蛋白(西格玛奥德里奇(上海)贸易有限公司)混合形成内水相(其中,牛血清白蛋白的用量为1g干酪乳酸菌LC2W菌落使用15g牛血清白蛋白),再与豆油搅拌形成W/O型乳液芯材(其中,豆油的用量为1g干酪乳酸菌LC2W菌落使用60mL豆油),搅拌速度为700rpm;
(3)壁材的制备:配制20g/L的乳清蛋白水溶液,持续搅拌2h后,6000rpm离心10min,保留上清液待用;配制15g/L的海藻酸钠水溶液,加热搅拌至完全溶解,冷却至室温,备用;将步骤(2)中的W/O型乳液芯材先与海藻酸钠水溶液混合,在慢速搅拌过程中再加入乳清蛋白水溶液,搅拌速度为300rpm,搅拌时间为20min,其中W/O型乳液芯材、海藻酸钠和乳清蛋白的重量比为1:4:4。
(4)Ca2+一次固化:配制浓度为10%(w/v)的CaCl2水溶液,并用盐酸调整其pH为4.0,在高压灭菌锅中杀菌,采用1mL 0.3mm孔径的针管吸取步骤(3)的混合液,滴加到上述CaCl2水溶液中(其中步骤(3)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.1~0.3:5~10),固化时间40min,搅拌速度300rpm,滴加速度为2-3滴/s,然后离心收集微胶囊,再用质量浓度为0.9%的NaCl水溶液清洗至中性、备用;
(5)纳晶纤维素涂层:将步骤(4)制备的微胶囊加入到灭菌后的质量浓度为1.5%的纳晶纤维素水溶液中(其中纳晶纤维素的用量为1g海藻酸钠使用6g晶纤维素),搅拌固化8min;
(6)Ca2+二次固化:将步骤(5)制备的混合液再分散到浓度为3%(w/v)的CaCl2水溶液(用盐水调节该CaCl2水溶液的pH为4.0)中(其中步骤(5)的混合液的体积与CaCl2水溶液的体积比为1:1~3,CaCl2与海藻酸盐的重量用量比为0.01~0.05:5~10),进行固化30min,搅拌速度400rpm,收集微胶囊,再用质量浓度为0.9%的NaCl水溶液清晰至中性,冷冻干燥3h,保藏备用。
微胶囊包埋的益生菌的形态研究:
利用扫描电镜(SEM)(Sirion 200FEI Electron Optics Company,USA)对实施例1-3的产品进行了形态观察,具体结果参见图2B、图2C和图2D。
同时为了更清楚地说明本发明的微胶囊包埋的益生菌的形态改进,还对仅用海藻酸钠作为壁材的微胶囊包埋的LC2W(对照)的形态进行了观察,具体结果参见图2A,其中海藻酸钠作为壁材的微胶囊包埋的益生菌是按照常规方法制备的。
比较从图2A与图2B-D明显可以看出仅仅用海藻酸钠作为壁材的微胶囊包埋的益生菌的表面呈不规则的形状,有细孔和裂缝。当这种微胶囊包埋的益生菌进入胃肠道中时,胆汁酸、消化酶等很容易进入微胶囊中杀伤益生菌,从而导致益生菌存活率降低。而本发明实施例1-3制备的微胶囊包埋的LC2W的表面比较紧实,因为纳晶纤维素(CNC)封闭了海藻酸盐壁材上的细孔和裂缝,当这种微胶囊包埋的益生菌进入胃肠道中时,这种封闭不仅能完全抵制胃液的渗透作用的缺陷,而且提高了微胶囊的隔酸和隔氧效果,保护益生菌免受胃酸侵蚀,在肠道酶和肠道微生物存在条件下定点释放能力增强。微胶囊包埋益生菌的粒径和包埋率的研究
利用上述测量微胶囊包埋益生菌的粒径的方法测得仅用海藻酸钠作为壁材的微胶囊包埋的LC2W湿微粒的粒径约1.08mm,冷冻干燥后的粒径约0.48mm;上述实施例1-3的步骤(5)中收集的湿微胶囊的粒径为0.9-1.18mm之间;收集的冷冻干燥的微胶囊的粒径为0.45-0.57mm之间。具体结果参见图3A和3B。
利用上述微胶囊包埋益生菌的包埋率测量方法得到仅用海藻酸钠作为壁材的微胶囊包埋的LC2W的湿微粒的包埋率为96.8%;冷冻干燥后的包埋率为96.8%~83.0%;上述实施例1-3的湿微胶囊的包埋率为92.31~98.74%之间;冷冻干燥的微胶囊的包埋率为83.24~88.12%之间。具体结果参见图3A和3B。
从以上结果可以看出,仅用海藻酸钠作为壁材的微胶囊包埋的LC2W与本发明的微胶囊包埋的LC2W的粒径和包埋率基本相当。
在不同温度条件下微胶囊包埋益生菌的活性研究
利用上述细胞活性测定方法在30℃、40℃、50℃和60℃条件下测定了上述未包埋的干酪乳酸菌LC2W以及实施例1~3所得微胶囊包埋干酪乳酸菌LC2W的益生菌的活性,具体结果如图4所示。结果说明随着温度的升高,本发明制备微胶囊包埋LC2W的活性减弱,但是减弱的速度明显比未包埋的干酪乳酸菌LC2W小很多。例如,在50℃条件下,未包埋的益生菌的活细胞数从109CFU/g下降到104CFU/g左右,而包埋后的益生菌的活细胞数仍然高于106CFU/g;仅用海藻酸钠作为壁材的微胶囊包埋的LC2W在各个测量温度下明显比本发明的微胶囊包埋干酪乳酸菌LC2W的活细胞数少。这可能是由于本发明的微胶囊具有隔氧效果,能有效保护益生菌免受周围环境的影响。微胶囊包埋益生菌的贮藏稳定性研究
利用上述细胞活性测定方法分别在4℃和25℃条件下测定了未包埋的干酪乳酸菌LC2W、仅用海藻酸钠作为壁材的微胶囊包埋的LC2W以及上述实施例1~3制得的微胶囊包埋干酪乳酸菌LC2W在120d内的益生菌在常规条件下的贮藏期内的活细胞数,结果分别如图5A和图5B所示。该结果说明了本发明的微胶囊包埋干酪乳酸菌LC2W的活细胞数都在107~108CFU/g左右,并随着贮藏时间的延长,益生菌的活细胞数没有显著性差异。未包埋的益生菌的活性随着贮藏时间的延长,活细胞数不断降低,在120d后下降到102CFU/g。仅用海藻酸钠作为壁材的微胶囊包埋的LC2W在各个测量时间点明显比本发明的微胶囊包埋干酪乳酸菌LC2W的活细胞数少。这可能是由于本发明的微胶囊具有隔氧效果,能有效保护益生菌免受周围环境的影响。
胃肠道中微胶囊包埋益生菌的活性研究
利用上述胃肠道中微胶囊包埋益生菌的活性测定方法测定了未包埋的干酪乳酸菌LC2W、仅用海藻酸钠作为壁材的微胶囊包埋的LC2W以及实施例1~3制得的微胶囊包埋干酪乳酸菌LC2W的益生菌的在胃肠道中的活性,结果分别参见图6A和图6B。
由图6A可知,未包埋的菌在模拟胃液中的存活率非常低,60min后的存活率不足一半,经过120min后,检测到不到四分之一的活菌;本发明实施例1~3的微胶囊的益生菌在模拟胃液处理120min后益生菌仍有四分之三以上是存活的;仅用海藻酸钠作为壁材的微胶囊包埋的LC2W在各个测量时间点明显比本发明的微胶囊包埋干酪乳酸菌LC2W的活细胞数少。这是由于经过两次Ca2+固化纳晶纤维素涂层的微胶囊结构致密,孔径更少,机械强度高,能较好地抵御胃液渗透,保持微胶囊的完整性,从而显著提高益生菌的存活率。
在肠道中的有效释放是发挥益生作用的首要条件,从图6B可知未包埋的干酪乳酸菌LC2W以及实施例1~3的微胶囊的益生菌在模拟胃液消化120min后,再经过肠液处理80min和120min后,活菌数仍达到105CFU/g,而未包埋的益生菌已经基本检测不到(对于益生菌来说,在人体中的实际情况是60min~120min是胃中的消化的时间,到200min~300min到达肠道);仅用海藻酸钠作为壁材的微胶囊包埋的LC2W在各个测量时间点明显比本发明的微胶囊包埋干酪乳酸菌LC2W的活细胞数少。这表明本发明的微胶囊的益生菌具有较好的肠溶性,并且在胃液中益生菌存活率较高(仍有80%的益生菌存活)。这可能是由于在胃液中,本发明的微胶囊能有效保护益生菌免受酸性损伤,而在肠液中,纳晶纤维素涂层的微胶囊明显膨胀,最终导致破裂释放。
实施例4
微胶囊包埋的双歧杆菌的制备
此实施例的微胶囊的制备和实施例3的步骤方法完全相同,不同之处仅在于将其中的干酪乳酸菌替换成双歧杆菌。
按照上面提到的胃肠道中微胶囊包埋益生菌的活性研究的具体方法,对该实施例制得的微胶囊包埋的双歧杆菌进行了研究,发现该微胶囊包埋的双歧杆菌具有较好的肠溶性,并且在胃液中益生菌存活率较高(仍有85%的益生菌存活)
实施例5
微胶囊包埋的发酵乳杆菌的制备
此实施例的微胶囊的制备和实施例3的步骤方法完全相同,不同之处仅在于将其中的干酪乳酸菌替换成发酵乳杆菌。
按照上面提到的胃肠道中微胶囊包埋益生菌的活性研究的具体方法,对该实施例制得的微胶囊包埋的发酵乳杆菌进行了研究,发现该微胶囊包埋的发酵乳杆菌具有较好的肠溶性,并且在胃液中益生菌存活率较高(仍有81%的益生菌存活)
实施例6
微胶囊包埋的嗜酸乳杆菌的制备
此实施例的微胶囊的制备和实施例3的步骤方法完全相同,不同之处仅在于将其中的干酪乳酸菌替换成嗜酸乳杆菌。
按照上面提到的胃肠道中微胶囊包埋益生菌的活性研究的具体方法,对该实施例制得的微胶囊包埋的嗜酸乳杆菌进行了研究,发现该微胶囊包埋的嗜酸乳杆菌具有较好的肠溶性,并且在胃液中益生菌存活率较高(仍有80%的益生菌存活)
以上所述的,仅为本发明的较佳实施例,并非用以限定本发明的范围,本发明的上述实施例还可以做出各种变化。即凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰,皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。
Claims (6)
1.一种可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述微胶囊包括W/O型芯材和壁材,
所述益生菌选自动物双歧杆菌、干酪乳杆菌、嗜酸乳杆菌和发酵乳杆菌中的一种或多种,
所述微胶囊包埋益生菌的制备方法包括以下步骤:
(1)益生菌的培养:将益生菌活化后,加入到培养基中传代培养,将益生菌培养液进行离心,收集益生菌的菌落;
(2)W/O型芯材的制备:将收集的菌落与水相混合形成内水相,再与油相混合搅拌形成W/O型芯材;
(3)微胶囊壁的制备:首先将步骤(2)的芯材先与海藻酸盐水溶液混合,然后在搅拌过程中向其中加入乳清蛋白水溶液,其中所述芯材、海藻酸盐和所述乳清蛋白的重量比例为1~2:3~5:3~5;
(4)Ca2+一次固化:步骤(3)的混合液在搅拌下滴加到浓度为5.0~10.0%w/v,pH为3.0~5.0的CaCl2水溶液中,室温下,固化20~40min,收集微胶囊,用质量浓度为0.5~1.5%NaCl水溶液清洗至中性;
(5)纳晶纤维素涂层:将步骤(4)制备的微胶囊搅拌下加入到质量浓度为0.3~2%的纳晶纤维素溶液中,室温下,固化1~10min;
(6)Ca2+二次固化:将步骤(5)的混合液在搅拌下分散到浓度为3~4%w/v,pH为3.0~5.0的CaCl2水溶液中进行固化,室温下,固化20~40min,收集微胶囊,用质量浓度为0.5~1.5%的NaCl水溶液清洗至中性,冷冻干燥,即得微胶囊包埋的益生菌。
2.根据权利要求1所述的可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述W/O型芯材的水相选自甘油和/或牛血清白蛋白。
3.根据权利要求1所述的可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述W/O型芯材的油相选自中链甘油三酯、玉米油和豆油中的一种或多种。
4.根据权利要求1所述的可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述微胶囊包埋的益生菌的制备方法的步骤(2)中益生菌与水相的重量比为5~10:95~90。
5.根据权利要求1所述的可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述微胶囊包埋的益生菌的制备方法的步骤(2)中水相选自甘油,油相选自中链甘油三酯,水相与油相的体积比为6:25,或者水相选自甘油,油相选自玉米油,水相与油相的体积比为3:10,或者水相选自牛血清白蛋白,油相选自豆油,水相与油相的重量体积比为1:4。
6.根据权利要求1所述的可在肠道定点释放的微胶囊包埋的益生菌,其特征在于,所述微胶囊包埋的益生菌的制备方法的步骤(5)中纳晶纤维素与海藻酸盐的重量用量比为3~5:0.5~1。
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