CN111685255B - 一种增强免疫功能的益生菌固体饮料及其制备方法 - Google Patents
一种增强免疫功能的益生菌固体饮料及其制备方法 Download PDFInfo
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- CN111685255B CN111685255B CN202010514048.XA CN202010514048A CN111685255B CN 111685255 B CN111685255 B CN 111685255B CN 202010514048 A CN202010514048 A CN 202010514048A CN 111685255 B CN111685255 B CN 111685255B
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Abstract
本发明涉及食品领域,公开了一种增强免疫功能的益生菌固体饮料及其制备方法。该益生菌固体饮料将瑞士乳杆菌冻干粉、嗜酸乳杆菌DDS®‑1冻干粉、乳双岐杆菌HN019冻干粉、接骨木莓提取物、蓝莓粉和赤藓糖醇及维生素C进行复配,具有较好的增强免疫功能。用于制备瑞士乳杆菌冻干粉的瑞士乳杆菌WHH2580具有良好的黏附特性和耐酸耐胆盐特性,并具有良好的增强免疫的功效;其灭活菌株同样可以通过提高调节机体的脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力,则使本发明的益生菌固体饮料稳定性更好,保质期更长,且在储存和运输过程中不需要冷藏,成本更低。
Description
技术领域
本发明涉及食品领域,尤其涉及一种增强免疫功能的益生菌固体饮料及其制备方法。
背景技术
随着城市化进程加快和居民不健康生活方式的流行,我国因免疫力低下所导致的各种疾病及亚健康人群急剧增多。免疫力低下导致机体不能正常发挥保护作用,极易受到细菌、病毒、真菌等感染,最直接的表现就是容易生病。因经常患病,加重了机体的消耗,不同程度的出现体质虚弱、营养不良、精神萎靡、疲乏无力、食欲降低、睡眠障碍等免疫力低下的表现,生病、***吃药便成了家常便饭。每次生病都要很长时间才能恢复,而且常常反复发作。长此以往导致身体和智力发育不良,还易诱发重大疾病。因此,增强免疫,防治免疫力低下是预防疾病的重要手段。
益生菌能够在人体肠道内发挥生理功能而有助于健康的有益微生物,它们被宿主摄入后黏附于肠道粘膜上皮细胞,定殖形成稳定菌群。经大量研究证明,一些乳酸菌种属可对宿主产生有益作用,不仅有助于维持肠道微生态的平衡,营养物质的吸收,还可以调节宿主免疫功能,促进肠道粘膜屏障功能完善以及增强抗感染的能力。
肠道是人体最大的免疫器官,人体50%以上的淋巴组织分布于肠黏膜,肠道微生物对于调节人体健康有着重要的作用。并且,益生菌可以通过抑制病原微生物的生长来平衡局部微生物群,或益生菌通过分泌各种酶和酸刺激肠道黏膜和肠上皮细胞代谢,进而增强局部和全身免疫反应。益生菌通过多种方式和信号途径调节机体免疫,主要的途径包括TLRs信号通路、NF-KB,MAPKs,STAT通路等,通过益生菌的调节,可激发肠道免疫,同时影响机体整体的免疫***。此外,乳杆菌还可通过调控免疫***、细胞因子、肠道渗透、肠道菌群平衡和细菌定植等方式,调节机体炎症。因此,从肠道微生物入手,尝试找到调节免疫力的新方法,以克服现有治疗药物以及治疗方法副作用的缺陷。
目前市面上宣称具有增强免疫的产品较多,临床常用的免疫增强药有化学合成药物左旋咪唑、异丙肌苷等,人或动物免疫产物胸腺素、转移因子、干扰素、白介素等,微生物来源的卡介苗及其他来源的生物多糖、中药有效成分等。这些药物调节免疫功能的作用机制有:①增强巨噬细胞和自然杀伤细胞的活性,提高非特异性免疫功能;②促进T淋巴细胞的***、增殖、成熟和分化,增强细胞免疫功能;③提高体液免疫功能;④诱导产生干扰素和某些细胞因子,激活免疫细胞发挥作用。但这些药品不同程度的有一些不良副作用,如左旋咪唑可引起恶心、呕吐、腹痛等胃肠道反应;卡介苗不良反应较多,可出现注射局部皮肤红肿、硬结及溃疡,偶见过敏性休克,甚至死亡。干扰素可引起流感样症状,部分患者还可出现骨髓抑制、心律失常、精神错乱、幻觉等症状。
近几年随着益生菌市场的普及,人们对益生菌的作用越来越关注。市面上的益生菌产品的种类大多数以片剂、滴剂、粉剂形式为主。粉剂益生菌又称益生菌冻干粉制剂,通过低温冷冻干燥技术加工而成,这是保护益生菌活性的一大重要保障。在冷冻干燥技术的保护下,益生菌能够保存十年甚至是更长的时间。可以最大程度的保证益生菌菌株的活性并直达肠道。益生菌固体饮料因为含水量低,可以长期在室温保存而保持较高的活菌效力。因此,从食疗的角度,从食品用微生物入手,搭配具有广泛群众认可度可增强免疫力的食品原料,开发一款安全、可靠、有效的增强免疫的益生菌固体饮料,定会具有良好的市场价值。
发明内容
为了解决上述技术问题,本发明提供了一种增强免疫功能的益生菌固体饮料及其制备方法。本发明将瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉、接骨木莓提取物、蓝莓粉和赤藓糖醇及维生素C进行复配,具有较好的增强免疫功能。
本发明的具体技术方案为:
所述瑞士乳杆菌冻干粉由瑞士乳杆菌和/或其突变体制得;所述瑞士乳杆菌命名为WHH2580,于2019年10月23日在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心保藏,其微生物保藏编号为:CGMCC No.18730。保藏单位地址为北京市朝阳区北辰西路1号院3号,微生物分类命名为瑞士乳杆菌Lactobacillus helveticus。其具有如SEQ IDNO.1所示的16s rRNA基因序列。所述突变体为对所述瑞士乳杆菌进行诱变、驯化、基因重组或者经自然突变而获得的突变体。
上述瑞士乳杆菌具备黏附特性,具备耐酸耐胆盐特性。
本发明配方中,以特定的瑞士乳杆菌冻干粉为主要功能物,嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉、接骨木莓提取物、蓝莓粉、赤藓糖醇及维生素C为辅料。其中,用于制备瑞士乳杆菌冻干粉的瑞士乳杆菌WHH2580是一种从新疆那拉提红山沟采集的酸奶中筛选得到的益生菌菌株,经过动物实验验证,其具有优良的黏附性能,可提高体外脾淋巴细胞增殖和脾淋巴细胞分泌白细胞介素IL-12,促进人结肠癌上皮细胞HT-29分泌白细胞介素IL-8;此外,在免疫低下动物模型中,该菌株还可显著提高小鼠脏器指数、促进脾淋巴细胞增殖和提高NK细胞活性。因此,该瑞士乳杆菌WHH2580具有良好的增强免疫的功效。虽然粘附不是菌株具备益生菌性质的先决条件,但因为许多原因,益生菌与肠粘膜的相互作用被认为至关重要。与肠粘膜的结合可以延长益生菌菌株存在于肠内的时间。这种与粘膜的相互作用使益生菌与肠免疫***紧密接触,使其有更好机会调节免疫应答。它还可以通过限制其在肠内留驻的能力来抵御肠道病原体。此外,在动物实验过程中,未发现实验动物死亡、精神不振、食欲不振等不良状态,说明该菌种具有较高的安全性。
具体而言,上述瑞士乳杆菌WHH2580具有以下优点:
(1)活体和非活体均具备优良的黏附特性:在人结肠癌上皮细胞HT-29细胞模型试验中表现出优良的黏附能力,单细胞黏附活体菌数达15.69±5.94个,单细胞黏附非活体菌数达11.59±4.02个,显著优于阳性对照菌株鼠李糖乳杆菌LGG和代田株LcS的黏附性能。该菌株可与肠道上皮细胞发生黏附,发挥生理功效。
(2)具备良好的耐酸耐胆盐特性:PBS pH3.0、PBS+0.3%pepsin pH2.0体系中3h降低一个数量级,0.1%Trypsin体系中3h降低0.8个数量级,0.3%BS体系中3h降低2.8个数量级,0.5%BS体系中3h降低3.6个数量级。良好的耐酸性使瑞士乳杆菌WHH2580在制备冻干粉和益生菌固体饮料的过程中,特别是在制备冻干粉时的发酵过程中,能适应更宽的pH范围,从而使本发明的益生菌固体饮料具有更高的活菌含量。
(3)在体外细胞试验中,可促进体外脾淋巴细胞增殖,不加诱导剂的增值倍数为1.912±0.087,加诱导剂的增值倍数为1.533±0.109。可促进体外脾淋巴细胞分泌白细胞介素IL-12;IL-12/IL-10比值与鼠李糖乳杆菌LGG无显差。在体外细胞试验中,可促进人结肠癌上皮细胞HT-29分泌白细胞介素IL-8;刺激分泌的IL-8显著高于商业菌株LGG。
(4)瑞士乳杆菌WHH2580菌株在体内免疫低下动物试验中,活性和非活性菌体均可显著提高小鼠脾淋巴细胞增殖和NK细胞活性。这能使本发明的益生菌固体饮料货架期更长,安全性更高,且在储存和运输过程中不需要冷藏,成本更低。
嗜酸乳杆菌-1,首次发现并被分离于1959年。欧洲食品***(EFSA)认为嗜酸乳杆菌菌种是安全并可广泛用于食品和膳食补充剂的,不需要限制及免于上市前的使用批准。嗜酸乳杆菌-1具有高耐酸性,可顺利通过胃部,进入肠道,更有利于定殖;在低pH值环境下有高抵抗性,能够抵抗胃部蛋白酶;对于胆汁盐和胰酶有高抵抗性,能够耐受十二指肠生理液;对于人体典型的肠道细胞Caco-2和HT-29有很强的粘附能力,能够在体外实验中广泛抑制一系列肠道致病菌。大量的动物和人体的临床研究,证实嗜酸乳杆菌-1对于消化,免疫和皮肤健康都有积极的效果。
乳双歧杆菌HN019最初从新西兰生产的酸奶中分离出来,并作为乳制品的一部分被摄入多年。乳双歧杆菌HN019是一种有据可查体现出显著益生菌功效的菌株。数项已发表的研究描述了该菌株的性质,特别是在免疫***调节领域。新西兰乳业研究所筛选了一系列广泛的(超过2000株)乳酸杆菌和双歧杆菌,作为潜在的益生菌备选菌株。乳双歧杆菌HN019凭借其体外耐胆汁和低pH环境的能力,被认为最具有益生菌潜力。研究表明乳双歧杆菌HN019在体外表现出对低pH的高耐受性和对胆汁盐的高耐受性,其无细胞上清液也显示出炎性标记物比率略有显著增加,表明乳双歧杆菌HN019具有免疫增强性。
接骨木莓,被称为“希波克拉底的药箱”,自古以来被用来治疗感冒、流感、关节炎等疾病,从希波克拉底时代起就一直被誉为治疗感冒发烧的最佳药物。接骨木莓其高含量的花青素,类黄酮组分,种类丰富的维生素和矿物质,在提高免疫力上表现优异,在国外已经被开发成提高免疫力的产品。根据美国草药协会的统计,2015年接骨木莓补充剂在美国市场销售比2014年增长了27.33%,并跃居畅销健康保健食品的前五。接骨木莓在西方市场的主要功能宣称为:提高免疫力,有效预防流感和呼吸道感染,改善细菌及病毒感染引发的炎症。接骨木莓已经开发成软糖,片剂,滴剂等类型的产品。将提高免疫力的益生菌与接骨木莓结合,开发提高免疫力的产品,符合当下中国市场的诉求,产品市场潜力巨大。
维生素可以增强人体免疫力。身体健康的人最好通过吃水果、蔬菜来吸收维生素,维生素因含有丰富的干扰素诱导剂而具有免疫作用。维生素C是人体不可缺少的,它可以降低毛细血管通透性,使之成为一个屏障,阻止病毒进入人体组织,保护机体器官。维生素C缺乏时,白细胞内维生素C含量减少,白细胞的战斗力减弱,人体易患病。
赤藓糖醇是一种天然的四碳多元醇,其甜味纯正,甜味特点与蔗糖非常接近,但是其能量热值非常低,仅为蔗糖的十分之一,是一种低代谢性的低热甜味剂,不能被人体消化,不会引起人体血糖和胰岛素变化。另外,本发明的益生菌固体饮料为以益生菌为核心成分,其具有一般益生菌组合物所具有的对水分、水分活度异常敏感的特点,组合物水分、水分活度越低,越有利于组合物中乳酸菌的活性稳定。组合物水分、水分活度与组合物中各组分自身含水量及吸湿性能有关,自身水分含量越低、吸湿性越低,越容易维持组合物的低水分、低水分活度。赤藓糖醇吸湿性极低,是糖醇及蔗糖等甜味剂中最小的,在温度20℃、相对湿度90%的环境中,放置5天,其吸湿增重仅2%,是非常理想的用于维持组合物低水分、低水分活度的食品原料,有利于益生菌固体饮料中瑞士乳杆菌WHH2580、嗜酸乳杆菌-1、乳双岐杆菌HN019的活性稳定。
蓝莓粉,源自北美进口浓缩汁,经严苛的工艺制程,喷雾干燥成粉。蓝莓属于低灌木小浆果果树,果实呈蓝色,果味酸甜,营养丰富。蓝莓中的主要抗氧化化合物是类黄酮。类黄酮可以明显的提高体内的抗氧化剂水平。蓝莓可增强人体对传染病的抵抗力。蓝莓含有相当多的钾,钾能帮助维持体内的液体平衡、正常的血压及心脏功能。抵抗自由基,减少氧自由基对细胞膜、DNA和其他细胞成分的损害,预防体内功能紊乱,可增强人自身的免疫抗病能力。
作为优选,益生菌固体饮料中按重量份包括瑞士乳杆菌冻干粉1-10份、嗜酸乳杆菌-1冻干粉1-10份、乳双岐杆菌HN019冻干粉1-10份、接骨木莓提取物2-11份、蓝莓粉10-40份、赤藓糖醇40-60份、维生素C 0.05-0.3份。
作为优选,所述瑞士乳杆菌冻干粉中的瑞士乳杆菌活菌数为1×107CFU/g-2×1012CFU/g。
作为优选,所述瑞士乳杆菌冻干粉的制备方法包括以下步骤:
1)培养基的制备;
2)菌株保护剂的制备;
3)以2%-10%接种量将瑞士乳杆菌和/或其突变体接种于发酵基质中发酵培养;
4)发酵结束后取发酵产物,离心;
5)离心产物与菌株保护剂混合;
6)冻干;
7)冻干产物粉碎、过筛,得到瑞士乳杆菌冻干粉。
本发明采用生物工程高密度发酵技术、冷冻干燥技术、粉碎过筛工艺制备瑞士乳杆菌冻干粉,获得的冻干粉具有活菌数高,且水分含量低、水分活度低,能保证其中的瑞士乳杆菌的活性稳定,此外还具有粒径分布合理、与其他物料的混合性能良好等特点。
作为优选,步骤1)中,所述培养基包括以下成分:脱盐乳清粉15-50g、酪蛋白水解物2-15g、胰蛋白胨3-15g、大豆蛋白胨7-20g、酵母粉3-6g、柠檬酸钠5-15g、磷酸氢二钾1-5g、硫酸镁0.4-0.8g、一水合硫酸锰0.2-0.5g,番茄汁5-30ml、水1000mL;所述培养基的pH为6.5±0.2。
作为优选,步骤2)中,所述菌株保护剂包括以下成分:脱脂乳20-150g/L,蔗糖30-100g/L,甘油5-45g/L。
作为优选,步骤3)中,发酵温度为34-45℃,发酵时间为6-18h。
作为优选,步骤3)中,发酵pH为4.0-6.0。
本发明采用的是高密度发酵工艺,发酵pH会影响瑞士乳杆菌的存活,因而需要对发酵pH进行调控。将pH控制在4.0-6.0范围内,能使发酵液中具有较高的活菌含量。
作为优选,步骤4)中,离心转速4,000-10,000rpm,离心时间3-15min。
作为优选,步骤5)中,所述离心产物与菌株保护剂以1:1-3的重量比混合。
作为优选,步骤7)中,过筛时,筛网选择10-80目标准筛。
一种增强免疫功能的益生菌固体饮料的制备方法,包括以下步骤:
(1)称取各原料备用;
(4)将步骤(3)所得混合半成品进行沸腾制粒,过筛,得到沸腾制粒半成品;
(6)将步骤(5)所得总混合半成品进行包装,即得增强免疫功能的益生菌固体饮料。
步骤(4)中,通过沸腾制粒技术进行制粒,可显著改善益生菌固体饮料的口感,若采用条包灌装机进行包装,则还可显著改善益生菌固体饮料的灌装性能。
作为优选,步骤(2)中,若各含量重量比小于1%的原料的重量之和小于总配方用料量的2%,则加入赤藓糖醇,使重量之和达到总配方用料量的2%,再进行混合。
作为优选,步骤(3)中,混合转速为15-35rpm,优选30rpm,混合时间为10-20min,优选15min。
作为优选,步骤(4)中,沸腾制粒过程采用粘合剂,所述粘合剂为纯水、玉米淀粉、麦芽糊精中的至少一种。
作为优选,步骤(4)中,沸腾制粒时,进风温度85-95℃,排风变频50%-80%,物料温度50-60℃,雾化压力2.5-3.5bar,喷浆供浆泵转速50-80rpm。
作为优选,步骤(4)中,过筛时,筛网选择15-40目标准筛。
作为优选,步骤(5)中,混合转速为15-35rpm,优选30rpm,混合时间为10-20min,优选15min。
作为优选,步骤(6)中,包装时,充入氮气,残氧量为3%-10%。
作为优选,步骤(7)中,包装所用包材,采用铝塑包材。
作为优选,步骤(1)~(6)中,操作过程全部在GMP车间中恒温恒湿环境中进行,优选为在十万级GMP车间中进行,温度为18-26℃,湿度为25-40%。
作为优选,步骤(6)中,所述益生菌固体饮料的水分含量为2-5%,水分活度为0.1-0.4aW。
本发明的有益效果是:
本发明将瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉、接骨木莓提取物、蓝莓粉、赤藓糖醇及维生素C进行复配。除可发挥瑞士乳杆菌WHH2580的功能外,嗜酸乳杆菌-1、乳双岐杆菌HN019、接骨木莓提取物、蓝莓粉可起到额外的辅助作用;赤藓糖醇可在混合、包装等过程中维持益生菌固体饮料的低水分、低水分活度,有利于瑞士乳杆菌WHH2580、嗜酸乳杆菌-1、乳双岐杆菌HN019的活性稳定;维生素C可提供良好的口味并补充人体所需的营养。
本发明中所采用的瑞士乳杆菌WHH2580具有良好的耐受性和黏附性,对免疫功能低下者具有增强免疫力作用,可用于增强免疫力的保健品或健康食品;此外,本发明灭活菌株同样可以通过提高调节机体的脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力,因此本发明的益生菌固体饮料稳定性更好,保质期更长,且在储存和运输过程中不需要冷藏,成本更低。
本发明将除瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉外的原料通过沸腾制粒技术进行制粒,可显著改善益生菌固体饮料口感,若采用条包灌装机进行包装,则还可显著改善益生菌固体饮料的灌装性能。
本发明所选用的瑞士乳杆菌冻干粉,采用生物工程恒pH高密度发酵技术、冷冻干燥技术、粉碎过筛工艺所得,具有活菌数高,低水分、低水分活度,粒径分布合理,与其他物料有良好的混合性能等特点。
附图说明
图1为瑞士乳杆菌WHH2580在显微镜下观察到的形态;
图2为瑞士乳杆菌WHH2580活体菌、非活体菌、商业菌株LGG和Lcs与HT-29细胞黏附效果图;
图3为实施例动物试验中各组BALB/c小鼠的体重变化图;
图4为实施例动物试验中各组BALB/c小鼠的脏器比值图;
图5为实施例动物试验中各组BALB/c小鼠的脾淋巴细胞转化数值图;
图6为实施例中各组BALB/c小鼠的NK细胞活性图。
具体实施方式
下面结合实施例对本发明作进一步的描述。
总实施例
一种增强免疫功能的益生菌固体饮料,包括瑞士乳杆菌冻干粉1-10份、嗜酸乳杆菌-1冻干粉1-10份、乳双岐杆菌HN019冻干粉1-10份、接骨木莓提取物2-11份、蓝莓粉20-40份、赤藓糖醇40-60份、维生素C 0.05-0.3份。所述瑞士乳杆菌冻干粉中的瑞士乳杆菌活菌数为1×107CFU/g-2×1012CFU/g。
所述瑞士乳杆菌冻干粉由瑞士乳杆菌和/或其突变体制得;所述瑞士乳杆菌命名为WHH2580,于2019年10月23日在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心保藏,其微生物保藏编号为:CGMCC No.18730。保藏单位地址为北京市朝阳区北辰西路1号院3号,微生物分类命名为瑞士乳杆菌Lactobacillus helveticus。所述突变体为对所述瑞士乳杆菌进行诱变、驯化、基因重组或者经自然突变而获得的突变体。
所述瑞士乳杆菌冻干粉的制备方法包括以下步骤:
1)培养基的制备;所述培养基包括以下成分:脱盐乳清粉15-50g、酪蛋白水解物2-15g、胰蛋白胨3-15g、大豆蛋白胨7-20g、酵母粉3-6g、柠檬酸钠5-15g、磷酸氢二钾1-5g、硫酸镁0.4-0.8g、一水合硫酸锰0.2-0.5g,番茄汁5-30ml、水1000mL;所述培养基的pH为6.5±0.2;
2)菌株保护剂的制备;所述菌株保护剂包括以下成分:脱脂乳20-150g/L,蔗糖30-100g/L,甘油5-45g/L;
3)以2%-10%接种量将瑞士乳杆菌和/或其突变体接种于发酵基质中发酵培养,发酵温度为34-45℃,发酵时间为6-18h,发酵过程pH控制于pH5.0-5.6;
4)发酵结束后取发酵产物,离心,离心转速4,000-10,000rpm,离心时间3-15min;
5)离心产物与菌株保护剂以1:1-3的重量比混合;
6)冻干;
7)冻干产物粉碎、过筛,筛网选择10-80目标准筛,得到瑞士乳杆菌冻干粉。
一种增强免疫功能的益生菌固体饮料的制备方法,包括以下步骤:
(1)称取各原料备用;
(2)将除瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉外的各含量重量比小于1%的原料混合均匀,得到混合后小料,待用;各含量重量比小于1%的原料的重量之和小于总配方用料量的2%时,加入赤藓糖醇,使其达到总配方用料量的2%,再进行混合;
(3)将除瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉外的其余原料与步骤(2)所得混合后小料一起混合均匀,即得混合半成品;混合转速为15-35rpm,优选30rpm,混合时间为10-20min,优选15min;
(4)将步骤(3)所得混合半成品进行沸腾制粒,过筛,得到沸腾制粒半成品;
沸腾制粒过程采用粘合剂,所述粘合剂为纯水、玉米淀粉、麦芽糊精中的至少一种;沸腾制粒时,进风温度85-95℃,排风变频50%-80%,物料温度50-60℃,雾化压力2.5-3.5bar,喷浆供浆泵转速50-80rpm;过筛时,筛网选择15-40目标准筛;
(5)将步骤(4)所得沸腾制粒半成品与瑞士乳杆菌冻干粉、嗜酸乳杆菌-1冻干粉、乳双岐杆菌HN019冻干粉外混合均匀,得到总混合半成品;混合转速为15-35rpm,优选30rpm,混合时间为10-20min,优选15min;
(6)将步骤(5)所得总混合半成品用条包灌装机包装,即得增强免疫功能的益生菌固体饮料成品。包装时应该充入氮气,残氧量为3%-10%;包装所用包材,采用铝塑包材;所述益生菌固体饮料的水分含量为2-5%,水分活度为0.1-0.4aW。
步骤(1)~(6)中,操作过程全部在十万级GMP车间中恒温恒湿环境中进行,温度为18-26℃,湿度为25-40%。
实施例1
本发明所提供的菌株经鉴定属于瑞士乳杆菌(Lactobacillus helveticus),命名为WHH2580,于2019年10月23日在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心保藏,其微生物保藏编号为:CGMCC No.18730。
本发明所提供的菌株是发明人从新疆那拉提红山沟采集的酸奶中筛选得到。
本发明瑞士乳杆菌2580的生物学性质如下:
形态学特征:在MRS琼脂培养基中生长状态为:在MRS琼脂培养基中生长形态为浅乳白色菌落,半透明、圆形、表面粗糙、边缘不整齐、平展。革兰氏染色呈典型阳性,显微镜下观察细胞呈长杆状,无鞭毛,不产芽孢,不运动(图1所示)。
培养特征:最适生长温度为37度,兼性厌氧,生长于MRS培养基中。
生理特征:使用API 50 CHL***,WHH2580菌株可利用6种碳源:D-葡萄糖、D-果糖、D-甘露糖、N-乙酰葡萄糖胺、D-乳糖和D-海藻糖。
生物学鉴定:针对16s rRNA基因序列测序,所得结果与NCBI GenBank数据库中进行同源性比对分析,结果显示该菌株为瑞士乳杆菌(Lactobacillus helveticus)。
基因序列如SEQ ID NO.1所示:
CCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTTGC
实施例2:菌株耐受性
瑞士乳杆菌WHH2580菌株的耐酸耐胆盐性能:
1耐酸性能:WHH2580菌株二代活化后的菌液20mL/管离心,pH7.2PBS洗两次。分别将菌泥置于20mL无菌PBS pH3.0、PBS+0.3%pepsin pH3.0和PBS pH7.2中,每一个体系进行两个重复。置于37℃,分别于0h、1h、2h、3h取样,进行活菌计数。结果如下表:
耐酸性实验结果显示:pH3.0在2h对该菌的存活性没有显著的影响,3h活菌数降低0.9个数量级。胃蛋白酶pH3.0在2h对该菌的存活性没有显著的影响,3h活菌数降低0.7个数量级。上述结果表明该菌有优良的耐酸性。
2耐胆盐性能:WHH2580菌株二代活化后的菌液20mL/管离心,pH7.2PBS洗两次。分别将菌泥置于20mL无菌PBS+0.1%Trypsin pH8.0、PBS+0.3%BS pH8.0、PBS+0.5%BSpH8.0和PBS pH7.2中,每一个体系进行两个重复。置于37℃,分别于0h、1h、2h、3h取样,进行活菌计数。结果如下表:
耐胆盐性实验结果显示:0.1%Trypsin体系中3h降低0.8个数量级,0.3%BS体系中3h降低2.8个数量级,0.5%BS体系中3h降低3.6个数量级。上述结果表明该菌有良好的耐胆盐性。
实施例3:菌株黏附性
益生菌通过黏附作用定殖于宿主肠上皮细胞,逐步形成生物膜并产生多种代谢产物,从而构成一层天然的保护屏障,对宿主产生多种生理功效。因此,益生菌黏附并定殖于宿主肠上皮细胞上是发挥其益生作用的前提。
采用HT-29细胞作为黏附性试验的细胞株,具体实验方法如下:
HT-29细胞传至第三代,采用0.25%的胰酶消化后1000rpm离心5min,加入5mL含10%胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)进行重悬,直至细胞呈单细胞悬液。取适量细胞用血球细胞计数板计数,用含10%胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)将细胞稀释至细胞悬液浓度为到1×106cells/mL,取1mL接种于已放置细胞爬片的12孔细胞培养板中,于37℃,5%CO2培养2天。
WHH2580菌株(鼠李糖乳杆菌LGG和代田株LcS为阳性对照菌株)二代活化后,取10mL菌液室温下4200g离心10min收集菌体,用10mL含10%胎牛血清的DMEM完全培养基中培养(不加双抗)重悬,调整菌浓度至2×108CFU/mL。取5mL菌株调整液95℃灭活5min。菌株调整液和灭活菌株调整液各取1mL接种于已放置细胞爬片的12孔细胞培养板中,各3个平行,于CO2培养箱中37℃孵育2h。
孵育结束后,缓慢吸去培养液,用PBS洗涤3次,用100%甲醇固定8min。取出细胞爬片静置20min,经革兰氏染色后,用中性树脂封片。于光学显微镜下进行观察。
黏附实验结果如下表:
瑞士乳杆菌WHH2580活体菌、非活体菌、商业菌株LGG和Lcs与HT-29细胞黏附效果附图2所示。
瑞士乳杆菌WHH2580活体菌和非活体菌的黏附能力显著高于阳性对照菌株鼠李糖乳杆菌LGG和代田株LcS。瑞士乳杆菌WHH2580非活体菌的黏附率稍低于其活体菌的黏附率,但非活体菌的黏附效果比活体菌好,HT-29细胞间的菌体较少,绝大部分非活体菌紧紧黏附在细胞上。
实施例4:菌株耐药性
瑞士乳杆菌WHH2580菌株抗生素的敏感性:
益生菌对抗生素的敏感性是检测菌株安全性的重要指标。为了检测WHH2580菌株的安全性,选取下列9种抗生素进行测试,检测WHH2580菌株对9种抗生素的敏感性,具体实验设计如下。
抗生素配制浓度是0.5ug/mL、1ug/mL、2ug/mL、4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL、128ug/mL、256ug/mL,实际作用浓度是0.125ug/mL、0.25ug/mL、0.5ug/mL、1ug/mL、2ug/mL、4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL。
无菌的96孔聚苯乙烯板中每个孔加入100μL培养基,2-11列按下表浓度标示每孔加50μL抗生素稀释液(ug/mL),1-11列每孔加入菌悬液50μL(菌液终浓度在105-106CFU/mL),密封后置37℃培养箱中24-48h,肉眼观察和测OD625。其中鼠李糖乳杆菌LGG和代田株LcS为阳性对照菌株。
实验结果如下表,MIC值ug/mL:
根据CLSI M4菌株对抗生素的敏感性的判断标准,得到下列结论。WHH2580菌株对达托霉素Daptomycin和克林霉素Clindamycin有抗性,对红霉素Erythromycin中性,对Penicillin、Ampicillin、Imipenem、Meropenem、Vancomycin和Linezolid敏感。LGG菌株对Imipenem、Meropenem、Vancomycin、Daptomycin、Clindamycin有抗性,对红霉素Erythromycin中性,对Penicillin、Ampicillin和Linezolid敏感。LcS菌株对Vancomycin和Daptomycin有抗性,对Imipenem、Meropenem和Erythromycin中性,对Penicillin、Ampicillin、Clindamycin和Linezolid敏感。
实施例5:菌株抑制病原菌
瑞士乳杆菌WHH2580菌株抑菌实验:
事先分装灭菌的1.5%素琼脂于无菌培养皿。摆放好牛津杯。灭菌1%琼脂的MRS培养基或NB培养基,50℃以下接种106-107CFU/mL浓度的五种指示菌(大肠埃希氏菌、金黄色葡萄球菌、福氏志贺氏菌、乙型副伤寒沙门氏菌、藤黄微球菌),振荡混匀后倾倒在之前倒好的有牛津杯的素琼脂培养基上。待以上培养基冷却凝固后,拔去牛津杯。在不同的小孔中分别加入100uL的相应菌液(WHH2580)和阳性对照(10g/mL多粘菌素B或1%Nisin),放入4℃冰箱中扩散7h。放入37℃培养16h后,观察并记录抑菌圈的大小。
实验结果如下表,表格中的数值为抑菌圈的大小cm:
实验结果显示WHH2580菌株对5种致病菌均有抑制效果,其对乙型副伤寒沙门氏菌、大肠埃希氏菌和福氏志贺氏菌的抑制作用和10g/mL多粘菌素B无明显差异,对金黄色葡萄球菌的抑制作用和1%Nisin无明显差异,稍低于其他对照菌株。
实施例6:菌株增强免疫力功能
1.体外脾淋巴细胞增殖:
处死小鼠,无菌取脾脏,用眼科剪将脾脏剪碎后,用无菌的玻璃注射器芯将其碾碎,经200目金属筛网过滤后,加入含10%胎牛血清的无菌Hank’s液,1000rpm,4℃离心5min,收获细胞。加入5倍于细胞体积的细胞裂解液,裂解5min,加入含10%胎牛血清的无菌Hank’s液终止裂解,1000rpm,4℃离心5min去上清。用含10%胎牛血清的RPMI-1640培养基洗涤,1000rpm,4℃离心5min。洗涤两次,沉淀重悬于5mL含10%胎牛血清的RPMI-1640培养基。细胞计数,调整细胞浓度为5×106cells/mL。
乳酸菌活化菌液4000rpm室温下离心10min去上清,PBS洗涤两次,加入适量含10%胎牛血清的RPMI-1640培养基,调整其OD600至0.50±0.10。将细胞悬液加入96孔细胞培养板,每个样品3个平行。设置调零组(细胞培养基),空白对照组(细胞悬液+细胞培养基,诱导剂组(细胞悬液+10μg/mL Con A),乳酸菌组(细胞悬液+10μg/mL Con A+菌悬液),置二氧化碳培养箱37℃培养72h。每孔加入20μL MTT溶液(2.5mg/ml),37℃显色4h后,1500rpm,4℃离心10min,吸出上清,再加入100μL DMSO,用酶标仪于490nm下测定吸光值。
数据分析:在Con A诱导和不诱导的情况下,均能显著促进脾淋巴细胞增殖的益生菌作为优选菌株。
结果如下表所示,在Con A诱导和不诱导的情况下,瑞士乳杆菌WHH2580脾淋巴细胞增殖倍数高于两株商业菌株。
2体外脾淋巴细胞细胞因子IL-10和IL-12测定
细胞取材、红细胞裂解、洗涤、细胞计数、益生菌培养同上述体外脾淋巴细胞增殖步骤。将细胞悬液加入96孔细胞培养板,每个处理5个重复,分为调零组(细胞培养基100μL),空白对照组(1×107cells/mL的细胞悬液100μl,细胞培养基100μl),益生菌组(1×107cells/mL的细胞悬液100μL,1×108cfu/mL菌悬液100μL),37℃5%CO2培养箱中培养24h。1500rpm离心10min,吸取上清,0.22μm滤膜过滤,采用BD试剂盒测定IL-10和IL-12含量。
IL-12/IL-10比值高的菌株可作为免疫调节潜力菌株。
结果如下表所示,瑞士乳杆菌WHH2580IL-12/IL-10比值与鼠李糖乳杆菌LGG接近,没有显差。
3体外人结肠癌细胞HT-29细胞因子IL-8测定
将生长融合至70%-80%的人结肠癌细胞HT-29进行消化,调整细胞浓度,1*106cell/孔接入24孔板,37℃培养,24h后换液。加入20ng/mL LPS处理6小时,再加入8*108CFU/mL菌液处理18小时后,3000r/min,5min离心得上清,用相应ELISA试剂盒检测白细胞介素IL-8的含量。
分泌IL-8高的菌株可作为免疫调节潜力菌株。
结果如下表所示:
菌株编号 | IL-8浓度(ng/mL) |
鼠李糖乳杆菌LGG | 2981±95 |
瑞士乳杆菌WHH2580 | 4021±145 |
瑞士乳杆菌WHH2580IL-8高于商业菌株LGG。
实施例7:动物试验
采取随机分组设计,将近交系BALB/C雄性小鼠(6-8周龄,16-20g),192只,适应期5天,随机分为8组,每组12只。动物饲养保持环境温度为21±2℃,湿度为30-70%,12h光照交替,自由饮水,自由摄入饲料。每三天换一次垫料,及时添水和饲料。分组如下:
对照组1:空白对照组,灌胃水;
对照组2:模型对照组,灌胃水+腹腔注射50mg/kg环磷酰胺造模;
对照组3:阳性药物组,灌胃40mg/kg盐酸左旋咪唑+腹腔注射50mg/kg环磷酰胺造模;
实验组1:灌胃本发明菌株菌悬液,灌胃剂量为2*108CFU/d;
实验组2:灌胃本发明菌株灭活菌悬液,灌胃剂量为2*108CFU/d;
其中灭活菌是通过80℃10min处理的。经平板培养,证实无活菌存在。
采用免疫功能低下模型动物进行试验。连续给予受试物38天,在第32天后开始给予免疫抑制剂。抑制剂前每周称一次体重,抑制剂后每天称一次体重。腹腔注射免疫抑制剂第5天开始检测,检测指标有脏器比,脾淋巴细胞增殖,NK细胞活性。
实验结果如下:
体重结果见图3。除空白对照外的各实验组的体重变化趋势类似,在注射完抑制剂的第一天体重下降,后面逐渐恢复。
脏器比结果见图4。模型组的胸腺/体重比和脾脏/体重比显著低于对照组。低剂量组和阳性药物组的胸腺/体重比显著高于模型组(P<0.05),高剂量组和死菌组的胸腺/体重比极显著高于模型组(P<0.001)。低剂量组、中剂量组、死菌组和阳性药物组的脾脏/体重比显著高于模型组(P<0.05)。
小鼠脾淋巴细胞增殖结果见图5。超低剂量组、低、中、高剂量组OD值显著高于模型组灭活菌组极显著高于模型组。表明WHH2580超低剂量、低、中、高剂量和灭活菌均能显著促进免疫低下小鼠脾淋巴细胞增殖。
小鼠NK细胞活性的测定结果见图6。中剂量组的NK细胞活性极显著高于模型组,灭活菌组的NK细胞活性显著高于模型组。表明WHH2580中剂量和灭活菌均能显著提高免疫低下小鼠NK细胞活性。
根据保健品功能评价方法,在免疫功能低下模型成立条件下,血液白细胞总数、细胞免疫功能(小鼠脾淋巴细胞转化实验,迟发型***反应实验)、体液免疫功能(抗体生成细胞检测,血清溶血素测定)、单核-巨噬细胞功能(小鼠碳廓清实验,小鼠腹腔巨噬细胞吞噬荧光微球实验)、NK细胞活性,任两个方面试验结果为阳性,判定该受试样品对免疫功能低下者具有增强免疫力作用。WHH2580菌株在细胞免疫功能和NK细胞活性两个方面试验结果为阳性,可判定WHH2580菌株对免疫功能低下者具有增强免疫力作用。
WHH2580灭活菌株同样可以通过提高调节机体的脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力。
本发明所提供的瑞士乳杆菌WHH2580菌株相关的产品可以是其活体形式,也可以是其非活体形式。目前对于非活体益生菌的研究相对较少。与活菌制剂相比,非活菌制剂有一些优势,如货架期更长,安全性更高,且在储存和运输过程中不需要冷藏,成本更低。
本发明所提供的瑞士乳杆菌WHH2580菌株高黏附性能是其发挥免疫功效的前提。应当理解的是,对于本领域普通技术人员来说,可以根据上述说明加以改进或变换,而所有这些改进和变换都应属于本发明所附权利要求。
实施例8
一种增强免疫功能的益生菌固体饮料,包括瑞士乳杆菌冻干粉2份、嗜酸乳杆菌-1冻干粉1份、乳双岐杆菌HN019冻干粉2份、接骨木莓提取物6份、蓝莓粉30份、赤藓糖醇58.95份、维生素C 0.05份。所述瑞士乳杆菌冻干粉中的瑞士乳杆菌活菌数为1.5×1010CFU/g。
所述瑞士乳杆菌冻干粉的制备方法包括:
1)培养基的制备:所述培养基配方为脱盐乳清粉15g、酪蛋白水解物13g、胰蛋白胨3g、大豆蛋白胨20g、酵母粉6g、柠檬酸钠5g、磷酸氢二钾3g、硫酸镁0.4g、一水合硫酸锰0.3g,番茄汁15ml、水1000mL;调整其pH为6.5±0.2;
2)菌株保护剂的制备,所述菌株保护剂配方为脱脂乳90g/L,蔗糖80g/L,甘油30g/L;
3)以5%接种量将瑞士乳杆菌WHH2580接种于发酵基质中进行发酵培养,发酵温度为40℃,发酵时间为12℃,发酵过程pH控制于pH5.0;
4)发酵结束后取发酵产物,离心,离心转速8,000rpm,离心时间10min;
5)离心产物与菌株保护剂以1:2的重量比混合;
6)冻干;
7)冻干产物粉碎、过筛,筛网选择30目标准筛,得到瑞士乳杆菌冻干粉。
一种增强免疫功能的益生菌固体饮料的制备方法,包括以下步骤:
(1)按照重量份配比称取各原料备用;
(2)将维生素C 0.05份与赤藓糖醇1.95份混合均匀,得到混合后小料;
(3)将接骨木莓提取物6份、蓝莓粉30份、赤藓糖醇57份与步骤(2)所得混合后小料一起混合均匀,即得混合半成品;混合转速为20rpm,混合时间为20min;
(4)将步骤(3)所得混合半成品进行沸腾制粒,过筛,得到沸腾制粒半成品;
沸腾制粒过程采用粘合剂,所述粘合剂为纯水;沸腾制粒时,进风温度85℃,排风变频80%,物料温度50℃,雾化压力2.5bar,喷浆供浆泵转速80rpm;过筛时,筛网选择20目标准筛;(5)将步骤(4)所得沸腾制粒半成品与瑞士乳杆菌冻干粉2份、嗜酸乳杆菌-1冻干粉1份、乳双岐杆菌HN019冻干粉2份混合均匀,得到总混合半成品;混合转速为20rpm,混合时间为20min;
(6)将步骤(5)所得总混合半成品用条包灌装机包装,即得增强免疫功能的益生菌固体饮料;包装时应该充入氮气,残氧量为5%;包装所用包材,采用铝塑包材。
步骤(1)~(6)中,操作过程全部在十万级GMP车间中恒温恒湿环境中进行,温度为26℃,湿度为40%。
所得益生菌固体饮料及其内容物进行关键指标检测,所得检测结果如下:
实施例9
一种增强免疫功能的益生菌固体饮料,包括瑞士乳杆菌冻干粉8份、嗜酸乳杆菌-1冻干粉2份、乳双岐杆菌HN019冻干粉5份、接骨木莓提取物10份、蓝莓粉30份、赤藓糖醇44.8份、维生素C 0.2份。所述瑞士乳杆菌WHH2580活菌数在5×1010CFU/g。
所述瑞士乳杆菌冻干粉的制备方法包括:
1)培养基的制备:所述培养基配方为脱盐乳清粉40g、酪蛋白水解物10g、胰蛋白胨8g、大豆蛋白胨15g、酵母粉5g、柠檬酸钠8g、磷酸氢二钾2g、硫酸镁0.6g、一水合硫酸锰0.4g,番茄汁20ml、水1000mL;调整其pH为6.5±0.2;
2)菌株保护剂的制备:所述菌株保护剂配方为脱脂乳30g/L,蔗糖50g/L,甘油20g/L;
3)以5%接种量将瑞士乳杆菌WHH2580接种于发酵基质中进行发酵培养,发酵温度为40℃,发酵时间为12℃,发酵过程pH控制于pH5.0;
4)发酵结束后取发酵产物,离心;离心转速8,000rpm,离心时间10min;
5)离心产物与菌株保护剂以1:2的重量比混合;
6)冻干;
7)冻干产物粉碎、过筛,筛网选择40目标准筛,得到瑞士乳杆菌冻干粉。
一种增强免疫功能的益生菌固体饮料的制备方法,包括以下步骤:
(1)按照重量份配比称取各原料备用;
(2)将维生素C 0.2份与赤藓糖醇1.8份混合均匀,得到混合后小料;
(3)将接骨木莓提取物10份、蓝莓粉30份、赤藓糖醇43份与步骤(2)所得混合后小料一起混合均匀,即得混合半成品;混合转速为20rpm,混合时间为20min;
(4)将步骤(3)所得混合半成品进行沸腾制粒,过筛,得到沸腾制粒半成品;
沸腾制粒过程采用粘合剂,所述粘合剂为纯水;沸腾制粒时,进风温度85℃,排风变频80%,物料温度50℃,雾化压力2.5bar,喷浆供浆泵转速80rpm;过筛时,筛网选择20目标准筛。
(6)将步骤(5)所得总混合半成品用条包灌装机包装,即得增强免疫功能的益生菌固体饮料;包装时应该充入氮气,残氧量为5%;包装所用包材,采用铝塑包材;
步骤(1)~(6)中,操作过程全部在十万级GMP车间中恒温恒湿环境中进行,温度为26℃,湿度为40%。
所得益生菌固体饮料及其内容物进行关键指标检测,所得检测结果如下:
本发明中所用原料、设备,若无特别说明,均为本领域的常用原料、设备;本发明中所用方法,若无特别说明,均为本领域的常规方法。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制,凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换,均仍属于本发明技术方案的保护范围。
序列表
<110> 杭州娃哈哈科技有限公司
杭州娃哈哈集团有限公司
<120> 一种增强免疫功能的益生菌固体饮料及其制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> 瑞士乳杆菌(Lactobacillus helveticus)
<400> 1
ccttcccgaa ggttaggcca ccggctttgg gcattgcaga cttccatggt gtgacgggcg 60
gtgtgtacaa ggcccgggaa cgtattcacc gcggcgttct gatccgcgat tactagcgat 120
tccagcttcg tgcagtcgag ttgcagactg cagtccgaac tgagaacagc tttcagagat 180
tcgcttgcct tcgcaggctc gcttctcgtt gtactgtcca ttgtagcacg tgtgtagccc 240
aggtcataag gggcatgatg acttgacgtc atccccacct tcctccggtt tatcaccggc 300
agtctcatta gagtgcccaa cttaatgctg gcaactaata acaagggttg cgctcgttgc 360
gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtcttag 420
cgtccccgaa gggaactcct aatctcttag gatggcacta gatgtcaaga cctggtaagg 480
ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat 540
tcctttgagt ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat gcgttagctg 600
cagcactgag aggcggaaac ctcccaacac ttagcactca tcgtttacgg catggactac 660
cagggtatct aatcctgttc gctacccatg ctttcgagcc tcagcgtcag ttgcagacca 720
gagagtcgcc ttcgccactg gtgttcttcc atatatctac gcattccacc gctacacatg 780
gagttccact ctcctcttct gcactcaaga aaaacagttt ccgatgcagt tcctcggtta 840
agccgagggc tttcacatca gacttattct tccgcctgcg ctcgctttac gcccaataaa 900
tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt tagccgtgac 960
tttctggttg attaccgtca aataaaggcc agttactacc tctatccttc ttcaccaaca 1020
acagagcttt acgatccgaa aaccttcttc actcacgcgg cgttgctcca tcagacttgc 1080
gtccattgtg gaagattccc tactgctgcc tcccgtagga gtttgggccg tgtctcagtc 1140
ccaatgtggc cgatcagtct ctcaactcgg ctatgcatca ttgccttggt aagccgttac 1200
cttaccaact agctaatgca ccgcggggcc atcccatagc gacagcttac gccgcctttt 1260
ataagctgat catgcgatct gctttcttat ccggtattag cacctgtttc caagtggtat 1320
cccagactat ggggcaggtt ccccacgtgt tactcaccca tccgccgctc gcgtccccag 1380
cgtcattacc gaagtaaatc tgctggttct gctcgctcga cttgc 1425
Claims (10)
1.一种增强免疫功能的益生菌固体饮料,其特征在于,包括瑞士乳杆菌冻干粉、嗜酸乳杆菌DDS®-1冻干粉、乳双岐杆菌HN019冻干粉、接骨木莓提取物、蓝莓粉、赤藓糖醇及维生素C;
所述瑞士乳杆菌冻干粉由瑞士乳杆菌制得;所述瑞士乳杆菌命名为WHH2580,于2019年10月23日在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心保藏,其微生物保藏编号为:CGMCC No.18730;保藏单位地址为北京市朝阳区北辰西路1号院3号,微生物分类命名为瑞士乳杆菌Lactobacillus helveticus。
2.如权利要求1所述的一种增强免疫功能的益生菌固体饮料,其特征在于,按重量份包括瑞士乳杆菌冻干粉1-10份、嗜酸乳杆菌DDS®-1冻干粉1-10份、乳双岐杆菌HN019冻干粉1-10份、接骨木莓提取物2-11份、蓝莓粉10-40份、赤藓糖醇40-60份、维生素C 0.05-0.3份。
3.如权利要求1或2所述的一种增强免疫功能的益生菌固体饮料,其特征在于,所述瑞士乳杆菌冻干粉中的瑞士乳杆菌活菌数为1×107CFU/g-2×1012CFU/g。
4.如权利要求1或2所述的一种增强免疫功能的益生菌固体饮料,其特征在于,所述瑞士乳杆菌冻干粉的制备方法包括以下步骤:
1)培养基的制备;
2)菌株保护剂的制备;
3)以2%-10%接种量将瑞士乳杆菌接种于发酵基质中发酵培养;
4)发酵结束后取发酵产物,离心;
5)离心产物与菌株保护剂混合;
6)冻干;
7)冻干产物粉碎、过筛,得到瑞士乳杆菌冻干粉。
5.如权利要求4所述的一种增强免疫功能的益生菌固体饮料,其特征在于:
步骤1)中,所述培养基包括以下成分:脱盐乳清粉15-50g、酪蛋白水解物2-15g、胰蛋白胨3-15g、大豆蛋白胨7-20g、酵母粉3-6g、柠檬酸钠5-15g、磷酸氢二钾1-5g、硫酸镁0.4-0.8g、一水合硫酸锰0.2-0.5g,番茄汁5-30ml、水1000mL;所述培养基的pH为6.5±0.2;和/或
步骤2)中,所述菌株保护剂包括以下成分:脱脂乳20-150g/L,蔗糖30-100g/L,甘油5-45g/L。
6.如权利要求4所述的一种增强免疫功能的益生菌固体饮料,其特征在于:
步骤3)中,发酵温度为34-45℃,发酵时间为6-18h;和/或
步骤3)中,发酵pH为4.0-6.0;和/或
步骤4)中,离心转速4,000-10,000rpm,离心时间3-15min;和/或
步骤5)中,所述离心产物与菌株保护剂以1:1-3的重量比混合;和/或
步骤7)中,过筛时,筛网选择10-80目标准筛。
7.一种制备如权利要求1~6之一所述增强免疫功能的益生菌固体饮料的方法,其特征在于,包括以下步骤:
(1)称取各原料备用;
(2)将除瑞士乳杆菌冻干粉、嗜酸乳杆菌DDS®-1冻干粉、乳双岐杆菌HN019冻干粉外的各含量重量比小于1%的原料混合均匀,得到混合后小料,待用;
(3)将除瑞士乳杆菌冻干粉、嗜酸乳杆菌DDS®-1冻干粉、乳双岐杆菌HN019冻干粉外的其余原料与步骤(2)所得混合后小料一起混合均匀,即得混合半成品;
(4)将步骤(3)所得混合半成品进行沸腾制粒,过筛,得到沸腾制粒半成品;
(5)将步骤(4)所得沸腾制粒半成品与瑞士乳杆菌冻干粉、嗜酸乳杆菌DDS®-1冻干粉、乳双岐杆菌HN019冻干粉混合均匀,得到总混合半成品;
(6)将步骤(5)所得总混合半成品进行包装,即得增强免疫功能的益生菌固体饮料。
8.如权利要求7所述的一种制备增强免疫功能的益生菌固体饮料的方法,其特征在于,步骤(2)中,若各含量重量比小于1%的原料的重量之和小于总配方用料量的2%,则加入赤藓糖醇,使重量之和达到总配方用料量的2%,再进行混合。
9.如权利要求7所述的一种制备增强免疫功能的益生菌固体饮料的方法,其特征在于:
步骤(3)中,混合转速为15-35rpm,混合时间为10-20min;和/或
步骤(4)中,沸腾制粒过程采用粘合剂,所述粘合剂为纯水、玉米淀粉、麦芽糊精中的至少一种;和/或
步骤(4)中,沸腾制粒时,进风温度85-95℃,排风变频50%-80%,物料温度50-60℃,雾化压力2.5-3.5bar,喷浆供浆泵转速50-80rpm;和/或
步骤(4)中,过筛时,筛网选择15-40目标准筛;和/或
步骤(5)中,混合转速为15-35rpm,混合时间为10-20min;和/或
步骤(6)中,包装时,充入氮气,残氧量为3%-10%;和/或
步骤(1)~(6)中,操作过程全部在GMP车间中恒温恒湿环境中进行,温度为18-26℃,湿度为25-40%。
10.如权利要求7所述的一种制备增强免疫功能的益生菌固体饮料的方法,其特征在于,步骤(6)中,所述益生菌固体饮料的水分含量为2-5%,水分活度为0.1-0.4aW。
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