CN117462602A - 一种益生菌复合剂及制备方法与应用 - Google Patents
一种益生菌复合剂及制备方法与应用 Download PDFInfo
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- CN117462602A CN117462602A CN202311814197.8A CN202311814197A CN117462602A CN 117462602 A CN117462602 A CN 117462602A CN 202311814197 A CN202311814197 A CN 202311814197A CN 117462602 A CN117462602 A CN 117462602A
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- parts
- powder
- probiotic
- fructo
- oligosaccharide
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Abstract
本发明公开了一种益生菌复合剂，涉及复合益生菌技术领域。本发明以重量份数计包括以下组分：低聚果糖10‑20份；水苏糖10‑20份；果汁粉4‑8份；益生菌组合物1‑3份。本发明可以调节肠道菌群，减少便秘、上呼吸道感染等健康问题发生率，辅助提高儿童抵抗力。
Description
技术领域
本发明涉及复合益生菌技术领域，更具体的说是涉及一种益生菌复合剂及制备方法与应用。
背景技术
幼儿群体由于免疫系统功能尚未健全、肠道菌群结构尚未稳定，导致胃肠功能和免疫功能更容易受到饮食、环境、季节等因素变化得以影响，从而引起一系列的腹泻、腹痛、便秘、感冒、湿疹、鼻炎等健康问题。幼儿时期肠道菌群的初期定殖可以抵抗病原体的定植繁殖、促进免疫系统的发育成熟和营养物质的代谢吸收。如果这一时期出现肠道菌群定殖延迟或构成发生紊乱，与儿童期乃至青春期的感染性疾病、过敏性疾病、自身免疫性疾病、内分泌疾病和肠道慢性炎症性疾病等密切相关。所以肠道正常菌群结构的平衡、稳定和协调是幼儿健康的标志。
益生菌指当摄入足够数量时，对宿主健康有益的活的微生物。幼儿时期，肠道菌群的建立和发育是一个动态的过程，受到分娩方式、喂养方式、环境因素、饮食结构和抗生素应用等因素的影响。在此阶段补充益生菌，有利于帮助尽快建立稳定的肠道菌群，增强抵抗力，减少感染发生风险，降低整体疾病发生几率。
目前，益生菌粉剂大多数采用冲水服用，但冲水后，产品里有菌粉特殊气味，影响儿童服用的积极性，但受产品特性影响，配方不可直接加入大量果粉（果粉水活度高，影响益生菌活菌存活率），也不适宜加入香精等矫味剂。直接食用时，儿童容易因过细的粉剂粉末增加呛咳风险。
益生菌的健康作用效果与其摄入量、产品类型、食用周期及不同人群等众多因素有关，其功效性评价也应建立在更精准的目标、科学的试验设计和规范的评价程序基础上。目前市面上的益生菌产品，有单菌株产品，也有多菌株组合产品。益生菌产品中多菌株的搭配使用是否产生协同增效作用，需要通过科学研究证实，而不是根据组合中单菌株的有益作用进行组合产品有益作用的推导。
目前，市场上绝大多数儿童益生菌产品均通过描述配方中单菌株的有益作用，试图将原料的有益作用与产品的有益功效做等同，而并无法提供人群针对性的、切实的产品有益作用支撑，从而导致市场益生菌法规监管混乱、产品良莠不齐等问题，不仅对消费者造成困扰，也对益生菌行业的良性发展产生负面影响。
因此，提供一种针对目标人群开展规范的临床试验，更真实反映产品对人体的健康作用的益生菌复合剂是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种益生菌复合剂及制备方法与应用。
为了实现上述目的，本发明采用如下技术方案。
一种益生菌复合剂，以重量份数计包括以下组分：低聚果糖10-20份；水苏糖10-20份；果汁粉4-8份；益生菌组合物1-3份。
进一步的，所述益生菌组合物以重量份数计包括以下组分：发酵粘液乳杆菌CECT5716 1-2份；动物双歧杆菌乳亚种HN019 3-5份；短双歧杆菌M-16V 2-4份；鼠李糖乳酪杆菌HN001 2-4份。
进一步的，所述果汁粉为山楂粉、接骨木莓粉、针叶樱桃粉、巴西梅粉、阿萨伊果粉、草莓粉、甜橙粉、葡萄粉、蓝莓粉、红树莓粉、黑醋栗粉中的任意一种。
进一步的，以重量份数计包括以下组分：
低聚果糖：水苏糖：山楂粉：益生菌组合物 =10：10：4：1。
一种益生菌复合剂的制备方法，包括以下步骤：
①按所述重量份数取益生元原料，先取3份低聚果糖用纯化水置于搅拌罐中配置成浓度50%的低聚果糖溶液，再将剩余的低聚果糖、水苏糖、山楂果粉进行混合5min，得混合物料；
②将混合物料置于沸腾制粒机中，将50%低聚果糖溶液从搅拌罐中抽入沸腾制粒机中；控制溶液的雾化及制粒物料的沸腾状态，使其处于最佳状态，当溶液用尽后观察物料状态，粒度、性状、质量符合工艺要求后启动干燥；
③干燥过程控制制粒物料粒度20-60，水分低于3%并且水活度低于0.15后停止加热干燥；
④将制粒物料过筛，取目数在20-60目之间的，不能通过20目的舍弃，通过60目的物料重量占比不得超过物料目数范围在20-60目的95%；
⑤将配方中的益生菌粉进行预混，预混时间5min；
⑥将益生菌粉与等量的制粒物料进行预混，预混时间5min；
⑦将剩余制粒物料与预混物料进行总混，混合时间30min；
⑧将总混好的物料进行内包装，得复合益生菌产品。
一种益生菌复合剂在调节肠道菌群、提高免疫力中的应用。
经由上述的技术方案可知，与现有技术相比，本发明的有益效果为：
本发明可以调节肠道菌群，减少便秘、上呼吸道感染等健康问题发生率，辅助提高儿童抵抗力；
本发明还可提高孩子自护力、促进营养吸收和生长发育。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 益生元的选择
1.取1.6g益生菌组合物（发酵粘液乳杆菌CECT5716 1份；动物双歧杆菌乳亚种HN019 3份；短双歧杆菌M-16V 2份；鼠李糖乳酪杆菌HN001 2份）落至80ml MRS肉汤培养基中培养16小时。
2.酶标仪检测培养后的菌液OD600浓度。
3.按1%比例将益生元添加至无葡萄糖MRS肉汤培养基中。
4.按2%的比例添加培养好的菌液至混合益生元的无葡萄糖MRS肉汤培养基中。
5.其中不添加益生元的无葡萄糖MRS肉汤培养基为阴性对照，添加葡萄糖的MRS肉汤培养基为阳性对照。
6.无葡萄糖MRS肉汤培养基及添加益生元且不添加菌液为空白对照。
7.将不同益生元的混合液及对照组添加至96孔板中培养48小时，每间隔2小时用酶标仪检测OD600浓度，重复平行3次。
8.将培养48小时所得的OD600值，按下列公式计算菌株在不同益生元生长的OD600值。
菌株生长OD600 = W2-W0-W1
其中：W1为阴性对照组OD600值，W0为空白对照组OD600值，W2为培养48小时后OD600值。
表1：菌株生长OD600
根据实验结果，益生元选择低聚果糖和水苏糖。
实施例2 益生菌复合剂口感优化
S1：
配方益生菌组合物1份（其中以重量份数计CECT5716 1份；动物双歧杆菌乳亚种HN019 3份；短双歧杆菌M-16V 2份；鼠李糖乳酪杆菌HN001 2份），水苏糖10份，低聚果糖10份，山楂果粉4份。
益生菌复合剂的制备方法包括以下步骤：
①取3份低聚果糖用纯化水置于搅拌罐中配置成浓度50%的溶液，再将剩余的低聚果糖、水苏糖、山楂果粉进行混合5min；
②将混合物料置于沸腾制粒机中，将50%低聚果糖溶液从搅拌罐中抽入沸腾制粒机中；控制溶液的雾化及制粒物料的沸腾状态，使其处于最佳状态，当溶液用尽后观察物料状态，粒度、性状、质量符合工艺要求后启动干燥；
③干燥过程控制制粒物料粒度20-60，水分低于3%并且水活度低于0.15后停止加热干燥；
④将制粒物料过筛，取目数在20-60目之间的，不能通过20目的舍弃，通过60目的物料重量占比不得超过物料目数范围在20-60目的95%；
⑤将配方中的益生菌粉进行预混，预混时间5min；
⑥将益生菌粉与等量的制粒物料进行预混，预混时间5min；
⑦将剩余制粒物料与预混物料进行总混，混合时间30min；
⑧将总混好的物料进行内包装，得益生菌复合剂产品。
S2：
配方益生菌组合物1份（菌种比例同S1），水苏糖10份，低聚果糖10份，山楂果粉4份。
益生菌复合剂的制备方法包括以下步骤：
①将配方中的益生菌粉进行预混，预混时间5min；
②将益生菌粉与等量的低聚果糖进行预混，预混时间5min；
③将剩余制粒物料与预混物料进行总混，混合时间30min；
④将总混好的物料进行内包装，得益生菌复合剂产品。
S3：
配方益生菌组合物1份（菌种比例同S1），水苏糖8份，低聚果糖15份，山楂果粉4份。
益生菌复合剂的制备方法包括以下步骤：
①取10份低聚果糖用纯化水置于搅拌罐中配置成浓度50%的溶液，再将剩余的低聚果糖、水苏糖、山楂果粉进行混合5min；
②将混合物料置于沸腾制粒机中，将50%低聚果糖溶液从搅拌罐中抽入沸腾制粒机中；控制溶液的雾化及制粒物料的沸腾状态，使其处于最佳状态，当溶液用尽后观察物料状态，粒度、性状、质量符合工艺要求后启动干燥；
③干燥过程控制制粒物料水分低于3%并且水活度低于0.15后停止加热干燥；
④将配方中的益生菌粉进行预混，预混时间5min；
⑤将益生菌粉与等量的制粒物料进行预混，预混时间5min；
⑥将剩余制粒物料与预混物料进行总混，混合时间30min；
⑦将总混好的物料进行内包装，得益生菌复合剂产品。
S4：
配方益生菌组合物1份（菌种比例同S1），水苏糖9份，低聚果糖13份，山楂果粉4份。
益生菌复合剂的制备方法包括以下步骤：
①取10份低聚果糖用纯化水置于搅拌罐中配置成浓度50%的溶液，再将剩余的低聚果糖、水苏糖、山楂果粉进行混合5min；
②将混合物料置于沸腾制粒机中，将50%低聚果糖溶液从搅拌罐中抽入沸腾制粒机中；控制溶液的雾化及制粒物料的沸腾状态，使其处于最佳状态，当溶液用尽后观察物料状态，粒度、性状、质量符合工艺要求后启动干燥；
③干燥过程控制制粒物料粒度20-60，水分低于4%后停止加热干燥；
④将制粒物料过筛，取目数在20-60目之间的，不能通过20目的舍弃，通过60目的物料重量占比不得超过物料目数范围在20-60目的95%；
⑤将配方中的益生菌粉进行预混，预混时间5min；
⑥将益生菌粉与等量的制粒物料进行预混，预混时间5min；
⑦将剩余制粒物料与预混物料进行总混，混合时间30min；
⑧将总混好的物料进行内包装，得益生菌复合剂产品。
分别取S1-S4的益生菌复合剂分装2g，将样品直接置于舌面，开启秒表计时，样品遇唾液迅速溶解，借吞咽动力，直至样品完全溶解化开，停止计时，并记录溶解时间，同时对直接服用过程的感官评价进行记录，具体结果如表2所示。数据表明本发明实施例的粉剂益生菌口腔速溶性好。
表2：口腔溶解时间
分别取S1-S4的益生菌复合剂，检测S1中乳酸菌的初始含量为1.2×1010CFU/g，S2-S4中乳酸菌初始含量分别为1.1×1010CFU/g、1.1×1010CFU/g、1.0×1010CFU/g，在高温条件为：30℃，75%RH环境下，模拟益生菌恶劣储存条件，测试3个月，按照GB4789.35-2016测试每克粉剂益生菌的乳酸菌菌数，计算菌的存活率（存活率%=存活菌数/初始含量总数*100%），结果如表3所示。
表3：各组粉剂益生菌在高温储存条件下的菌株存活情况
实施例3 益生菌复合剂对学龄前儿童肠道菌群和免疫功能的调节作用
1.试验目的
（1）评估益生菌复合剂对学龄前儿童上呼吸道感染的预防及改善作用；
（2）评估益生菌复合剂对学龄前儿童肠道免疫功能及肠道微生态的影响；
（3）评估益生菌复合剂对学龄前儿童粪便布里斯托评分的影响；
（4）评估益生菌复合剂对学龄前儿童睡眠的影响。
2.试验设计及食用方法：本次试验为单中心、双盲、随机、平行对照试验，采用自身和组间两种对照设计，评估140名符合入组标准的学龄前儿童经过洗脱期后，每日餐后食用益生菌复合剂（2×1010CFU/天）或安慰剂，持续24周，对上呼吸道感染的相关影响。
3.样本采集及观察指标
（1）记录及量表指标：在食用产品前后，对以下信息进行记录和计算：上呼吸道感染（定义为病毒性呼吸道感染、感冒、喉炎、咳嗽不伴随其他相关症状或由上呼吸道感染引起的肺炎）发生率、症状持续天数、病假天数、出勤记录、就医治疗情况、住院天数、用药记录、粪便布里斯托评分、匹兹堡睡眠质量指数。其中粪便布里斯托评分根据布里斯托大便分类法进行分类打分，匹兹堡睡眠质量指数按美国匹兹堡大学1988年编制的匹兹堡睡眠质量指数量表条目项目进行评分；
（2）实验室检测指标：在食用产品前后，对以下指标进行留样检测：毛发微量元素检测、粪便检测、肠道菌群16sRNA分析、唾液sIgA检测。
4.试验结果
（1）益生菌复合剂对学龄前儿童上呼吸道感染的预防及改善作用
对比产品组和对照组志愿者上呼吸道感染整体发病率发现，24周试验期间，产品组与对照组分别有24人和39人发生上呼吸道感染，产品组相较于对照组的风险优势比为0.42（95%置信区间0.21,0.82），表明产品组发生上呼吸道感染的风险比对照组低58%，组间差异显著（p=0.012），结果如表4所示。
表4：试验期间上呼吸道感染发生率
注：描述性统计量表示为频数（百分比），组间差异比较采用逻辑回归模型，差异表示为优势比（95%置信区间），优势比Odds Ratio<1表明事件发生风险低于对照组。*： p<0.05。
对比产品组和对照组志愿者上呼吸道感染相关次要指标发现（表5），24周试验期间，产品组24人均发生1次感染，对照组有3人发生2次感染，其余36人发生1次感染。产品组因上呼吸道感染接受抗生素治疗的人数占总人数的比例显著低于对照组（p=0.021），无人因此住院，而对照组有2人因病住院。与对照组相比，产品组上呼吸道感染者的症状持续天数平均减少1.74天、接受抗生素治疗天数平均减少1.2天、病假天数平均减少1.84天，组间差异显著。
表5：上呼吸道感染相关次要指标
注：描述性统计量表示为均值± 标准差，组间差异比较采用单因子方差分析。*：p<0.05。a描述性统计量表示为中位数（第一四分位数，第三四分位数），组间差异比较采用Kruskal Wallis检验。
（2）益生菌复合剂对学龄前儿童肠道免疫功能及肠道微生态的影响
短链脂肪酸，是肠道细菌代谢膳食纤维、淀粉、蛋白质等物质而产生的主要代谢产物，对维持肠道正常生理功能有重要意义。而钙卫蛋白，是一种钙和锌结合的胞质蛋白，由肠道中性粒细胞释放，主要与抗感染、免疫调节有关，粪便中钙卫蛋白的水平降低，可视为肠道炎症状态改善。同时，sIgA作为重要的免疫分子，在肠道黏膜免疫中起到关键作用，其含量与黏膜免疫功能正相关。
在不同干预期，通过粪便短链脂肪酸含量、钙卫蛋白水平、sIgA水平，对比产品组和对照组志愿者肠道免疫功能发现，干预后12周及24周，产品组志愿者粪便中总短链脂肪酸含量较基线明显升高，且显著高于对照组；粪便钙卫蛋白含量比基线明显降低，显著低于对照组（p <0.0001）；粪便sIgA含量比基线明显升高，且显著高于对照组（p <0.0001），结果如表6所示。由此，说明益生菌复合剂有助于维持学龄前儿童肠道正常生理功能，对肠道黏膜免疫功能有一定的增强作用。
表6：粪便检测结果
注：描述性统计量表示为均值±标准差，组间差异比较采用单因子方差分析。*： p<0.05。
采用16sRNA测序技术，对比产品组志愿者试验前后粪便肠道菌群中各菌属相对丰度变化情况，结果如表7所示。24周试验结束后，双歧杆菌属Bifidobacterium、乳杆菌属Lactobacillus、阿克曼氏菌属Akkermansia相对丰度均较基线明显提高，而普雷沃氏菌属Prevotella、拟杆菌属Bacteroides相对丰度则较基线明显降低，说明益生菌复合剂能够调节学龄前儿童肠道菌群，提高有益菌相对丰度、下调条件致病菌相对丰度。
表7：产品组志愿者肠道菌群菌属相对丰度变化
（3）益生菌复合剂对学龄前儿童粪便布里斯托评分的影响
基线时两组志愿者的三日平均排便次数、粪便性状及颜色无显著差异（表8）。干预后12周及24周，产品组的平均每天排便次数比基线显著增加（p=0.004），显著高于对照组（p<0.0001）；粪便含水量显著提升，粪便性状平均评分由肠状、有干裂变得较柔软光滑（p<0.0001），粪便颜色显著变浅（p=0.001，p=0.0004），显著优于对照组（p<0.0001）。干预结束后1周（第25周），产品组的排便情况各指标仍显著优于自身基线和对照组。而对照组的排便情况各指标在试验期间均无明显变化，说明益生菌复合剂能够辅助学龄前儿童每天规律排便，软化干结粪便。
表8：布里斯托排便表（三日平均排便频率、粪便性状及颜色）
注：除特别注释外，描述性统计量表示为均值±标准差，组间差异比较采用方差分析。a描述性统计量表示为中位数（第一四分位数，第三四分位数），组间差异比较采用Kruskal Wallis检验。
（4）益生菌复合剂对学龄前儿童睡眠的影响
对比产品组和对照组志愿者睡眠情况得知，两组志愿者的睡眠情况在基线无显著差异（表9）。试验第12周，产品组的睡眠质量得分（p=0.003）、睡眠障碍得分（p=0.013）和匹兹堡睡眠指数总分（p=0.0001）比基线显著下降，表明睡眠情况有显著改善，其中睡眠障碍和匹兹堡睡眠指数总分显著优于对照组（p=0.004）。24周试验期后，以上指标进一步改善，均显著优于对照组（p<0.0001）；此外，入睡时间得分（p=0.0002）和日间功能障碍得分（p<0.0001）也比基线显著下降，表明入睡困难（p=0.005）及时间日间功能障碍（p<0.0001）显著改善。对照组的睡眠指数评分在试验期间无显著变化。
表9：匹兹堡睡眠质量指数
注：除特别注释外，描述性统计量表示为均值±标准差，组间差异比较采用单因子方差分析。*： p<0.05。a描述性统计量表示为中位数（第一四分位数，第三四分位数），组间差异比较采用Kruskal Wallis检验。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.一种益生菌复合剂，其特征在于，以重量份数计包括以下组分：低聚果糖10-20份；水苏糖10-20份；果汁粉4-8份；益生菌组合物1-3份；
所述益生菌组合物以重量份数计包括以下组分：发酵粘液乳杆菌CECT5716 1-2份；动物双歧杆菌乳亚种HN019 3-5份；短双歧杆菌M-16V 2-4份；鼠李糖乳酪杆菌HN001 2-4份。
2.根据权利要求1所述的一种益生菌复合剂，其特征在于，所述果汁粉为山楂粉、接骨木莓粉、针叶樱桃粉、巴西梅粉、阿萨伊果粉、草莓粉、甜橙粉、葡萄粉、蓝莓粉、红树莓粉、黑醋栗粉中的任意一种。
3.根据权利要求2所述的一种益生菌复合剂，其特征在于，以重量份数计包括以下组分：
低聚果糖：水苏糖：山楂粉：益生菌组合物 =10：10：4：1。
4.权利要求1所述一种益生菌复合剂的制备方法，其特征在于，包括以下步骤：
①按所述重量份数取益生元原料，先取3份低聚果糖用纯化水置于搅拌罐中配置成浓度50%的低聚果糖溶液，再将剩余的低聚果糖、水苏糖、山楂果粉进行混合5min，得混合物料；
②将混合物料置于沸腾制粒机中，将50%低聚果糖溶液从搅拌罐中抽入沸腾制粒机中；控制溶液的雾化及制粒物料的沸腾状态，使其处于最佳状态，当溶液用尽后观察物料状态，粒度、性状、质量符合工艺要求后启动干燥；
③干燥过程控制制粒物料粒度20-60，水分低于3%并且水活度低于0.15后停止加热干燥；
④将制粒物料过筛，取目数在20-60目之间的，不能通过20目的舍弃，通过60目的物料重量占比不得超过物料目数范围在20-60目的95%；
⑤将配方中的益生菌粉进行预混，预混时间5min；
⑥将益生菌粉与等量的制粒物料进行预混，预混时间5min；
⑦将剩余制粒物料与预混物料进行总混，混合时间30min；
⑧将总混好的物料进行内包装，得复合益生菌产品。
5.一种益生菌复合剂在调节肠道菌群、提高免疫力中的应用。
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