CN110279118A - 一种复合益生菌组合物、复合益生菌冻干粉胶囊及制备方法 - Google Patents
一种复合益生菌组合物、复合益生菌冻干粉胶囊及制备方法 Download PDFInfo
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- CN110279118A CN110279118A CN201910516435.4A CN201910516435A CN110279118A CN 110279118 A CN110279118 A CN 110279118A CN 201910516435 A CN201910516435 A CN 201910516435A CN 110279118 A CN110279118 A CN 110279118A
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- Prior art keywords
- compound probiotic
- freeze
- dried powder
- bacterium
- lactobacillus
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Classifications
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- A—HUMAN NECESSITIES
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种复合益生菌组合物、复合益生菌冻干粉胶囊及制备方法。一种复合益生菌组合物,包括鼠李糖乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种菌和干酪乳杆菌,以单菌培养至稳定期的菌液,按体积比50~60∶25~35∶1∶1~2混合。相比于市面上的益生菌冻干粉制品,本发明复合益生菌组合物配方通过研究获得了特殊的菌种数量配比,使得菌体在肠道定植的数量达到最佳化。制备成肠溶复合益生菌冻干粉胶囊后,可用于补充人体所需的益生菌,平衡肠道菌群平衡,有助于预防和治疗某些源自细菌、病毒、真菌和原生动物的疾病,还对人体起到去除有害物质,清除体内毒素和净化体内环境等作用。
Description
技术领域
本发明涉及益生菌食品加工技术领域,特别是涉及一种复合益生菌组合物、复合益生菌冻干粉胶囊及制备方法。
背景技术
益生菌的最大特点是活菌,世卫组织提出的官方最低活菌数需达到106~107 cfu/g,因此对于益生菌这种干燥热敏性制品和需要保持生物活性的物质。冷冻干燥技术对生物组织和细胞结构和特征的损伤较小,使其快速进入休眠状态,有效保护了益生菌这类许多热敏性制品有效成份的稳定性。
益生菌能够调节机体胃肠道正常菌群、保持微生态平衡,提高食物消化率和生物价,降低血清胆固醇,控制内毒素,抑制肠道内腐败菌生长繁殖和腐败产物的产生,制造营养物质,刺激组织发育,从而对机体的营养状态、生理功能、细胞感染、药物效应、毒性反应、免疫反应、肿瘤发生、衰老过程和突然的应急反应等产生作用。而其中乳酸菌在体外研究中发现,其依靠细胞的应急和pH环境条件可以吸收基因毒性和致癌原。
比如,公开号为CN109432136A的中国发明专利公开了一种益生菌组合制剂及其制备方法和应用。其由下述重量份的原料制得:鼠李糖乳杆菌5-15份,副凯氏乳酸杆菌8-16份,植物乳杆菌2-7份,发酵乳酸杆菌7-15份,瑞士乳杆菌1-8份,发酵乳杆菌10-15份,干酪乳杆菌8-17份,乳酸乳球菌12-16份,唾液乳酸杆菌3-8份,龙根菌7-11份,LA-5嗜酸乳杆菌3-15份,BB-12雷特氏B 菌7-15份,硅酸钙5-8份,甘油4-10份,阿拉伯胶4-8份,壳聚糖1-5份。
公开号为CN106820156A的中国发明专利公开了一种包含乳铁蛋白、菊粉、菠萝蛋白酶、白萝卜提取物的综合益生菌产品,该产品中富含嗜酸乳杆菌、婴儿双歧杆菌、长双歧杆菌、短双歧杆菌、两歧双歧杆菌、鼠李糖乳杆菌、保加利亚乳杆菌、植物乳杆菌、干酪乳杆菌、副干酪乳杆菌、嗜热链球菌等肠道益生菌。
然而,上述现有技术中,配方中虽然还有多种益生菌,但益生菌相互之间协同性较差,食用效果不佳。
发明内容
本发明要解决的技术问题是针对现有菌制品食用效果不佳,开发出一种复合益生菌组合物,并制备成复合益生菌冻干粉胶囊。
一种复合益生菌组合物,包括鼠李糖乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种菌和干酪乳杆菌,以单菌培养至稳定期的菌液,按体积比50~60∶ 25~35∶1∶1~2混合。优选的,鼠李糖乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种菌和干酪乳杆菌,以单菌培养至稳定期的菌液,按体积比54∶27∶1∶4/3 混合。
本发明中,所述的益生菌菌种包括鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(Lacto bacterium acidophilus)、德式乳杆菌保加利亚亚种菌(Lactobacillusbulgaricus)、干酪乳杆菌(Lactobacillus paracasei)等。
本发明又提供了一种复合益生菌冻干粉胶囊,包含所述的复合益生菌组合物,益生菌的总浓度为益生菌的总浓度为1.0×108~1.0×109cfu/g。
优选的,所述的复合益生菌冻干粉胶囊,使用肠溶胶囊密封包装。这样可以保证益生菌安全通过胃,到达肠道处再被释放出来。更优选的,使用脱脂奶粉作为保护剂。进一步优选的,所述复合益生菌冻干粉胶囊的制备方法,包括以下步骤:
(1)培养、混合益生菌;
(2)分离获得湿菌体;
(3)将湿菌体与脱脂奶粉混合并加入无菌水制得菌液,菌液中脱脂奶粉的质量浓度为8.0%~12.0%;
(4)将菌液真空冷冻干燥获得所述复合益生菌冻干粉;
(5)包装后获得所述复合益生菌冻干粉胶囊。
其中,所述真空冷冻干燥方法为:0~8℃环境冷藏30min,-25~-15℃1h, -85~-75℃1h,分步冷却后,真空冷冻干燥14h。
优选的,使用肠溶胶囊密封包装所述复合益生菌冻干粉。
采用真空冷冻干燥技术对生物组织和细胞结构和特征的损伤较小,使其快速进入休眠状态,有效保护了益生菌这类许多热敏性制品有效成份的稳定性。
本发明还提供了所述复合益生菌冻干粉胶囊在作为调节肠道功能的保健食品中的应用。
本发明的优点和有益效果:
相比于市面上的益生菌冻干粉制品,本发明复合益生菌组合物配方通过研究获得了特殊的菌种数量配比,使得菌体在肠道定植的数量达到最佳化。制备成肠溶复合益生菌冻干粉胶囊后,可用于补充人体所需的益生菌,平衡肠道菌群平衡,有助于预防和治疗某些源自细菌、病毒、真菌和原生动物的疾病,还对人体起到去除有害物质,清除体内毒素和净化体内环境等作用。本发明中所含益生菌的配方能够促成其之间正相互作用,在保证其发挥各自保健效果的同时增大了单独培养的益生菌活菌数,体现了该配方的独特优越性。
附图说明
图1为单菌最大稳定浓度检测结果图。
图2为不同混合配方的菌体培养结果图,其中菌种A:鼠李糖乳杆菌,菌种B:嗜酸乳杆菌,菌种C:德式乳杆菌保加利亚亚种,菌种D:干酪乳杆菌。
图3为不同甘油浓度下菌体存活率检测结果图。
图4为不同麦芽糊精浓度下菌体存活率检测结果图。
图5为不同脱脂奶粉浓度下菌体存活率检测结果图。
图6为冻干粉在人工肠液中的剩余活菌数检测结果图。
具体实施方式
微生物培养基所用为GYP培养基。
各菌株均来源于中国普通微生物菌种保藏管理中心(CGMCC)。
实施例1
1、实验原料
(1)菌种:嗜酸乳杆菌、干酪乳杆菌、德式乳杆菌保加利亚亚种菌、鼠李糖乳杆菌;
(2)培养基:GYP;
(3)冷冻干燥保护剂:脱脂奶粉。
2、实验方法
(1)配制GYP液体培养基,在121℃高压灭菌锅中灭菌20min。分别从4℃保存的平板培养基中将实验原料中提到的四种菌株的单菌落转接至5mL GYP 液体培养基37℃,静置培养24h,活化菌种;
(2)将种子培养液以1∶50的比例转接至100mL GYP液体培养基,37℃扩大培养24h,混匀后取样稀释涂平板,37℃培养48h,观察并统计菌数;
(3)通过单菌菌液浓度计算混合培养加入菌液的需求量,从单菌培养24h 的菌液中取样,按一定比例菌液混合,保证混合菌液中各种菌的初始菌浓度相同,并且保证总接种量为250μL,转入5mL GYP液体培养基,37℃培养24h;
(4)对混合培养的菌液进行稀释涂平板,测定菌数,从而分析四种菌株之间的相互作用。
3、实验结果
(1)4种单菌在5ml GYP液体培养基里培养24h后的稳定浓度如图1;
(2)为确保菌体种类的丰富性,将候选菌种中三种菌株菌数等比例搭配与四种菌的等比例进行测试比较,四种搭配设置如表1所示,其中A表示鼠李糖乳杆菌,B表示嗜酸乳杆菌,C表示德式乳杆菌保加利亚亚种菌,D表示干酪乳杆菌。将单菌培养至稳定期的菌液混合,保证每种配方混菌中各种菌株浓度相同。结果如图2所示。混合培养的菌液中BCD搭配的菌液菌体浓度最高,达 3.2×108CFU/mL左右,而ABCD搭配的菌液菌体浓度较初始体系增幅最大,达到了5.4倍左右,表现出了最强的正相互作用,最有利于混合菌体的生长。ABCD 菌数等比例混合的配方既满足了菌株的多样性又最能表现最强的菌活性,故作为本次试验最优结果。
表1四种配方的设置方案
实施例2
1、实验原料
(1)菌种:嗜酸乳杆菌、干酪乳杆菌、德式乳杆菌保加利亚亚种菌、鼠李糖乳杆菌;
(2)培养基:GYP;
(3)冷冻干燥保护剂:脱脂奶粉、甘油、麦芽糊精。
2、实验方法
(1)配制GYP液体培养基,在121℃高压灭菌锅中灭菌20min。按实施例 1的方法,培养并制备其中所述最优配方混菌菌液。
(2)将混菌菌液按2%比例转接至5mL GYP液体培养基37℃,静置培养 24h,活化菌种;
(3)将种子培养液以1∶50的比例转接至100mL GYP液体培养基,37℃扩大培养24h;
(4)将培养液部分以稀释涂平板的方式用于测定菌体数量,余下的部分进行冷冻干燥;
(5)将菌液充分并适当离心,取出湿菌体加入脱脂奶粉(高温水浴以杀菌并冷却),配制成液体。脱脂奶粉浓度分别为4%,6%,8%,10%,12%(质量分数);
(6)置于0~8℃环境冷藏30min,-25~-15℃1h,-85~-75℃1h,分步冷却后将固态混合物平铺于冷冻干燥机中处理14h,制得冻干粉;
(7)将冻干粉稀释涂平板测定菌体数量,与原菌液菌体数量进行比较,得出冷冻干燥存活率,得出最优脱脂奶粉浓度;
(8)重复步骤(5),将冷冻干燥保护剂分别换成不同浓度的甘油、麦芽糊精,得出保护效果最好的浓度,并进行分析比较。
3、实验结果
(1)当甘油添加量超过4%时,菌悬液水分含量较高,粘度较大,难以干燥为粉末状的微胶囊,而是粘稠状,影响细胞的存活。因此,如图3,甘油添加量4%时,微胶囊菌剂的活菌率为34.98%,达到最大值。在本实验中,甘油确实能起到保护的作用,但冻干后整体呈现的颜色,状态其冷冻干燥后菌体的外观与要求不符,因此不选用甘油作保护剂材料;
(2)如图4,在麦芽糊精添加量为14%时,其对酵母菌的保护效果差异显著(p<0.05),活菌率达到最大值,存活率约31.8%,若继续提高浓度,其产品活菌率下降。所以,选取14%为麦芽糊精的较优添加量;
(3)如图5,在脱脂奶粉添加量为10%时,其对酵母菌的保护效果已经达到最大值,存活率约33.5%,菌剂活菌率显著(p<0.05)高于其余各添加量,所以选择10%为脱脂奶粉较优添加量。
通过比较,脱脂奶粉的保护效果优于麦芽糊精,甘油不符合产品要求,故最终选用8.0%~12.0%(质量分数)的脱脂奶粉作为冷冻干燥保护剂。
实施例3
1、实验原料
(1)肠溶胶囊;
(2)人工胃液(pH=2.0):取稀盐酸16.4mL,胃蛋白酶10g,加水稀释成 1000mL。
2、实验方法
(1)配制足量的人工胃液,随机取肠溶胶囊使其充分浸没于人工胃液中;
(2)定时观察记录肠溶胶囊的完整性,检定其是否能有效抵御胃液强酸的破坏。
3、实验结果
在处理30min后,肠溶胶囊的结构外观仍具完整性,没有明显的变化。在 30min内可保证胶囊已从胃进入肠道,从而保证了菌粉能顺利进入肠道,不受胃液的破坏。
实施例4
1、实验原料
(1)菌种:嗜酸乳杆菌、干酪乳杆菌、德式乳杆菌保加利亚亚种菌、鼠李糖乳杆菌;
(2)培养基:GYP;
(3)冷冻干燥保护剂:脱脂奶粉;
(4)人工肠液(pH=6.8):取磷酸二氢钾6.8g,加水500mL使溶解,并调节pH值为6.8;另取胰酶10g,加水溶解;将两液混合后,加水至1000mL。
2、实验方法
(1)取部分冻干粉进行菌体数目的测定:用减量法分别称取0.5g的冻干粉 10ml人工肠液中,在37℃分别培养0.5h、1h、1.5h;
(2)将处理后的菌液转接至固体平板37℃培养48h,测定活菌数。
3、实验结果
将冻干粉放入人工肠液中,测定经不同时间后剩余活菌数,结果见图6。由此可知,在人工肠液中冻干粉内活菌数随时间不断减少。在前1.5h保持下降的趋势,但仍能在1.5h时保持较高的存活数,而存活率较低,为2.04%,但总活菌数为7.34×106cfu/g。
Claims (9)
1.一种复合益生菌组合物,其特征在于,包括鼠李糖乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种菌和干酪乳杆菌,以单菌培养至稳定期的菌液,按体积比50~60∶25~35∶1∶1~2混合。
2.如权利要求1所述的复合益生菌组合物,其特征在于,鼠李糖乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种菌和干酪乳杆菌,以单菌培养至稳定期的菌液,按体积比54∶27∶1∶4/3混合。
3.一种复合益生菌冻干粉胶囊,其特征在于,包含如权利要求1所述的复合益生菌组合物,益生菌的总浓度为1.0×108~1.0×109cfu/g。
4.如权利要求3所述的复合益生菌冻干粉胶囊,其特征在于,使用肠溶胶囊密封包装。
5.如权利要求3所述的复合益生菌冻干粉胶囊,其特征在于,使用脱脂奶粉作为保护剂。
6.如权利要求5所述复合益生菌冻干粉胶囊的制备方法,其特征在于,包括以下步骤:
(1)培养、混合益生菌;
(2)分离获得湿菌体;
(3)将湿菌体与脱脂奶粉混合并加入无菌水制得菌液,菌液中脱脂奶粉的质量浓度为8.0%~12.0%;
(4)将菌液真空冷冻干燥获得所述复合益生菌冻干粉;
(5)包装后获得所述复合益生菌冻干粉胶囊。
7.如权利要求6所述的制备方法,其特征在于,所述真空冷冻干燥方法为:0~8℃环境冷藏30min,-25~-15℃1h,-85~-75℃1h,分步冷却后,真空冷冻干燥14h。
8.如权利要求6所述的制备方法,其特征在于,使用肠溶胶囊密封包装所述复合益生菌冻干粉。
9.如权利要求3~5任一所述复合益生菌冻干粉胶囊在作为调节肠道功能的保健食品中的应用。
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