CN104450562A - 一种产胆盐水解酶副干酪乳杆菌大枣益生菌片的制备方法 - Google Patents
一种产胆盐水解酶副干酪乳杆菌大枣益生菌片的制备方法 Download PDFInfo
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Abstract
本发明涉及一种产胆盐水解酶并具有降低血清胆固醇功效的大枣益生菌片的制备方法,适用于功能性益生乳酸菌活菌制剂等微生态制剂的生产。本发明大枣益生菌片的配方是由副干酪乳杆菌KL1-Liu益生菌菌粉与大枣粉、低聚果糖、木糖醇、蔗糖、柠檬酸、微晶纤维素、麦芽糊精等辅料组成;本发明利用产胆盐水解酶的副干酪乳杆菌(Lactobacillus paracasei)KL1-Liu CGMCC No.7029,经活化、扩大培养、高密度发酵、离心浓缩、加入冻干保护剂、预冻和冷冻干燥等工序制备益生菌菌粉;将经过粉碎、过筛的辅料与益生菌菌粉混合后,压制成大枣益生菌片。本发明制作的大枣益生菌片具有良好的贮藏稳定性,4℃冷藏活菌保质期至少1年以上,即可发挥产品降低血清胆固醇的保健功效,又可发挥大枣药食同源的营养与保健功效。本发明制作的大枣益生菌片原料来源方便,工艺简单,制作周期短,操作简单,适于工业化生产。
Description
技术领域
本发明涉及一种产胆盐水解酶并具有降低血清胆固醇功效的大枣益生菌片的制备方法,适用于功能性益生乳酸菌活菌制剂(片剂型、粉剂型)等微生态制剂的生产。
背景技术
本发明涉及的副干酪乳杆菌(Lactobacillus paracasei)KL1-Liu是从藏灵菇中分离筛选得到的一种产胆盐水解酶并具有降低血清胆固醇作用及调节肠道菌群平衡等功效的益生乳酸菌。
益生菌(Probiotics)是指一类通过肠道定殖作用改善宿主特定部位微生态平衡并兼有若干其他有益生理功能的微生物。它们通过改善肠道微生物生态平衡,控制肠道感染,降低血清胆固醇水平,提高乳糖不耐症人对乳糖的利用,并能刺激特异性或非特异性免疫反应,具有免疫增强的作用。益生菌制品的活菌应该代谢稳定,活性较强,通过消化道后大量存活,进入肠道后发挥益生菌作用。目前用于生产益生菌类产品的优良菌种有双歧杆菌属、乳杆菌属、乳球菌属中的乳酸乳球菌乳酸亚种等。
大枣作为药食同源应用已有2000多年的历史,它具有补虚益气,养血安神,健脾和胃等功效,主要用于中气不足、脾胃虚弱、体倦乏力、食少便溏、血虚萎黄等病症的治疗。近年来药理研究发现,大枣中含有多种生物活性物质,如大枣多糖、黄酮类、皂苷类、三萜类、生物碱类、环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)、三楂酸、维生素C、维生素P、B族维生素等,对人体有多种保健防病功效。大枣中含有的多糖可作为免疫促进剂,具有控制细胞分裂和分化、调节细胞的生长与衰老,以及具有清除人体内氧自由基的作用;大枣中含有的皂苷类、三萜类化合物具有抗肿瘤的生物活性;其含有的山楂酸亦具有抗癌作用;大枣中含有的cAMP具有改善人体微循环,扩张冠状动脉,增加脑和心脏的供血量,减慢心律,降低心肌耗氧量而改善缺血心肌的代谢,故可防治心脑血管疾病;此外,cAMP还可调节细胞的分裂增殖,有利于肿瘤细胞向正常细胞方向转化,故有抗癌作用;大枣中富含的维生素C具有对抗自由基的侵袭,抗氧化、抗衰老等作用;维生素P对健全毛细血管、维持血管壁弹性,抗动脉粥样硬化很有益;大枣中含有的B族维生素能保护心脏、减轻疲劳、增强心肌活力、降低血压;大枣中的黄酮类化合物具有镇静、催眠和降压作用。
本发明所用副干酪乳杆菌KL1-Liu是利用选择培养技术从藏灵菇中筛选得到高产胆盐水解酶和胞外多糖的副干酪乳杆菌(Lactobacillus paracasei)KL1-Liu。研究发现,该菌株具有较强耐受胃肠道逆环境(胃酸和胆盐)能力,可以定殖于肠道内发挥益生作用;动物实验研究显示,该菌株产生的胆盐水解酶能够显著降低或调节血清胆固醇水平,其产生的胞外多糖具有显著抑 制人结肠癌细胞的增殖作用;动物实验研究显示,该菌株还具有调节肠道菌群平衡、预防便秘、抗脂质过氧化及护肝等功能。课题组系统研究揭示了该菌株降低血清胆固醇的机理是缘于副干酪乳杆菌KL1-Liu代谢产生大量高活力的胆盐水解酶(Bile Salt Hydrolase,BSH)。BSH是肠道中的双歧杆菌属、乳杆菌属、乳球菌属中的多种乳酸细菌在生长过程中产生的代谢产物。大量实验证明,胆盐水解酶能在肝肠循环中将结合胆盐分解,产生氨基酸与溶解度较低的游离胆盐。后者能与胆固醇结合形成沉淀复合物而排出体外,从而降低血清总胆固醇的含量。同时测定该菌株全长基因组序列,为副干酪乳杆菌KL1-Liu菌株应用奠定理论基础。
益生菌对于人体的保健功效早已被世界所公认,益生菌产品已经成为保健食品的首选。该产品既具有丰富的营养价值,又具有特殊的生理功能,集营养与保健于一身。益生菌片产品是干燥制品的典型形式之一,具有可直接咀嚼或吞服、食用携带方便、溶出和吸收速度较快等特点。
近年来随着现代科技水平和食品加工技术的发展,国内外相继出现了一些关于益生菌类产品的报道,例如陕西科技大学申请的“一种益生菌片的制备方法”,专利申请号为:CN101843337 A;陕西农产品加工技术研究院申请的“一种益生菌羊奶片及其制备方法”,专利申请号为:CN102742659 A;王世义申请的“复合活性益生菌奶片”,专利申请号为:CN102450331A。刘秀河等人研制的益生菌AB奶片的原始活菌数能达到109CFU/g;周海军和樊兆武研制的益生菌奶片初始活菌数均只有106CFU/g左右。虽然上述专利和文献有关于利用益生菌制备片剂型食品的报道,但是关于利用分离筛选自藏灵菇中的产胆盐水解酶并具有降低血清胆固醇作用的副干酪乳杆菌KL1-Liu,以及利用大枣与益生菌相结合制备大枣益生菌片的方法尚未见国内外相关文献报道和专利报道。
发明内容
本发明的目的是提供一种利用产胆盐水解酶并具有降低血清胆固醇功效的副干酪乳杆菌KL1-Liu制备大枣益生菌片的方法。
本发明是通过以下技术方案实现的:
本发明的大枣益生菌片配方组成(W/W,质量分数):副干酪乳杆菌KL1-Liu益生菌菌粉0.1%~1.0%[活菌数为(1.0~5.0)×1011CFU/g],大枣粉40%~50%,低聚果糖10%~20%,木糖醇4%~10%,蔗糖0.25%~5%,柠檬酸0.25%~3%,硬脂酸镁0.5%~5.5%,微晶纤维素10%~20%,麦芽糊精25%~30%。
所述方法中,副干酪乳杆菌(Lactobacillus paracasei)KL1-Liu分离筛选自吉林、呼和浩特市普通家庭的藏灵菇(又称开菲尔粒),并已于2012年12月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏号为:CGMCCNo.7029(菌株KL1-Liu)。在含有碳酸钙和纳他霉素的MRS选择性培养基上,副干酪乳杆菌(Lactobacillus paracasei)KL1-Liu菌株的菌落大小2~3mm,菌落 有较高黏性,表面粗糙如雪花状,边缘不规则,无光泽,扁平,灰白色,半透明;个体形态呈杆状,长短不等,短链状排列,G+无芽孢杆菌。
所述方法中,副干酪乳杆菌KL1-Liu益生菌菌粉为副干酪乳杆菌KL1-Liu活菌体与复合冻干保护剂的混合物。
所述方法中,副干酪乳杆菌KL1-Liu益生菌菌粉的制备步骤如下:
(1)采用立式高速冷冻离心机将经过扩大培养与高密度发酵的副干酪乳杆菌KL1-Liu发酵液于4℃条件下,6000r/min离心10~20min,弃上清液,收集得到菌泥。
(2)在灭菌冻干瓶中,按离心前发酵液1/10体积的量,加入10.5%~20.5%麦芽糊精+11.5%~1.5%脱脂奶粉,与收集的菌泥混合均匀后,得到浓缩发酵剂。
上述方法中,冻干保护剂组合配方为(W/V,质量体积比浓度):10.5%~20.5%麦芽糊精+11.5%~1.5%脱脂奶粉(伊利牌高蛋白高钙脱脂奶粉)。
(3)将浓缩发酵剂于-40℃预冻10h至完全冻结状态,得到预冻发酵剂;利用美国6L LABCONCO真空冷冻干燥机将预冻发酵剂于-55℃、真空度0.14mBar的条件下,冻干48h至完全干燥状态,得到副干酪乳杆菌KL1-Liu益生菌菌粉。
所述方法中,副干酪乳杆菌KL1-Liu大枣益生菌片的制备步骤如下:
(1)采用鼓风干燥箱将大枣于70℃烘干72h后自然晾干、去核备用。
上述方法中,大枣由甘肃新远泰生态农业科技有限公司提供。
(2)采用中药粉碎机分别将去核大枣、木糖醇、蔗糖、柠檬酸等辅料进行破碎,过80目筛子备用。
上述方法中,80目筛子由鸿玗筛网有限公司提供;中药粉碎机由江南欧川电机厂提供,型号为Hc-280。
(3)采用三维多向运动混合机将大枣益生菌片配方中的各成分,在转速为10~20r/min,电机功率为0.5KW条件下混合均匀,制成大枣益生菌片粗粉。
上述方法中,三维多向运动混合机由上海天凡制药机械厂提供,型号为HD20。
(4)采用单冲式压片机将大枣益生菌片粗粉在最大压力为18KN,压片直径为15~20mm,每片重1g,硬度4~8N条件下,压制成产胆盐水解酶副干酪乳杆菌KL1-Liu大枣益生菌片。
上述方法中,单冲式压片机由上海天凡制药机械厂提供,型号为DP50。
所述方法中,大枣益生菌片中的主要菌种为藏灵菇源副干酪乳杆菌KL1-Liu益生菌。该菌粉具有良好的降低血清胆固醇功效,并有整肠作用,可在肠道内建立一个有利于健康长寿的微生态平衡系统,促进消化吸收,并有改善便秘作用,可以预防消费者的心血管疾病、胃肠道疾病等病症。
上述得到的一种产胆盐水解酶并具有降低血清胆固醇功效的副干酪乳杆菌KL1-Liu制备大枣益生菌片的方法及其产品,以及专用生产菌株属于本发明保护 范围。
附图说明
产胆盐水解酶副干酪乳杆菌大枣益生菌片制备工艺流程图说明如下:
(1)副干酪乳杆菌KL1-Liu菌株益生菌:分离筛选自吉林、呼和浩特市普通家庭的藏灵菇。(2)菌株活化、扩大培养与高密度发酵:将活化好的副干酪乳杆菌KL1-Liu菌株经改良MRS培养基扩大培养与高密度发酵,得到较高活菌数的发酵液。(3)离心浓缩与添加保护剂:将KL1-Liu菌株发酵液于4℃条件下,6000r/min离心10~20min,弃上清液,收集得到菌泥;在灭菌冻干瓶中,按离心前发酵液1/10体积的量,加入10.5%~20.5%麦芽糊精+11.5%~1.5%脱脂奶粉,与收集的菌泥混合均匀后,得到浓缩发酵剂。(4)预冻与真空冷冻干燥:将浓缩发酵剂于-40℃预冻10h至完全冻结状态,得到预冻发酵剂;利用美国6LLABCONCO真空冷冻干燥机将预冻发酵剂于-55℃、真空度0.14mBar的条件下,冻干48h至完全干燥状态,得到副干酪乳杆菌KL1-Liu益生菌菌粉。(5)添加辅料:大枣粉40%~50%,低聚果糖10%~20%,木糖醇4%~10%,蔗糖0.25%~5%,柠檬酸0.25%~3%,硬脂酸镁0.5%~5.5%,微晶纤维素10%~20%,麦芽糊精25%~30%。(6)大枣干燥与去核:采用鼓风干燥箱将大枣于70℃烘干72h后自然晾干、去核备用。(7)辅料破碎与过筛:采用中药粉碎机分别将去核大枣、蔗糖、柠檬酸、木糖醇等辅料进行破碎,并采用80目的筛子过筛。(8)大枣益生菌片粗粉的制备:采用三维多向运动混合机将益生菌菌粉与辅料混合均匀制成大枣益生菌片的粗粉,混合使用转速为10~20r/min。(9)大枣益生菌片的制备:压片采用单冲式压片机的最大压力为18KN,压片直径为15~20mm,每片重1g,硬度4~8N。(10)大枣益生菌片包装与贮藏:将大枣益生菌片分装于密封袋或塑料瓶中,压膜封口或压盖封口后,于4℃冰箱贮藏,产品活菌保质期为一年。
本发明制作的大枣益生菌片原料来源方便,工艺简单,制作周期短,操作简单,对设备要求较低,成品大枣益生菌片活菌数量明显高于同类产品,适于工业化生产。
具体实施方式
下述实施例对本发明作进一步说明而不限制本发明的保护范围。
下述实施例中的实验方法,如无特别说明,均为常规方法。
下述实施例中的百分含量,如无特别说明,均为质量体积比浓度(W/V)。
实施例1、产胆盐水解酶副干酪乳杆菌KL1-Liu大枣益生菌片的制备方法
(1)副干酪乳杆菌KL1-Liu菌株的活化、扩大培养与高密度发酵
将活化好的副干酪乳杆菌KL1-Liu菌株经改良MRS培养基扩大培养与高密度发酵,得到活菌数为(1.0~5.0)×1010CFU/mL的发酵液。
(2)副干酪乳杆菌KL1-Liu菌株的离心浓缩与冷冻干燥
将发酵液于4℃条件下,6000r/min离心10~20min,弃上清液,收集得到菌泥;在灭菌冻干瓶中,按离心前发酵液1/10体积的量,加入10.5%~20.5%麦芽糊精+11.5%~1.5%脱脂奶粉,与收集的菌泥混合均匀后,得到浓缩发酵剂;将浓缩发酵剂于-40℃预冻10h至完全冻结状态,得到预冻发酵剂;利用美国6LLABCONCO真空冷冻干燥机将预冻发酵剂于-55℃、真空度0.14mBar的条件下,冻干48h至完全干燥状态,得到副干酪乳杆菌KL1-Liu益生菌菌粉。
(3)益生菌菌粉中副干酪乳杆菌KL1-Liu活菌数量的检测
称取25g益生菌菌粉放入含有225mL灭菌生理盐水中,采用拍击式匀质器以8 000~10 000r/min的速度处理1min,充分振荡后,制成10-1的均匀稀释液;再以9mL灭菌生理盐水做10倍递增稀释至10-11;取10-9~10-11的稀释液各1mL置于无菌平皿中,倒入溶化并冷却至46℃的改良MRS培养基约15mL,迅速轻轻旋动平皿,使培养基与菌液充分混匀,每个稀释度3次重复。同时将改良MRS培养基注入加有1mL无菌生理盐水的灭菌平皿内作空白对照。待培养基凝固后,翻转平板,置(36±1)℃温箱中培养(48±2)h,待菌落长出后即可计数。其平板菌落计数结果显示,每1g益生菌菌粉中副干酪乳杆菌KL1-Liu的活菌数量为(1.0~5.0)×1011CFU。
(4)大枣干燥与去核:采用鼓风干燥箱将大枣于70℃烘干72h后自然晾干、去核备用。
(5)辅料破碎与过筛
采用中药粉碎机分别将去核大枣、木糖醇、蔗糖、柠檬酸等辅料进行破碎,过80目筛子备用。目的是使益生菌菌粉与辅料更易混合均匀,压片时颗粒均匀容易成型。
(6)大枣益生菌片粗粉的制备
将大枣粉40%~50%,低聚果糖10%~20%,木糖醇4%~10%,蔗糖0.25%~5%,柠檬酸0.25%~3%,硬脂酸镁0.5%~5.5%,微晶纤维素10%~20%,麦芽糊精25%~30%等辅料与添加量为0.1%~1.0%的副干酪乳杆菌KL1-Liu益生菌菌粉[活菌数为(1.0~5.0)×1011CFU/g],采用转速为10~20r/min,电机功率为0.5KW的三维多向运动混合机充分混合均匀,制成大枣益生菌片粗粉。
(6)大枣益生菌片的制备
采用单冲式压片机将混合均匀的大枣益生菌片粗粉压制成大枣益生菌片,单冲式压片机最大压片压力为18KN,压片直径为15~20mm,每片重1g,硬度4~8N。
(7)大枣益生菌片的感官评价
评定小组由专业评价人员构成(即由受过专业感官评价培训的人员组成),共有15人,采用描述性的检测方法分别对样品的质地、口感、风味、外观、色泽、进行细致的描述,评分标准被分为三档:好、中、差三个档次,以感官评 价的结果作为大枣益生菌片配方正交试验的评定标准。大枣益生菌片的感官评定标准及评价指标见表1。
表1 副干酪乳杆菌大枣益生菌片的感官评定标准
根据表1感官评定标准作为评价大枣益生菌片感官品质的试验指标,通过正交试验确定了副干酪乳杆菌大枣益生菌片的较优组合配方,以较优组合配方制作的大枣益生菌片产品,其内质结构细密均匀且断面组织紧密,口感幼滑不粘牙,有大枣特有的香气,外形完整、光滑、薄厚均匀、边缘整齐,色泽均匀成枣黄色,其感官评定分数最高。
(8)大枣益生菌片中副干酪乳杆菌KL1-Liu活菌数量的检测
称取25g大枣益生菌片放入含有225mL灭菌生理盐水中,采用拍击式匀质器以8000~10000r/min的速度处理1min,充分振荡后,制成10-1的均匀稀释液。再以9mL灭菌生理盐水做10倍递增稀释至10-11,取10-9~10-11的稀释液各1mL置于无菌平皿中,倒入溶化并冷却至46℃的改良MRS培养基约15mL,迅速轻轻旋动平皿,使培养基与菌液充分混匀,每个稀释度3次重复。同时将改良MRS培养基注入加有1mL无菌生理盐水的灭菌平皿内作空白对照。待培养基凝固后,翻转平板,置(36±1)℃温箱中培养(48±2)h,待菌落长出后即可计数。其平板菌落计数结果显示,每1g大枣益生菌片中副干酪乳杆菌KL1-Liu的活菌数量为(1.0~5.0)×109CFU,见表2所示。
(9)大枣益生菌片中副干酪乳杆菌KL1-Liu活菌保质期试验
将副干酪乳杆菌KL1-Liu大枣益生菌片置于4℃冰箱贮藏,每隔2个月采用平板菌落计数法检测大枣益生菌片中副干酪乳杆菌KL1-Liu的活菌数量,检测方法同上述(8)的操作步骤。其平板菌落计数结果见表2。
表2 大枣益生菌片中副干酪乳杆菌KL1-Liu活菌保质期结果
由表1可见,大枣益生菌片中副干酪乳杆菌KL1-Liu的活菌数检测结果为4.94×109CFU/g,符合益生菌产品的活菌数量大于106CFU/g的标准。在冷藏4℃环境下放置一年后,在相同方法条件下测得益生菌片的活菌数为7.5×108CFU/g,显示该产品的活菌数量仍然大于106CFU/g的标准,益生菌的活菌数量明显高于同类产品。说明本发明制作的副干酪乳杆菌KL1-Liu大枣益生菌片在4℃冷藏条件下活菌保质期至少1年以上,表明该大枣益生菌片具有良好的贮藏稳定性。
采用上述发酵生产技术、冻干工艺和大枣益生菌片制备工艺所得到的产胆盐水解酶副干酪乳杆菌KL1-Liu大枣益生菌片的活菌数量均可达到109CFU/g以上,4℃贮藏1年副干酪乳杆菌KL1-Liu的活菌数量达到108CFU/g以上,大于106CFU/g的标准,仍可发挥降低血清胆固醇的保健作用。本发明制作的大枣益生菌片原料来源方便,工艺简单,制作周期短,操作简单,对设备要求较低,适于工业化生产。利用产胆盐水解酶副干酪乳杆菌KL1-Liu益生菌株与大枣结合,开发制备成大枣益生菌片,即可发挥产品降低血清胆固醇的保健功效,又可发挥大枣药食同源的营养与保健功效,具有广阔的市场应用前景。
本专利所属项目1:科技部国家科技重大专项“抗病转基因羊新品种培育”子课题“抗病转基因羊扩繁体系的建立/抗病转基因羊抗病、生产性能及安全评价”
项目编号:2013ZX08008-005
项目起止时间:2013.01-2013.12
项目负责人:刘慧
本专利所属项目2:“十二五”农村领域国家科技计划课题“动物源食品HACCP体系建设及致病菌高通量检测技术”
项目编号:2012BAD28B02-01
项目起止时间:2012.01-2015.12
项目负责人:孔保华
本专利所属项目3:科技部农业科技成果转化资金项目“利用藏灵菇源产胆盐水解酶乳酸菌生产功能性酸奶技术转化”
项目编号:2013GB2A000006
项目起止时间:2013.09-2015.08
项目负责人:白永强 。
Claims (2)
1.副干酪乳杆菌(Lactobacillus paracasei)KL 1-Liu CGMCC No.7029。
2.一种利用产胆盐水解酶副干酪乳杆菌(Lactobacillus paracasei)KL1-LiuCGMCC No.7029菌株制备大枣益生菌片的方法,其特征在于:(1)大枣益生菌片配方组成:包括副干酪乳杆菌KL1-Liu益生菌菌粉0.1%~1.0%,大枣粉40%~50%,低聚果糖10%~20%,木糖醇4%~10%,蔗糖0.25%~5%,柠檬酸0.25%~3%,硬脂酸镁0.5%~5.5%,微晶纤维素10%~20%,麦芽糊精25%~30%;(2)KL1-Liu益生菌菌粉的制备:将经过扩大培养与高密度发酵的副干酪乳杆菌KL1-Liu发酵液于4℃条件下,6000r/min离心10~20min,弃上清液,收集得到菌泥;在灭菌冻干瓶中,按离心前发酵液1/10体积的量,加入10.5%~20.5%麦芽糊精+11.5%~1.5%脱脂奶粉,与收集的菌泥混合均匀后,得到浓缩发酵剂;将浓缩发酵剂于-40℃预冻10h至完全冻结状态,得到预冻发酵剂;利用美国6LLABCONCO真空冷冻干燥机将预冻发酵剂于-55℃、真空度0.14mBar的条件下,冻干48h至完全干燥状态,得到副干酪乳杆菌KL1-Liu益生菌菌粉;(3)大枣益生菌片的制备:采用鼓风干燥箱将大枣于70℃烘干72h后自然晾干、去核备用;采用中药粉碎机分别将去核大枣、木糖醇、蔗糖、柠檬酸等辅料进行破碎,过80目筛子备用;采用三维多向运动混合机将大枣益生菌片配方中的各成分,在转速为10~20r/min,电机功率为0.5KW条件下混合均匀,制成大枣益生菌片粗粉;采用单冲式压片机将大枣益生菌片粗粉在最大压力为18KN,压片直径为15~20mm,每片重1g,硬度4~8N条件下,压制成产胆盐水解酶副干酪乳杆菌KL1-Liu大枣益生菌片。
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