CN111635875A - 一种长双歧杆菌cz70及其制备活菌型黑莓果浆的方法 - Google Patents
一种长双歧杆菌cz70及其制备活菌型黑莓果浆的方法 Download PDFInfo
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Abstract
本发明公开了一种长双歧杆菌CZ70,其保藏号为CGMCC No.19838;已于2020年05月19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。本发明的长双歧杆菌CZ70长双歧杆菌拥有较高的耐酸性,可以存活状态到达大肠,采用其制备的活性益生菌黑莓果浆营养丰富,与凝结芽孢杆菌JY69可以共生协调,互不拮抗,将产品保质期从市场活菌产品的30天延长至90天。30天后产品的对DPPH抗氧化率>45%。产品中添加低聚木糖,作为益生菌的食物,起到刺激双歧杆菌生长与活性,从而改善人体肠道环境。
Description
技术领域
本发明涉及一种长双歧杆菌CZ70及其制备活菌型黑莓果浆的方法。
背景技术
目前市场上的发酵产品多为使用脱脂奶粉为发酵基料的长保质期(一年)灭菌型产品。少数活菌型产品同样使用脱脂奶粉为发酵基料且保质期较短(30日以内),这下活菌型产品大部分使用好氧性乳酸菌商业菌株,而这些商业菌株多为不耐酸菌株,耐酸性乳酸菌株只能到达小肠而不能以存活状态到达大肠,而益生菌对保证大肠正常的水分吸收与代谢功能对润肠通便起到关键性作用。当前还未见以黑莓果浆为基料添加长双歧杆菌的活菌型黑莓果浆产品。
发明内容
本发明要解决的技术问题是克服现有技术中未有关于黑莓果浆为基料添加长双歧杆菌的活菌型黑莓果浆产品报道的现状,提供一种长双歧杆菌CZ70及其制备活菌型黑莓果浆的方法。
为了解决上述技术问题,本发明提供了如下的技术方案:
一种长双歧杆菌Bifidobacterium longum CZ70,其保藏号为CGMCC No.19838;已于2020年05月19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,保藏地址为北京市朝阳区北辰西路1号院3号。
长双歧杆菌Bifidobacterium longum CZ70拥有较高的耐酸性,可以存活状态到达大肠,起到增加有益菌,减少有害菌和有害物质,增强免疫力,预防疾病的作用;黑莓被誉为“生命之果”,含有花青素,SOD,有机硒和18中氨基酸(包括人体8种必需氨基酸)以及对人体至关重要的6种矿物质元素(钾,钙,钠,镁,锌,铁),在机体能量供给,修复细胞,新陈代谢以及延年益寿方面发挥着重要作用。
因此可将长双歧杆菌Bifidobacterium longum CZ70应用于发酵饮品的制备中。特别是利用长双歧杆菌CZ70发酵生产活性益生菌黑莓果浆。
另外益生元(低聚木糖)是一种膳食补充剂,作为益生菌的食物,起到刺激双歧杆菌生长与活性,从而改善人体肠道环境。
一种活性益生菌黑莓果浆的制备方法,包括以下步骤:
S1、制备基料果浆,将黑莓果浆进行杀菌冷却;
S2、制备长双歧杆菌CZ70的冻干粉,每克冻干粉中CZ70活菌含量≥1011;
S3、将长双歧杆菌CZ70的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中CZ70的活菌含量≥108;
S4、搅拌均匀后均质,冷藏保存。
进一步的,冷藏保存的温度为5~10℃。优选的,在均质前,加入0.5~1%的低聚木糖。
进一步的,为了提高活菌数,在S4之前将凝结芽孢杆菌JY69的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中JY69的活菌含量≥105。凝结芽孢杆菌JY69,其保藏号为CGMCCNo.18454;已于2019年09月02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,该菌株在公开号为110692719A的发明专利“一种凝结芽孢杆菌JY69及其制备活菌型混合莓果浆的方法”中已有报道,因此本专利不涉及JY69菌株的生物材料的保藏。凝结芽孢杆菌JY69和厌氧性长双歧杆菌CZ70可以共生协调,互不拮抗,二者均具有较强的耐受性,可以确保有足够多的活菌数在大肠内存活。
厌氧性长双歧杆菌CZ70拥有较强的耐酸性并能以存活状态到达大肠从而发挥有效作用,而凝结芽孢杆菌BCJY69菌株拥有较强的耐酸和耐热性,能将产品保质期提高到90天(0-10℃保存)。当前还未见以黑莓果浆为发酵基料添加能以存活状态到达大肠的长双歧杆菌以及能延长产品保质期的耐热性凝结芽孢杆菌的长保质期活菌型功能性黑莓果浆产品。
本发明所达到的有益效果是:本发明的长双歧杆菌CZ70长双歧杆菌拥有较高的耐酸性,可以存活状态到达大肠,起到增加肠道有益菌,减少有害菌,增强免疫力,预防疾病的作用;本发明制备的活性益生菌黑莓果浆中富集了黑莓中氨基酸、矿物质元素、花青素、黄酮,酚酸、维生素等多种营养成分,同时添加了低聚木糖益生元,作为益生菌的食物,起到刺激益生菌生长与活性,从而改善人体肠道健康,增强免疫力,预防疾病的作用。另外,凝结芽孢杆菌JY69和厌氧性长双歧杆菌CZ70可以共生协调,互不拮抗,二者均具有较强的耐受性,可以确保有足够多的活菌数在大肠内存活,而且协同凝结芽孢杆菌JY69的超强的耐热性(95度15分钟),可将产品保质期从市场活菌产品的30天延长至90天。30天后产品的对DPPH抗氧化率>45%。益生元(低聚木糖)是一种膳食补充剂,作为益生菌的食物,起到刺激双歧杆菌生长与活性,从而改善人体肠道环境。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1是Bifidobacterium longum CZ70的系统发育树;
图2是长双歧杆菌CZ70的耐胃液试验结果;
图3为长双歧杆菌CZ70的耐胆汁试验结果。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例
长双歧杆菌Bifidobacterium longum CZ70的分离方法
从我国湖南长沙一带的传统发酵的臭豆腐(油煎前)中分离样品,取1克上述样品放入灭菌试管中,接种于10毫升在121℃15分钟高压灭菌后冷却至37℃的牛奶中,置于厌氧菌培养盒中培养(37℃48h)。再用灭菌接种环挑取划线接种于MRS琼脂培养基中,将其放入厌氧培养盒中,置于37℃条件下培养48小时,形成菌落后,用接种环挑取菌落,接种于MRS培养液中,置于37℃厌氧条件下培养24小时,待菌株生长好后,再次划线接种于MRS琼脂培养基中,37°下厌氧培养48小时,记录观察菌落形态和革兰氏染色细胞形态特征,并进行过氧化酶实验,分离出在所述试验中呈阴性的杆菌,所述杆菌为长双歧杆菌。将所述长双歧杆菌进一步纯化培养,并进行低温保存。
长双歧杆菌Bifidobacterium longum CZ70的鉴定
经生理生化初步鉴定,CZ70菌株呈杆状,革兰氏染色阳性,接触酶阴性,能在37℃和42℃条件下生长,能利用葡萄糖和果糖,初步鉴定为长双歧杆菌(Bifidobacteriumlongum)其生理生化特征见表1.
表1其生理生化特征
注:+表示阳性;-表示阴性;w表示轻微利用
BLAST同源性比较结果
将所得16S rRNA测得序列,在NCBI网站中进行同源性搜索,JY70菌株与Bifidobacterium longum subsp longum mcc1110的16S rRNA相似度高达100%。
从Genbank中选择了菌株的16S rRNA基因序列,用Clustal X软件进行序列比对(alignment),然后利用MEGA3.1生物学软件构建系统发育树,所构建的系统发育树见图1。JY70与Bifidobacterium longum subsp longum mcc1110亲缘关系最近,因此,进一步证明,JY70菌株为Bifidobacterium longum。将其命名为Bifidobacterium longum JY70。
为长双歧杆菌Bifidobacterium longum CZ70的凝乳性试验结果为与对照相比,在37℃培养48小时后培养液的pH值达到3.5,该菌株分泌乳酸,具备凝乳作用。
序列分析与系统树的构建
采用序列图谱软件Chromas,参照正,反向序列图谱,对序列人工校对。
从GenBank
从Genbank中选择了菌株的16S rRNA基因序列,用Clustal X软件进行序列比对(alignment),然后利用MEGA3.1生物学软件构建系统发育树,所构建的系统发育树见图1。JY70与Bifidobacterium longum subsp longum mcc1110亲缘关系最近,因此,进一步证明,CZ70菌株为Bifidobacterium longum。将其命名为Bifidobacterium longum CZ70。
长双歧杆菌CZ70的耐胃液试验结果
将长双歧杆菌CZ70,嗜酸乳杆菌、干酪乳杆菌干酪亚种(LCSC)、嗜热链球菌在pH3的条件下分别培养0h,1h,2h,3h,4h后测定活菌数。结果如图2所示,BLCZ70表现出良好的耐酸性,在pH3时培养3h后仍能保持89.33%的活菌数,与其他商业菌株相比,其对酸的耐受性也表现出一定的优势。
将CZ70、嗜酸乳杆菌、干酪乳杆菌干酪亚种(LCSC)、嗜热链球菌在不同的胆汁浓度下培养24小时后测定活菌数。图3为长双歧杆菌CZ70的耐胆汁试验结果;图3种可以看出,CZ70在0.3%及0.6%的胆汁浓度下,均保持良好的生长状态,相比较嗜酸乳杆菌、LCSC及嗜酸乳杆菌均表现出一定的优势。在1%的胆汁浓度下,BLCZ70仍能获得超过106的活菌数,表明该菌具有良好的耐胆汁特性,为其在小肠及大肠中发挥作用奠定了基础。
实施例1一种活菌型黑莓果浆的制造方法,它包括以下步骤:
S1、制备基料果浆,将黑莓果浆进行杀菌冷却;
S2、制备长双歧杆菌CZ70的冻干粉,每克冻干粉中CZ70活菌含量≥1011;
S3、将长双歧杆菌CZ70的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中CZ70的活菌含量≥108;
S4、加入0.5~1%的低聚木糖,搅拌均匀后均质,5~10℃保存。
初始微生物指标:
长双歧杆菌CZ70(cfu/mL)>1.0×108
0~10℃下冷藏90天后微生物指标:
长双歧杆菌含量>1.0×105,霉菌、大肠杆菌和肠道致病菌(沙门氏菌、志贺氏菌、金黄色葡萄球菌)无检出;
浆体细腻均匀,粘稠度适中,无分层洗水。
实施例2
一种活性益生菌黑莓果浆的制备方法,包括以下步骤:
S1、制备基料果浆,将黑莓果浆进行杀菌冷却;
S2、制备长双歧杆菌CZ70的冻干粉,每克冻干粉中CZ70活菌含量≥1011
S3、将长双歧杆菌CZ70的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中CZ70的活菌含量≥108;
将凝结芽孢杆菌JY69的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中JY69的活菌含量≥105,凝结芽孢杆菌JY69的保藏号为CGMCC No.18454;
S4、加入0.5~1%的低聚木糖,搅拌均匀后均质,5~10℃保存。
已于2019年09月02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,该菌株在公开号为110692719A的发明专利“一种凝结芽孢杆菌JY69及其制备活菌型混合莓果浆的方法”中已有报道,因此本专利不涉及JY69菌株的生物材料的保藏。凝结芽孢杆菌JY69和厌氧性长双歧杆菌CZ70可以共生协调,互不拮抗,二者均具有较强的耐受性,可以确保有足够多的活菌数在大肠内存活。
初始微生物指标:长双歧杆菌CZ70(cfu/mL):>1.0×108,凝结芽孢杆菌JY69(cfu/mL):>1.0×105;
0~10℃下冷藏90天后微生物指标:
长双歧杆菌含量:大于1.0×106,凝结芽孢杆菌JY69(cfu/mL):>1.0×103;霉菌、大肠杆菌和肠道致病菌(沙门氏菌、志贺氏菌、金黄色葡萄球菌)无检出;
浆体细腻均匀,粘稠度适中,无分层洗水。
本发明制备的活性益生菌黑莓果浆的在0~10℃下可以保存90天,而且益生菌可以存活状态到达肠道,这样可以分泌乳酸,改善肠道菌群,促进通便和提高免疫力的功效,活菌型黑莓果浆中还含有黑莓,蓝莓,树莓,桑葚果浆,4种益生菌长双歧杆菌CZ70、保加利亚乳酸杆菌、嗜热链球菌、和双歧杆菌,还含有低聚异麦芽糖和低聚木糖两种益生元;制备方法简单,可最大限度的保留黑莓的风味和营养成分,无异味,极大地满足对健康生活有较高追求的人的需要。
低聚木糖可以有效提高活性益生菌黑莓果浆的悬浮稳定性,大大减少分层沉淀,有效提高产品的储藏稳定性,明显改善产品口感,长双歧杆菌CZ70和凝结芽孢杆菌JY69可明显改善产品口感,提高产品的营养吸收,特别是功能成分的吸收,提升了产品的档次,增加了产品的核心竞争力。
以下通过具体的临床试验例说明本发明产品的效果:
以实施例1制得的活性益生菌黑莓果浆为供试品,选严重便秘患者20人试用。
受试者每天服用30-45mL,服用实施例一个月后便秘明显改善或康复者18人,占90%,有一定程度上改善的1人,占5%,无效果的为1人,占5%。结果表明本发明产品具有良好的改善肠道菌群,促进通便的效果,并且上述试用人员均未产生任何副作用及不良反应。
以实施例2制得的蓝莓酵素为供试品,选有皮肤干燥,发黄的青年自愿者20人试用,年龄为25~35岁。受试者每天睡前服用此酵素15-20mL,服用1个月后
统计结果表明:显效(皮肤变白,更加光滑细腻,痤疮明显淡化。)的16人,占80%;
有效(皮肤光滑的为1人,占5%;完全无效果的为1人,占5%。
因此,本发明产品具有改善人体肠道,促进营养物质吸收,从而增强免疫力,从而使得人体更健康,养颜益容的功效,并且上述试用人员均未产生任何副作用及不良反应。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种长双歧杆菌CZ70,其保藏号为CGMCC No.19838;已于2020年05月19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。
2.如权利要求1所述的长双歧杆菌CZ70,其特征在于,所述的长双歧杆菌CZ70,可存活状态到达大肠,可分泌乳酸。
3.一种权利要求1所述的长双歧杆菌CZ70在发酵饮品中的应用。
4.如权利要求3所述的应用,其特征在于,利用长双歧杆菌CZ70发酵生产活性益生菌混合果浆。
5.一种活性益生菌黑莓果浆的制备方法,其特征在于,包括以下步骤:
S1、制备基料果浆,将黑莓果浆进行杀菌冷却;
S2、制备长双歧杆菌CZ70的冻干粉,每克冻干粉中CZ70活菌含量≥1011;
S3、将长双歧杆菌CZ70的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中CZ70的活菌含量≥108;
S4、搅拌均匀后均质,冷藏保存。
6.如权利要求5所述的活性益生菌黑莓果浆的制备方法,其特征在于,S4之前将凝结芽孢杆菌JY69的冻干粉接种到黑莓果浆中,每毫升黑莓果浆中JY69的活菌含量≥105。
7.如权利要求5所述的活性益生菌黑莓果浆的制备方法,其特征在于,冷藏保存的温度为5~10℃。
8.如权利要求5所述的活性益生菌黑莓果浆的制备方法,其特征在于,在均质前,加入0.5~1%的低聚木糖。
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CN116286535B (zh) * | 2023-03-27 | 2024-01-30 | 湖南绿韵数字化健康科技有限公司 | 一种含益生菌的胶原蛋白肽组合物及其应用 |
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