CN102056489B - 新型发酵乳及其应用 - Google Patents
新型发酵乳及其应用 Download PDFInfo
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Abstract
本发明提供一种对促进健康有益的发酵乳。用复合微生物群对乳原料进行发酵得到发酵乳,所述复合微生物群包括:(1)开菲尔乳杆菌(Lactobacillus kefiri)、以及(2)选自Kazachstania turicensis、单孢酿酒酵母(Kazachstania unispora)及马克斯克鲁维酵母菌(Kluyveromyces marxianus)中的至少一种。优选地,用至少包括开菲尔乳杆菌P-IF菌株、开菲尔乳杆菌P-B1菌株、Kazachstania turicensis P-Y3菌株、单孢酿酒酵母(Kazachstania unispora)P-Y4菌株及马克斯克鲁维酵母菌P-Y5菌株的复合微生物群,对乳原料进行发酵得到发酵乳。
Description
技术领域
本发明涉及一种发酵乳,所述发酵乳是用含有乳酸菌及酵母菌的复合微生物群对乳原料进行发酵制得,本发明还涉及含有该发酵乳作为有效成分的抗菌剂,以及含有该发酵乳的饮食品。
背景技术
众所周知,在酸奶等乳酸菌发酵产物中,含有细菌素(bacteriocins)等抗菌物质。且人们认为,通过抗菌性,抑制对肠内环境造成不良影响的肠内细菌的生长,促进对肠内环境有益的肠内细菌的生长。
关于乳酸菌对肠内细菌的抗菌性,下述专利文献1中描述了为抑制大肠菌(E.coli)O157:H7及其他病原菌的发生率及繁殖,将开菲尔乳杆菌等生成乳酸的菌施用于动物。
专利文献1:特表2006-501311号公报
发明内容
但是,上述专利文献1中描述的是用来对反刍动物肠内病原菌的发生进行抑制的技术,并非有助于促进人体健康、诸如改善肠内环境等的技术。
因此,本发明的目的在于提供一种发酵乳,所述发酵乳对给人体肠内环境造成不良影响的肠内细菌具有很强的抗菌性,且有利于促进健康,诸如改善肠内环境等。
本发明如下所述。
[1]一种发酵乳,其是用复合微生物群对乳原料进行发酵得到的发酵乳,所述复合微生物群包括(1)开菲尔乳杆菌(Lactobacilluskefiri)、以及(2)选自Kazachstania turicensis、单孢酿酒酵母(Kazachstania unispora)及马克斯克鲁维酵母菌(Kluyveromycesmarxianus)中的至少一种。
[2]上述[1]中所述的发酵乳,所述开菲尔乳杆菌为开菲尔乳杆菌P-IF(保藏号FERM BP-10896)。
[3]上述[1]或[2]所述的发酵乳,将乳原料用复合微生物群进行发酵得到的发酵乳,所述复合微生物群至少包括开菲尔乳杆菌P-IF(保藏号FERM BP-10896)、开菲尔乳杆菌P-B1(保藏号FERMBP-11115)、Kazachstania turicensis P-Y3(保藏号FERM BP-11116)、单孢酿酒酵母菌P-Y4(保藏号FERM BP-11117),以及马克斯克鲁维酵母P-Y5(保藏号FERM BP-11118)。
[4]含有上述发酵乳作为有效成分的抗菌剂。
[5]含有上述发酵乳作为有效成分的肠内环境改善剂。
[6]含有上述发酵乳的饮食品。
本发明的发酵乳,能够抑制对肠内环境造成不良影响的肠内细菌的生长。因此,通过经口摄取,能够谋求促进健康诸如改善肠内环境等。另外,本发明的抗菌剂通过其抗菌活性,能够抑制对肠内环境造成不良影响的肠内细菌的生长、促进对肠内环境有益的肠内细菌的生长。并且,本发明还能够提供有助于促进健康诸如改善肠内环境等的饮食品。
附图说明
图1为“P-IF菌”的电子显微镜下的照片。
具体实施方式
本发明所使用的微生物为,乳杆菌属的开菲尔乳杆菌,酵母菌属的Kazachstania turicensis、单孢酿酒酵母菌以及马克斯克鲁维酵母。优选为,开菲尔乳杆菌P-IF(Lactobacillus kefiri P-IF)(保藏号:FERMBP-10896,保藏机构:独立行政法人产业技术综合研究所专利生物保藏中心茨城县筑波市东1丁目1号1中央第6)(以下,也将其称为“P-IF菌”)、开菲尔乳杆菌P-B1(Lactobacillus kefiri P-B1)(保藏号:FERM BP-11115,保藏机构:独立行政法人产业技术综合研究所专利生物保藏中心茨城县筑波市东1丁目1号1中央第6)(以下,也将其称为“P-B1菌”)、Kazachstania turicensis P-Y3(保藏号:FERM BP-11116,保藏机构:独立行政法人产业技术综合研究所专利生物保藏中心茨城县筑波市东1丁目1号1中央第6)(以下,也将其称为“P-Y3菌”)、单孢酿酒酵母菌P-Y4(Kazachstania unisporaP-Y4)(保藏号:FERM BP-11117,保藏机构:独立行政法人产业技术综合研究所专利生物寄托中心茨城县筑波市东1丁目1号1中央第6)(以下,也将其称为“P-Y4”)、以及马克斯克鲁维酵母P-Y5(Kluyveromyces marxianus P-Y5)(保藏号:FERM BP-11118,保藏机构:独立行政法人产业技术综合研究所专利生物寄托中心茨城县筑波市东1丁目1号1中央第6)(以下,也将其称为P-Y5)。
分别通过琼脂培养基培养后挑取单菌落菌株来分离“P-IF菌”、“P-B1菌”、“P-Y3菌”、“P-Y4菌”及“P-Y5菌”,并且进行了DNA序列分析用来鉴定其种属。具体地说,对于菌株“P-IF菌”及“P-B1菌”,测定其基因组DNA上的核糖体RNA基因16SrDNA的核苷酸序列,并进行同源性分析。另外,对菌株“P-Y3菌”、“P-Y4菌”及“P-Y5菌”,测定其基因组DNA上的核糖体RNA基因26SrDNA-D1/D2区域的核苷酸序列,并进行同源性分析。结果表明,“P-IF菌”的核苷酸序列与开菲尔乳杆菌的核苷酸序列显示出99.6%的同源性,因此推定“P-IF菌”属于开菲尔乳杆菌。同样地,“P-B1菌”与开菲尔乳杆菌的核苷酸序列显示出99.6%的同源性,因此推定“P-B1菌”属于开菲尔乳杆菌。并且,“P-Y3菌”与Kazachstaniaturicensis的核苷酸序列显示出100%的同源性,因此推定“P-Y3菌”属于Kazachstania turicensis。另外,“P-Y4菌”与单孢酿酒酵母的核苷酸序列显示出100%的同源性,由此推定“P-Y4菌”属于单孢酿酒酵母。再者,“P-Y5菌”与克斯克鲁维酵母的核苷酸序列显示出100%的同源性,因此推定“P-Y5菌”属于克斯克鲁维酵母菌。
下面对“P-IF菌”的微生物学性质做更详细地说明。
表1列出了“P-IF菌”的微生物学性质
表1
并且,表2列出了“P-IF菌”的同化(資化)特性。
表2
如上所述,测定了“P-IF菌”的16S rDNA核苷酸序列,利用微生物的16S rDNA核苷酸序列的数据库,进行同源性分析,其与开菲尔乳杆菌的序列显示出99.6%的同源性,因此推定“P-IF菌”属于开菲尔乳杆菌。另外,上述微生物学特性及同化特性也与开菲尔乳杆菌的相应特性一致。
但是,在半乳糖作为糖原的利用上,其与现有的开菲尔乳杆菌(Lactobacillus kefiri)有所不同。并且,若在试管中进行液体培养,仅仅使该试管稍微震动,就会如同香槟酒一样产生二氧化碳。这也是在现有开菲尔乳杆菌(Lactobacillus kefiri)中没有发现的特征。此外,如图1所示,在电子显微镜下观察时,与通常为沿纵向平面***生长不同,“P-IF菌”具有与其他菌粘附组成三维结构的特征。这表明菌体表面的糖链等结构不同。
同时表明,“P-IF菌”在MRS液体培养基中的发酵过程中,pH达到4.3的低值,仍继续生长,由此表明其具有耐胃酸性,可存活直至到达肠道。
以上表明,“P-IF菌”虽为属于开菲尔乳杆菌(Lactobacillus kefiri)的微生物,但其为具有特异形态特征的新菌株。
并且,关于“P-B1菌”、“P-Y3菌”、“P-Y4菌”及“P-Y5菌”的微生物学性质,分别具有与同种微生物相同的微生物学性质。
分离的“P-IF菌”、“P-B1菌”、“P-Y3菌”、“P-Y4菌”及“P-Y5菌”均可以通过公知的方法进行培养,例如,乳酸菌“P-IF菌”以及“P-B1菌”,可通过用市售的MRS培养基“Lactobacilli MRS Broth”(商品名,Difeo公司制产品),进行厌氧静置培养。并且,酵母菌“P-Y3菌”、“P-Y4菌”及“P-Y5菌”,例如,可采用市售的YM培养基“葡萄糖1%、蛋白胨0.5%、麦芽提取物0.3%、酵母提取物0.3%、琼脂2%”(Difco公司制产品),进行厌氧静置培养。
本发明的发酵乳,其为用复合微生物群对乳原料进行发酵制得,所述复合微生物群包括(1)开菲尔乳杆菌菌株、及(2)选自Kazachstania turicensis、单孢酿酒酵母及马克斯克鲁维酵母菌中的至少1种。所述开菲尔乳杆菌优选为菲尔乳杆菌P-IF(保藏号:FERMBP-10896)。
并且,更优选的是,将乳原料用至少含有开菲尔乳杆菌P-IF(保藏号:FERM BP-10896)、开菲尔乳杆菌P-B1(保藏号:FERMBP-11115)、Kazachstania turicensis P-Y3(保藏号:FERM BP-11116)、单孢酿酒酵母P-Y4(保藏号:FERM BP-11117)及马克斯克鲁维酵母P-Y5(保藏号:FERM BP-11118)的复合微生物群进行发酵制得。
作为乳原料例如有,牛奶、乳清、发酵乳、乳酸菌饮料、脱脂乳、脱脂奶粉、配方奶粉、全脂奶粉、浓缩乳、脱脂浓缩乳、炼乳、脱脂炼乳、加糖炼乳、加糖脱脂炼乳等。
本发明中,对乳原料的发酵,例如,可以将接种有由上述复合微生物群构成的酵母的乳原料,通过公知的发酵培养条件进行发酵,但优选为先在24~26℃的条件下发酵20~28小时,然后再在20~22℃的条件下发酵20~28小时、然后将温度降至10~12℃进行42~54小时熟化的方法。或者,作为其他培养方法有,在24~26℃条件下开始初始培养后,在20~28小时内将该培养温度直降到20~22℃,然后在20~22℃下进行20~28小时的追加发酵后,在10~12℃下保持42~54小时使其熟化的方法。这样做,可将上述复合微生物群中含有的各微生物共存培养,容易产生有效成分。
本发明中,可以将进行了上述发酵的发酵乳不经过杀菌处理直接使用,也可以经过杀菌处理后再使用。或者,还可以将从进行了上述发酵的发酵乳中,通过离心分离过滤等分离除去菌体部分后的溶液部分作为本发明的发酵乳。或不限制发酵乳的其它形态。
经口摄取本发明的发酵乳时,对每天所摄入的量没有特别的限制,但以固体成分换算,优选为0.01(mg/kg体重)~10(g/kg体重),更优选为0.05(mg/kg体重)~1(g/kg体重)。
本发明的发酵乳可应用于医药品、健康食品、加工食品等各种领域。并且对制剂的形态没有特别的限制。可采用适当的公知方法,制成液剂、糖浆、凝胶、胶囊、粉剂、片剂、颗粒剂、糖锭剂等制剂使用。并且,本发明的发酵乳还可加入到各种饮食品使用。
本发明的发酵乳,如后述实施例所示,通过其抗菌作用,可作为抗菌剂的有效成分来使用。并且,特别是对肠内细菌的厌氧性菌群的、被称为坏细菌的梭状芽孢杆菌属微生物等,有很强的抗菌效果,因此可作为肠内环境改善剂的有效成分来使用。
实施例
下面通过一些实施例对本发明进行具体说明,但这些实施例并不限定本发明的范围。
制备例1发酵乳的制备
将接种含有菌株“P-IF菌”、“P-B1菌”、“P-Y3菌”、“P-Y4菌”及“P-Y5菌”的复合微生物群构成的酵母的乳原料(乳清),先在25℃的条件下发酵24小时,之后在21℃条件下发酵24小时、然后将温度降至11℃熟化48小时。
实施例1抗菌活性实验
对由上述制备例1制得的发酵乳进行抗菌活性研究。具体地说,在过滤去除菌体的发酵乳(1L)中,加入等体积的正丁醇充分混合后静置一夜。然后通过离心将丁醇层和水层分别进行回收。然后将丁醇层减压浓缩干燥。然后将其溶于少量的去离子水中,经玻璃过滤器过滤后,用截留分子量1kDa的透析管对去离子水进行透析。将低分子截留部分的透析外液进行回收,减压浓缩后冷冻干燥。
将得到的正丁醇萃取-低分子截留部分的冷冻干燥物,按10mg/ml溶解于去离子水中,用1/10N的乳酸调节pH值为4.5后,用ADVANTEC公司制的滤纸(8mm)吸收25μl或50μl。
另一方面,将人粪便的一部分用无菌生理盐水进行稀释后,用GAM肉汤(日水制药公司制),在简易厌氧培养罐“Anaeropackkennki”(商品名,三菱天然气化学公司制)进行厌氧培养。24小时后,从GAM肉汤中取少量放入培养皿中,通过加入平板计数琼脂培养基(Merck公司制)进行混合稀释的混释法,制成平板。
在含有来自人粪便的细菌的琼脂平板培养基中,放入吸收了各检测试样的滤纸,然后再在35℃下进行24小时培养,观察抑菌圈。结果如下表3所示。
比较例1
在牛奶中加入市售的粉末克菲尔A(作为克菲尔种菌,在各家庭中添加到牛奶中,进行发酵后食用的类型)后,在25℃条件下发酵18小时。在上述发酵液中加入等体积的正丁醇,按照与实施例1相同的方法,制得正丁醇萃取-低分子截留部分的冷冻干燥物,进行了抗菌活性试验。其结果如下表3所示。
比较例2
在市售的乳酸菌饮料B中加入等体积的正丁醇,按照与实施例1相同的方法,制得正丁醇萃取-低分子截留部分的冷冻干燥物,进行了抗菌活性试验。其结果如下述表3所示。
比较例3
在市售的酸奶C中加入等体积的正丁醇,按照与实施例1相同的方法,制得正丁醇萃取-低分子截留部分的冷冻干燥物,进行了抗菌活性试验。其结果如下述表3所示。
表3
括号内为被滤纸吸收的量
-:阴性;+:弱阳性(轮廓分明的抑菌圈)
++:中阳性(轮廓分明的抑菌圈,含有φ8mmDisc的抑菌圈的φ在10mm以下)
+++:强阳性(轮廓分明的抑菌圈,含有φ8mmDisc的抑菌圈的φ在10mm以上)
如表3所示,能够确认本发明的发酵乳具有很强的抑菌活性(实施例1)。另一方面,作为对照的市售发酵乳制品没有显示抑菌圈的形成,不能确认抗菌活性(比较例1-3)。并且,尽管结果中没有列出,但能够确认的是,将“P-IF菌”接种到MRS液体培养基中,在25℃下培养48-72小时,繁殖至1010-1011cfu/ml后,将其中一部分接种到3ml脱脂乳培养基(在90质量份去离子水中加入10质量份脱脂奶粉,混合、121℃/15分钟灭菌,配制成的培养基)中,在25℃条件下进行5天厌氧静置培养,使其发酵得到的培养物也有若干抑菌活性。因此可知,用“P-IF菌”对乳原料发酵,是体现上述抑菌活性的主要原因。
用于这次试验的来自人粪便的菌为肠内细菌的厌氧性菌群、含有被称为坏细菌的梭状芽孢杆菌属微生物。本发明的发酵乳对这些菌群具有很强的抑菌作用,因此具有更为优秀的改善肠内环境的作用。
保藏号
(1)保藏号:FERM BP-10896:开菲尔乳酸杆菌P-IF
(2)保藏号:FERM BP-11115:开菲尔乳酸杆菌P-B1
(3)保藏号:FERM BP-11116:Kazachstania turicensis P-Y3
(4)保藏号:FERM BP-11117:单孢酿酒酵母P-Y4
(5)保藏号:FERM BP-11118:马克斯克鲁维酵母P-Y5
受理单位记入栏
国际事务局记入栏
Claims (6)
1.一种发酵乳,其特征在于,其是用复合微生物群对乳原料进行发酵得到的发酵乳;所述复合微生物群包括:
(1)开菲尔乳杆菌P-IF(Lactobacillus kefiri P-IF)(保藏号:FERMBP-10896),以及
(2)选自Kazachstania turicensis、Kazachstania unispora及马克斯克鲁维酵母菌(Kluyveromyces marxianus)中的至少1种。
2.根据权利要求1所述的发酵乳,其特征在于,其是将乳原料用复合微生物群进行发酵制得;所述复合微生物群至少含有开菲尔乳杆菌P-IF(Lactobacillus kefiri P-IF)(保藏号:FERM BP-10896)、开菲尔乳杆菌P-B1(Lactobacillus kefiri P-B1)(保藏号:FERMBP-11115)、Kazachstania turicensis P-Y3(保藏号:FERM BP-11116)、Kazachstania unispora P-Y4(保藏号:FERM BP-11117)及马克斯克鲁维酵母P-Y5(Kluyveromyces marxianus P-Y5)(保藏号:FERMBP-11118)。
3.含有权利要求1或2所述发酵乳作为有效成分的抗梭状芽孢杆菌制剂。
4.含有权利要求1或2所述发酵乳作为有效成分的肠内环境改善剂。
5.含有权利要求1或2所述发酵乳的饮食品。
6.开菲尔乳杆菌P-IF(Lactobacillus kefiri P-IF)(保藏号:FERMBP-10896)。
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KR102393420B1 (ko) * | 2019-10-30 | 2022-05-02 | 명지대학교 산학협력단 | 산업배지로 배양된 케피어를 함유하는 이유자돈 면역증진용 기능성 사료 첨가제 및 이의 제조방법 |
WO2021171061A1 (es) * | 2020-02-25 | 2021-09-02 | Universidad de Valparaíso | Grupo de microorganismos compuesto por lactobacillus sp. cepa k03d08, bacillus sp. cepa k03b01 y kazachstania sp. cepa k03k02g y sus composiciones; un proceso de obtención de un derivado lácteo libre de caseína que contenga ácidos grasos de cadena corta y ácidos grasos de cadena corta hidroxilados generados por el metabolismo del grupo de microorganismos |
EP4140311A1 (en) * | 2021-08-25 | 2023-03-01 | Technische Universität Berlin | Method to produce a non-kefir grain based synthetic kefir-like fermented product and method for kefir-grain free cultivation of kefir microorganism cultures |
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JP2005312424A (ja) * | 2004-04-30 | 2005-11-10 | Nippon Kefia Kk | ケフィアグレインを用いた発酵産物の製造方法及び当該方法によって得られる発酵産物 |
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WO2009151020A1 (ja) | 2009-12-17 |
CN102056489A (zh) | 2011-05-11 |
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US20110123640A1 (en) | 2011-05-26 |
KR101399712B1 (ko) | 2014-06-20 |
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JPWO2009151020A1 (ja) | 2011-11-17 |
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