CN107109345A - 威克汉姆酵母属微生物培养物 - Google Patents
威克汉姆酵母属微生物培养物 Download PDFInfo
- Publication number
- CN107109345A CN107109345A CN201680004685.4A CN201680004685A CN107109345A CN 107109345 A CN107109345 A CN 107109345A CN 201680004685 A CN201680004685 A CN 201680004685A CN 107109345 A CN107109345 A CN 107109345A
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- culture
- fragrance
- yeast
- culture medium
- brunswick durham
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Classifications
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Abstract
本发明提供一种大量含有苯甲酸乙酯,具有比化学合成品更复杂且更新鲜的水果样香味的培养物。一种培养物,其为将属于威克汉姆酵母属的微生物在含有乳成分的培养基中进行培养而获得的。
Description
技术领域
本发明涉及一种具有良好香气的培养物及其用途。
背景技术
在现有技术中已知有使用各种产生香气成分的酵母、例如酿酒酵母等而获得具有良好香气的培养物的方法,该培养物被用作饮食品(专利文献1~3)。
作为香气成分之一的苯甲酸乙酯在化学上可由苯甲酸与乙醇合成。该化合物已知为杨桃的香气成分之一,在工业上被用作果实香味的人工香料。还已知有使用含有苯甲酸乙酯作为香气成分的酿酒酵母的培养物,但其含量较少,为0.006~0.009ppm(非专利文献1)。
此外,皮杰普氏威克汉姆酵母(Wickerhamomyces pijperi)是从皮杰普氏毕赤氏酵母(Pichia pijperi)再分类的一种酵母,非专利文献2中记载了将该酵母在酵母提取物-甘露醇(yeast extract-mannitol)培养基、酵母-蛋白胨-葡萄糖(yeast-peptone-dextrose,YPD)培养基中进行培养,但所获得的培养物的特性不明,对于能够在含有乳成分的培养基中进行培养及其培养物的香气完全未知。
现有技术文献
专利文献
专利文献1:日本专利特开平5-49465号公报
专利文献2:日本专利特开2001-103959号公报
专利文献3:日本专利特开2012-55286号公报
非专利文献
非专利文献1:Journal of the institute of Brewing,116,190-196,2010
非专利文献2:Microbiology,155,3142-3148,2009
发明内容
发明所要解决的课题
上述的苯甲酸乙酯等香气成分的化学合成品的香气单调,作为香料用组合物而言未必令人满意。因此,需求一种能够利用在化妆品或饮食品、医药品,具有嗜好性优于化学合成品的香味,且能够用作香料用组合物的组合物。此外,喜好水果样香味的消费者也很多,需求提供水果样的新颖香味。
本发明的课题在于提供一种组合物及其用途,该组合物为能够利用在香料用组合物、化妆品、饮食品及医药品等的新颖组合物,并且大量含有苯甲酸乙酯,具有较苯甲酸乙酯的化学合成品更复杂且更新鲜的水果样香味。
用于解决课题的手段
因此,本发明的发明人为了开发出具有良好香气的组合物而进行了各种研究,结果完全意外地发现,在将威克汉姆酵母属的微生物在含有乳成分的培养基中进行培养时,获得大量含有作为香气成分已知的苯甲酸乙酯,且具有较苯甲酸乙酯的化学合成品更复杂且更新鲜的水果样香气的培养物,该培养物作为可用在香料用组合物、化妆品、饮食品、医药品等的培养物是有用的,从而完成了本发明。
即,本发明提供以下的[1]~[10]。
[1]一种培养物,其为将属于威克汉姆酵母属的微生物在含有乳成分的培养基中进行培养而获得的。
[2]如[1]所述的培养物,其中,含有乳成分的培养基为含有乳清的培养基。
[3]如[1]或[2]所述的培养物,其中,属于威克汉姆酵母属的微生物为皮杰普氏威克汉姆酵母。
[4]如[1]~[3]中任一项所述的培养物,其中,属于威克汉姆酵母属的微生物为皮杰普氏威克汉姆酵母NBRC1290和/或皮杰普氏威克汉姆酵母NBRC1887。
[5]如[1]~[4]中任一项所述的培养物,其中,含有乳成分的培养基是还含有苯甲酸和/或其盐的培养基。
[6]一种香料用组合物,其含有[1]~[5]中任一项所述的培养物。
[7]一种化妆品,其含有[1]~[5]中任一项所述的培养物。
[8]一种饮食品,其含有[1]~[5]中任一项所述的培养物。
[9]一种医药品,其含有[1]~[5]中任一项所述的培养物。
[10]一种对化妆品、饮食品或医药品赋予香味的方法,其特征在在:配合[1]~[5]中任一项所述的培养物。
发明的效果
本发明的培养物大量含有苯甲酸乙酯,且具有较苯甲酸乙酯的化学合成品更复杂且更新鲜的水果样香味,因此,不仅作为香料用组合物和化妆品有用,作为饮食品或医药品所使用的培养物也是有用的。
具体实施方式
本发明的培养物为将属于威克汉姆酵母属的微生物在含有乳成分的培养基中进行培养而获得的培养物。
作为属于威克汉姆酵母属的微生物,可列举:皮杰普氏威克汉姆酵母(Wickerhamomyces pijperi)、异常威克汉姆酵母(Wickerhamomyces anomalus)、牛威克汉姆酵母(Wickerhamomyces bovis)、腊宝威克汉姆酵母(Wickerhamomyces rabaulensis)、汉普夏威克汉姆酵母(Wickerhamomyces hampshirensis)、斯特拉斯堡威克汉姆酵母(Wickerhamomyces strasburgensis)、赛道威威克汉姆酵母(Wickerhamomycessydowiorum)、lynferdii威克汉姆酵母(Wickerhamomyces lynferdii)、西弗威克汉姆酵母(Wickerhamomyces ciferrii)、钱巴德威克汉姆酵母(Wickerhamomyces chambardii)、森林威克汉姆酵母(Wickerhamomyces silvicola)、双孢威克汉姆酵母(Wickerhamomycesbisporus)、赤杨威克汉姆酵母(Wickerhamomyces alni)、加拿大威克汉姆酵母(Wickerhamomyces canadensis)、指甲威克汉姆酵母(Wickerhamomyces onychis)、土壤威克汉姆酵母(Wickerhamomyces edaphicus)、掷孢威克汉姆酵母(Wickerhamomycespatagonicus)等。其中,更优选皮杰普氏威克汉姆酵母。进而,在皮杰普氏威克汉姆酵母中,进一步优选保藏在NBRC(NITE Biological Resource Center,日本技术评价研究所生物资源中心)的NBRC1290、NBRC1887。此外,这些微生物可单独使用,也可以组合多种而使用。需要说明的是,在旧分类中分类为皮杰普氏毕赤氏酵母(Pichia pijperi)而新分类为皮杰普氏威克汉姆酵母的酵母,包括在本申请的皮杰普氏威克汉姆酵母中。
在本说明书中,所谓“乳成分”是指含有牛乳、山羊乳、绵羊乳等兽乳的生乳、加热乳、脱脂乳粉、全脂乳粉或者生奶油、乳清等源自乳的成分的物质。
作为含有乳成分的培养基,只要为含有乳成分的培养基即可,也可以进一步含有其他成分。作为该其他成分,没有特别限定,优选为葡萄糖、半乳糖、乳糖、果糖等糖,苯甲酸和/或其盐。作为糖,从被微生物同化的方面考虑,优选为葡萄糖。此外,从添加后提高微生物的苯甲酸乙酯的产生量的方面考虑,优选为苯甲酸和/或其盐,作为苯甲酸的盐,更优选苯甲酸钠和苯甲酸钾等苯甲酸碱金属盐,进一步优选苯甲酸钠。因此,作为苯甲酸和/或其盐,优选为添加苯甲酸和/或苯甲酸钠。
作为乳成分,乳清由于会提高微生物的苯甲酸乙酯的产生量,因此特别优选。所谓“乳清”是指从牛乳中去除乳脂肪成分、酪蛋白而获得的物质。例如,乳清可以作为将乳成分利用微生物进行发酵时的培养上清而获得,优选为含有乳糖、半乳糖等糖、氨基酸、乳酸、蛋白质等的物质。此外,作为乳清,特别优选将乳成分利用乳酸菌和/或双歧杆菌进行培养而获得的培养上清。此处,作为乳酸菌和/或双歧杆菌,可列举:干酪乳杆菌、马里乳杆菌、嗜酸乳杆菌、德氏乳杆菌保加利亚亚种、瑞士乳杆菌等乳杆菌属细菌;嗜热链球菌等链球菌属细菌;乳酸乳球菌乳亚种、乳酸乳球菌乳脂亚种等乳球菌属细菌;粪肠球菌等肠球菌属细菌;或者短双歧杆菌、两歧双歧杆菌、长双歧杆菌等双歧杆菌属细菌。
培养基中的乳成分的含量没有特别限定,以固态物成分换算,优选为1~50质量%(以下,简称为“%”),更优选为2~10%。此外,从微生物的苯甲酸乙酯的产生量及微生物的增殖能力的方面考虑,苯甲酸和/或其盐的含量在含有乳成分的培养基中优选为0.001~0.1%,更优选为0.01~0.05%。进而,从微生物的增殖能力的方面考虑,葡萄糖的含量优选为0.1~10%,更优选为1~5%。
属于威克汉姆酵母属的微生物的培养温度没有特别限定,从培养物的香味的方面考虑,优选为15~30℃,进一步优选为20~30℃。培养时间优选为8小时以上,进一步优选为24~32小时。此外,在将苯甲酸和/或其盐添加至培养基中的情况下,优选为24~48小时,进一步优选为32~48小时。作为培养方法,可列举:搅拌培养、静置培养、振荡培养、中和培养等。
本发明的培养物中包含1.0ppm以上的苯甲酸乙酯,具有较苯甲酸乙酯的化学合成品更复杂且更新鲜的水果样香气。苯甲酸乙酯的含量优选为1.0~50ppm,更优选为1.0~30ppm。
此外,本发明的培养物中也可以包含苯甲酸乙酯以外的其他香气成分。作为其他香气成分,可列举异戊醇等香气成分。
本发明的培养物大量含有苯甲酸乙酯,具有且较苯甲酸乙酯的化学合成品更复杂且更新鲜的如水果般的良好香气,因此,可在香料用组合物、化妆品、饮食品和医药品等中利用。在化妆品领域,工业上可利用的源自生物的香料较少,天然香料的需求高于合成香料,因此能够适宜地在化妆品中利用。
对于本发明的培养物而言,可以将培养物原样配合在香料用组合物、化妆品、饮食品和医药品等中,但优选配合经过滤的培养物。此外,也可通过冷冻干燥法或者蒸馏法等对培养物进行浓缩,并将该浓缩物配合在香料用组合物、化妆品、饮食品和医药品等中。
作为化妆品,可列举:香水、科隆香水、淡香水等芳香化妆品;化妆水、乳液、润肤露、乳霜、面膜、美容液等基础化妆品,洗发液或护发素等头发用化妆品;浴液等浴用化妆品;粉底等化妆用化妆料;防晒霜等特殊化妆品等。作为饮食品,可列举:各种清凉饮料、起泡酒、啤酒、清酒、点心类、冰点心类、冰淇淋、发酵乳等乳制品等。此外,作为医药品,可列举:霜剂、软膏、凝胶等外用剂等。这些化妆品、饮食品、医药品中的本发明培养物的含量以固态物成分换算优选为0.01~10%,更优选0.1~1%。
实施例
下面,列举实施例详细地说明本发明。
实施例1
(1)菌株及预前培养(pre-preculture)
(i)使用菌株
皮杰普氏威克汉姆酵母YIT8095(NBRC 1290)、皮杰普氏威克汉姆酵母YIT12779(NBRC 1887)
比较例:马克思克鲁维酵母(Kluyveromyces marxianus)YIT12612(NBRC 0260)
(ii)预前培养
预前培养:向2ml的酵母及霉菌(Yeast and Mold,YM)用培养基(1%葡萄糖、0.5%蛋白胨、0.3%酵母提取物、0.3%麦芽提取物)中接种20%甘油冷冻保存株20μL,在30℃、160rpm下振荡培养24小时。
(2)菌所产生的香气成分的分析
(i)前培养(preculture)
前培养:向包含1%的葡萄糖的2ml含有乳清的培养基中接种预前培养中所获得的培养液20μL,在30℃、160rpm下振荡培养24小时。
作为上述含有乳清的培养基,使用了将嗜热链球菌(Streptococcusthermophilus)YIT2084(FERMBP-10879)在3%脱脂乳粉培养基中进行培养而获得的培养物的上清。对该上清进行分析的结果为包含1.1%的乳糖、0.4%的半乳糖、0.4%的乳酸,pH值为4.0。
(ii)培养
培养:向包含1%的葡萄糖的5ml含有乳清的培养基中接种前培养液50μL,在30℃、160rpm下振荡培养24小时。
(iii)香气成分的分析
培养完毕后,试样在8000rpm下离心分离5分钟,取2ml的上清装入20ml的小瓶中,并通过顶空气相色谱-质谱法(Headspace gas chromatography mass spectrometry,HS-GC-MS)鉴定培养物中的香气成分(表1、表2)。此外,所鉴定的香气成分的定量利用HS-GC-火焰离子化检测器(Flame Ionization Detector,FID)而进行(表3、表4)。将结果示于表5。此外,基于下述基准进行了培养液的香味的感官检验。将其结果示于表5。
(感官检验的评价基准)
5:强烈地发出复杂且新鲜的水果样香味。
4:发出复杂且新鲜的水果样香味。
3:略微发出复杂且新鲜的水果样香味。
2:几乎不发出复杂且新鲜的水果样香味。
1:不发出复杂且新鲜的水果样香味。
[表1]
HS条件
[表2]
GC-MS条件
[表3]
HS条件
[表4]
GC条件
[表5]
化合物(ppm) | NBRC1290 | NBRC1887 | 马克思克鲁维酵母NBRC0260 |
醇类 | |||
异戊醇 | 10.07 | 10.79 | 29.07 |
乙基酯类 | |||
苯甲酸乙酯 | 1.14 | 1.24 | 0.00 |
感官检验的评价 | 5 | 5 | 1 |
从NBRC1290株与NBRC1887株的培养液强烈地感觉到复杂且新鲜的水果样香味,对香气成分进行分析,并将各化合物的含量加以比较,结果特征为苯甲酸乙酯的含量较多。作为与这些培养液的比较,对将另一种酵母(马克思克鲁维酵母NBRC0260,Kluyveromycesmarxianus NBRC0260株)在含有乳清的培养基中进行培养而获得的培养液的香气成分进行分析、评价,结果如蔷薇般的香味强烈,但几乎不发出水果样香味,不含有苯甲酸乙酯。
对NBRC1290株和NBRC1887株的培养液、以及与培养液中的苯甲酸乙酯等量的化学合成品单独的香味进行感官检验,结果,对于NBRC1290株和NBRC1887株的培养液,感觉到化学合成品不会发出的香味的复杂感,是优于化学合成品的香味。
(3)菌的香气成分产生量根据培养基的差异
使用NBRC1290株和NBRC1887株进行了实验。
(i)前培养
前培养:向包含1%的葡萄糖的2ml含有乳清的培养基、2ml的YPD培养基(1%酵母提取物、2%蛋白胨、2%葡萄糖)、2ml的YM培养基(1%葡萄糖、0.5%蛋白胨、0.3%酵母提取物、0.3%麦芽提取物)中,分别接种预前培养中所获得的培养液20μL,在30℃、160rpm下振荡培养24小时。
(ii)培养
培养:向包含1%的葡萄糖的2ml含有乳清的培养基、2ml的YPD培养基、2ml的YM培养基中,分别接种前培养中所获得的培养液5ml,在30℃、160rpm下振荡培养24小时。
(iii)香气成分的含量的分析
培养完毕后,利用HS-GC-FID对培养物中的香气成分的含量进行分析(表3、表4)。将结果示于表6。此外,以与实施例1(2)相同的基准进行了培养物的香味的感官检验。将其结果示于表6。
[表6]
从在含有乳清的培养基中培养NBRC1290株与NBRC1887株而获得的培养液强烈地感觉到复杂且新鲜的水果样香味,培养液大量含有苯甲酸乙酯。另一方面,在YM培养基、YPD培养基中培养上述菌株而获得的培养液中有时也含有苯甲酸乙酯,但其含量较少,几乎不发出复杂且新鲜的水果样香味。
(4)菌的香气成分产生量根据培养条件的差异
使用NBRC1887株进行了实验。
(i)前培养
前培养:向包含1%的葡萄糖的2ml的含有乳清的培养基中接种预前培养中所获得的培养液20μL,在30℃、160rpm下振荡培养24小时。
(ii)培养
培养:向包含1%的葡萄糖的100ml的含有乳清的培养基中接种前培养液1ml,在15℃、20℃、25℃、30℃的培养条件下,在160rpm下进行振荡培养。此外,在培养了0、24、32、48、56小时的时候回收样品。
(iii)苯甲酸乙酯的含量的分析
利用HS-GC-FID对各培养物中的苯甲酸乙酯的含量进行分析(表3、表4)。将结果示于表7。此外,基于下述基准进行培养物的香味的感官检验。将其结果示于表7。
(感官检验的评价基准)
◎:强烈地发出复杂且新鲜的水果样香味。
○:发出复杂且新鲜的水果样香味。
△:略微发出复杂且新鲜的水果样香味,但复杂感较弱。
▲:其他香气成分的香味较强,香味的特征发生变化,发出如日本酒般的香味。
×:不发出复杂且新鲜的水果样香味。
[表7]
苯甲酸乙酯(ppm)/感官检验的评价 | 0小时 | 24小时 | 32小时 | 48小时 | 54小时 |
15℃ | 0/× | 0.73/△ | 2.48/△ | 2.24/△ | 2.18/△ |
20℃ | 0/× | 3.04/◎ | 2.54/○ | 2.06/▲ | 1.90/▲ |
25℃ | 0/× | 2.48/○ | 1.89/○ | 1.65/▲ | 1.67/▲ |
30℃ | 0/× | 2.23/○ | 1.72/○ | 1.26/▲ | 1.51/▲ |
在培养温度为15℃的情况下,虽然通过微生物的发酵而产生苯甲酸乙酯,但其他香气成分的香味较弱,培养物的香味的复杂感较弱。另一方面,在20~30℃下进行培养而获得的培养物发出复杂且新鲜的水果样香味,香味良好。此外,若培养时间达到48小时以上,则因微生物的发酵,苯甲酸乙酯以外的香气成分的产生量会上升,因此其他香气成分的香味变强,香味的特征发生变化,发出如日本酒般的香味。因此,作为培养时间,宜为24~32小时。
(5)将苯甲酸或其盐配合至培养基中时的苯甲酸乙酯含量的差异使用NBRC1887株进行了实验。
(i)前培养
前培养:向包含1%的葡萄糖的2ml含有乳清的培养基中接种预前培养中所获得的培养液20μl,在30℃、160rpm下振荡培养24小时。
(ii)培养
培养:向包含1%的葡萄糖的100ml含有乳清的培养基中,以成为0.01%的方式配合苯甲酸钠。此外,同样地,向包含1%的葡萄糖的100ml含有乳清的培养基中,分别以成为0.01%的方式配合苯甲酸,以成为0.01%、0.05%、0.1%的方式配合苯丙氨酸,以成为0.01%、0.05%的方式配合对羟基甲基苯。进而,作为对照,也准备了不配合添加物的包含1%的葡萄糖的100ml含有乳清的培养基。向各培养基中接种前培养液1ml,在30℃、160rpm下进行振荡培养。此外,在培养了0、24、32、48小时的时候回收样品。
(iii)苯甲酸乙酯的含量的分析
利用HS-GC-FID对各培养物中的苯甲酸乙酯的含量进行分析(表3、表4)。将结果示于表8。
[表8]
各培养液中的苯甲酸乙酯含量(ppm)
在配合有苯甲酸或苯甲酸钠的情况下,在培养了32~48小时的时候,培养物中的苯甲酸乙酯的含量显著增加。在配合有其他物质的情况下,培养物中的苯甲酸乙酯的含量几乎未增加。
制造例1
以表9的组成将(5)与(6)混合,向其中添加(1)~(4)并充分地搅拌,从而制备了化妆水。该化妆水发出复杂且新鲜的水果样香味。其中,作为培养液,使用了以下物质,即,向包含1%的葡萄糖的含有乳清的培养基100ml中接种实施例1(4)的前培养中所获得的前培养液1ml,在20℃、24小时、160rpm下进行振荡培养而获得培养液,对该培养液进行过滤而获得的物质。
[表9]
原料 | 使用量(%) | |
(1) | 乙醇 | 5.0 |
(2) | 1,3-丁二醇 | 2.0 |
(3) | 聚氧乙烯硬化蓖麻油 | 0.05 |
(4) | 对羟基苯甲酸甲酯 | 0.1 |
(5) | 培养液 | 10.0 |
(6) | 蒸馏水 | 使整体成为100的量 |
制造例2
以表10的组成,对(10)添加(7)~(9)并加温,在80℃下添加(1)~(6)并进行乳化,冷却至室温,而制备了乳液。该乳液发出复杂且新鲜的水果样香味。其中,培养液使用了与制造例1相同的培养液。
[表10]
原料 | 使用量(%) | |
(1) | 硬脂酸 | 2.0 |
(2) | 液体石蜡 | 5.0 |
(3) | 角鲨烷 | 2.0 |
(4) | 山梨糖醇酐单硬脂酸酯 | 0.05 |
(5) | 聚氧乙烯(20)山梨糖醇酐单硬脂酸酯 | 2.0 |
(6) | 对羟基苯甲酸丁酯 | 0.05 |
(7) | 甘油 | 2.0 |
(8) | 对羟基苯甲酸甲酯 | 0.1 |
(9) | 培养液 | 3.0 |
(10) | 蒸馏水 | 使整体成为100的量 |
制造例3
以表11的组成,对(12)添加(9)~(11)并加温,在80℃下添加(1)~(8)并进行乳化,冷却至室温,而制备了乳霜。该乳霜发出复杂且新鲜的水果样香味。其中,培养液使用了与制造例1相同的培养液。
[表11]
原料 | 使用量(%) | |
(1) | 液体石蜡 | 23.0 |
(2) | 凡士林 | 7.0 |
(3) | 鲸蜡醇 | 1.0 |
(4) | 硬脂酸 | 2.0 |
(5) | 蜂蜡 | 2.0 |
(6) | 山梨糖醇酐单硬脂酸酯 | 3.5 |
(7) | 聚氧乙烯(20)山梨糖醇酐单硬脂酸酯 | 2.5 |
(8) | 对羟基苯甲酸丁酯 | 0.05 |
(9) | 1,3-丁二醇 | 1.0 |
(10) | 对羟基苯甲酸甲酯 | 0.1 |
(11) | 培养液 | 3.0 |
(12) | 蒸馏水 | 使整体成为100的量 |
Claims (10)
1.一种培养物,其特征在于:
其为将属于威克汉姆酵母属的微生物在含有乳成分的培养基中进行培养而获得的。
2.如权利要求1所述的培养物,其特征在于:
含有乳成分的培养基为含有乳清的培养基。
3.如权利要求1或2所述的培养物,其特征在于:
属于威克汉姆酵母属的微生物为皮杰普氏威克汉姆酵母。
4.如权利要求1~3中任一项所述的培养物,其特征在于:
属于威克汉姆酵母属的微生物为皮杰普氏威克汉姆酵母NBRC1290和/或皮杰普氏威克汉姆酵母NBRC1887。
5.如权利要求1~4中任一项所述的培养物,其特征在于:
含有乳成分的培养基是还含有苯甲酸和/或其盐的培养基。
6.一种香料用组合物,其特征在于:
其含有权利要求1~5中任一项所述的培养物。
7.一种化妆品,其特征在于:
其含有权利要求1~5中任一项所述的培养物。
8.一种饮食品,其特征在于:
其含有权利要求1~5中任一项所述的培养物。
9.一种医药品,其特征在于:
其含有权利要求1~5中任一项所述的培养物。
10.一种对化妆品、饮食品或医药品赋予香味的方法,其特征在于:配合权利要求1~5中任一项所述的培养物。
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