JP6429513B2 - Effective use of sugar alcohol mixture - Google Patents
Effective use of sugar alcohol mixture Download PDFInfo
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- JP6429513B2 JP6429513B2 JP2014138375A JP2014138375A JP6429513B2 JP 6429513 B2 JP6429513 B2 JP 6429513B2 JP 2014138375 A JP2014138375 A JP 2014138375A JP 2014138375 A JP2014138375 A JP 2014138375A JP 6429513 B2 JP6429513 B2 JP 6429513B2
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- maltitol
- candida utilis
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- sorbitol
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- 150000005846 sugar alcohols Chemical class 0.000 title description 19
- 239000000203 mixture Substances 0.000 title description 2
- 239000000845 maltitol Substances 0.000 claims description 44
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 claims description 44
- 235000010449 maltitol Nutrition 0.000 claims description 44
- 229940035436 maltitol Drugs 0.000 claims description 44
- 241000235646 Cyberlindnera jadinii Species 0.000 claims description 32
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 23
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 23
- 239000000600 sorbitol Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 26
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 26
- 229960002920 sorbitol Drugs 0.000 description 22
- 235000000346 sugar Nutrition 0.000 description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 19
- 239000008103 glucose Substances 0.000 description 19
- 239000002609 medium Substances 0.000 description 17
- 230000012010 growth Effects 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 210000005253 yeast cell Anatomy 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 239000007222 ypd medium Substances 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 3
- 241000235058 Komagataella pastoris Species 0.000 description 3
- 244000253911 Saccharomyces fragilis Species 0.000 description 3
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 3
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010006326 Breath odour Diseases 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 201000006082 Chickenpox Diseases 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241001518243 Gluconobacter frateurii Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241001123227 Saccharomyces pastorianus Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000006364 Torula Species 0.000 description 1
- 206010046980 Varicella Diseases 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000001013 cariogenic effect Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、糖アルコールであるマルチトールとソルビトールを糖源（炭素源）として酵母を培養する、酵母菌体の製造法に関する。さらには、糖アルコール製造工程液を培地に用いて酵母を培養する、酵母菌体の製造法に関する。 The present invention relates to a method for producing yeast cells, wherein yeast is cultured using maltitol and sorbitol, which are sugar alcohols, as sugar sources (carbon sources). Furthermore, it is related with the manufacturing method of a yeast cell body which culture | cultivates yeast using a sugar alcohol manufacturing process liquid for a culture medium.
近年、肥満や生活習慣病の予防や、虫歯や口臭の予防といったオーラルケアへの関心が高まり、低カロリー・非齲蝕性などの機能性を持つ糖アルコールが、各種シュガーレス食品の原料として注目され、需要が伸びている。糖アルコールはトウモロコシやイモの澱粉などを原料として得られる種々の糖、水飴に水素を添加して製造されるが、その製造過程でマルチトールやソルビトールを多く含む混合液を生じる。このうちソルビトールは、これを資化できる微生物は多種の糖や糖アルコールを酸化する能力に長けているGluconobactor属細菌などに限定される。また、もう一つの主要成分であるマルチトールに至っては、微生物により資化できるといった報告はされていない。 In recent years, interest in oral care such as prevention of obesity and lifestyle-related diseases and prevention of tooth decay and bad breath has increased, and sugar alcohols that have low calorie and non-cariogenic functions have attracted attention as raw materials for various sugarless foods. , Demand is growing. Sugar alcohol is produced by adding hydrogen to various sugars obtained from corn or potato starch as a raw material, or chickenpox. In the production process, a mixture containing a large amount of maltitol and sorbitol is produced. Among these, sorbitol is limited to microorganisms that can assimilate this, such as bacteria belonging to the genus Gluconobactor, which have excellent ability to oxidize various sugars and sugar alcohols. There has been no report that maltitol, which is another main component, can be assimilated by microorganisms.
ところで他方、調味料等の原料となる酵母菌体を取得するために、酵母の培養が多く行われているが、その培地に添加する糖としてはグルコースが一般的であり、それが酵母菌体生産コストの一部を占めている。 On the other hand, in order to obtain yeast cells that are raw materials for seasonings and the like, yeast is often cultured, but glucose is generally used as a sugar to be added to the medium. It accounts for part of the production cost.
本発明は、マルチトール又はソルビトールを糖源として酵母菌体の製造を行うこと、及びマルチトールやソルビトールを含む混合液を糖源に用いて、酵母菌体の製造を行うことを課題とする。 An object of the present invention is to produce yeast cells using maltitol or sorbitol as a sugar source, and to produce yeast cells using a mixed solution containing maltitol or sorbitol as a sugar source.
本発明者は、糖アルコール廃液の主要な成分であるマルチトールやソルビトールを、食用酵母であるキャンディダ・ユーティリス（Candida utilis）が資化できること、すなわちそれらを糖源として酵母菌体の培養ができることを見出し、本発明に到達した。
すなわち本発明は、
（１） マルチトールまたはソルビトールをキャンディダ・ユーティリス（Candida utilis）に資化させることを特徴とする、キャンディダ・ユーティリス（Candida utilis）の培養方法、
（２） 上記（１）の培養方法により得られた、キャンディダ・ユーティリス（Candida utilis）の菌体
を提供するものである。
The present inventor is able to assimilate maltitol and sorbitol, which are main components of sugar alcohol waste liquor, by edible yeast, Candida utilis, that is, culturing yeast cells using them as a sugar source. We have found out that we can do it and have reached the present invention.
That is, the present invention
(1) A method for culturing Candida utilis, characterized in that maltitol or sorbitol is assimilated by Candida utilis.
(2) The present invention provides a cell body of Candida utilis obtained by the culture method of (1) above.
本発明により、これまで微生物による資化が困難とされていたマルチトールやソルビトールを、キャンディダ・ユーティリスの培養糖源として用いうるようになった。これにより、糖アルコール製造工程で生じる、マルチトールやソルビトールを含む混合液でも、培地用グルコースの代替をすることが可能となる。さらに、マルチトールやソルビトールを資化できる微生物がほとんど無いことから、他の糖源を用いた場合と比較して酵母の培養において最も問題となる汚染のリスクを軽減できる。 According to the present invention, maltitol and sorbitol, which have been difficult to assimilate by microorganisms, can be used as a cultured sugar source for Candida utilis. This makes it possible to replace glucose for the medium with a mixed solution containing maltitol and sorbitol produced in the sugar alcohol production process. Furthermore, since there are few microorganisms that can assimilate maltitol and sorbitol, the risk of contamination, which is the most problematic in yeast culture, can be reduced compared to the case of using other sugar sources.
以下、本発明を詳細に説明する。
本発明において用いるマルチトール、ソルビトールは、糖アルコールに属する化合物である。
工業的に糖アルコールを製造する過程において、マルチトールやソルビトールを含む混合液が生じる。
本発明は、酵母の一種であるキャンディダ・ユーティリス（Candida utilis）に、マルチトール、ソルビトールを資化させるものである。
Hereinafter, the present invention will be described in detail.
Maltitol and sorbitol used in the present invention are compounds belonging to sugar alcohols.
In the process of industrially producing sugar alcohol, a mixed solution containing maltitol and sorbitol is produced.
In the present invention, maltitol and sorbitol are assimilated by Candida utilis, which is a kind of yeast.
酵母には、Saccharomyces属、Schizosaccharomyces属、Candida属、Yarrowia属、Pichia属、Hansenula属、Kluyveromyces属等の種類が知られている。
いわゆる食用酵母には、通称ビール酵母（Saccharomyces pastorianus, Saccharomyces cerevisiae）、パン酵母（Saccharomyces cerevisiae）、トルラ酵母（Candida utilis）と呼ばれるものがある。そのうち調味料用の酵母エキスに一般的に用いられている酵母菌株には、サッカロミセス・セレビシエ（Saccharomyces cerevisiae）とキャンディダ・ユーティリス（Candida utilis）がある。
Known yeasts include the genera Saccharomyces, Schizosaccharomyces, Candida, Yarrowia, Pichia, Hansenula, and Kluyveromyces.
So-called edible yeasts include what are commonly called brewer's yeast (Saccharomyces pastorianus, Saccharomyces cerevisiae), baker's yeast (Saccharomyces cerevisiae), and torula yeast (Candida utilis). Among them, yeast strains commonly used for seasoning yeast extracts include Saccharomyces cerevisiae and Candida utilis.
本発明は、上述した多種多様の酵母菌株のうち、食用酵母であるキャンディダ・ユーティリス（Candida utilis）に、マルチトール及びソルビトールを資化する能力があることを見出したものである。本発明に用いるキャンディダ・ユーティリス菌株は、特に遺伝子組み換えや形質転換等を行う必要はなく、一般的に用いられている菌株でよい。 The present invention has been found that among the various yeast strains described above, the edible yeast Candida utilis has the ability to assimilate maltitol and sorbitol. The Candida utilis strain used in the present invention is not particularly required to be genetically modified or transformed, and may be a commonly used strain.
本発明においては、マルチトール、ソルビトールを、キャンディダ・ユーティリスの培養の際に、糖源の一部または全部として、培地に添加することができる。
添加する糖アルコールは、販売されている精製品のマルチトール、ソルビトールでもよいし、糖アルコール製造工程から得られたマルチトールとソルビトールを含む混合液でもよい。マルチトールとソルビトールを含む混合液を糖源として用いる場合、マルチトールとソルビトールの混合比は任意でよく、含有量も培地組成時に調整できるため、任意でよい。
In the present invention, maltitol and sorbitol can be added to the medium as part or all of the sugar source during the cultivation of Candida utilis.
The sugar alcohol to be added may be maltitol and sorbitol, which are commercially available products, or a mixed solution containing maltitol and sorbitol obtained from the sugar alcohol production process. When a mixed solution containing maltitol and sorbitol is used as a sugar source, the mixing ratio of maltitol and sorbitol may be arbitrary, and the content can be adjusted at the time of medium composition, and thus may be arbitrary.
本発明で培養に使用する培地組成には、これらの糖源の他に、窒素源、硫黄源、リン酸塩、ミネラルが含まれていればよい。 In addition to these sugar sources, the medium composition used for culture in the present invention only needs to contain a nitrogen source, a sulfur source, a phosphate, and a mineral.
本発明において、糖アルコールの資化性を有する菌株とは、酵母を糖アルコール含有培地、例えば、SM培地（0.67w/v%イーストニトロゲンベース、2w/v％マルチトール、ｐHを６．５に調製） にて3日間培養したとき、菌体の生育が見られたもののことを言う。 In the present invention, a strain having an assimilability of sugar alcohol refers to a yeast containing a sugar alcohol-containing medium such as SM medium (0.67 w / v% yeast nitrogen base, 2 w / v% maltitol, pH 6.5). In this case, the growth of the cells was observed when cultured for 3 days.
以下に実施例および比較例を用いて、本発明を具体的に説明する。
本発明はこれらに限定されるものではない。
The present invention will be specifically described below with reference to examples and comparative examples.
The present invention is not limited to these.
＜マルチトールを資化できる菌株＞
＜実施例１＞
Candida utilis NBRC0988を５ ｍｌのYPD培地（酵母エキス１ｗ/ｖ％、ポリペプトン２ｗ/ｖ％、グルコース２ｗ/ｖ％）で一晩前培養した培養液を、１００ｍｌのSD培地（0.67w/v%イーストニトロゲンベース、2w/v%グルコース、pHを6.5に調整）とSM培地（0.67w/v%イーストニトロゲンベース、2w/v％マルチトール、ｐHを６．５に調整）にそれぞれ０．５ｖ/ｖ％植菌し、３０℃、２２５ｒｐｍで培養した。SD培地の糖源はグルコース、SM培地の糖源はマルチトールである。それぞれについて、菌体増殖の指標として培養液のOD６００を測定し、その経時変化をプロットしたグラフを図１Aに示す。
<Strain capable of assimilating maltitol>
<Example 1>
Candida utilis NBRC0988 was precultured overnight in 5 ml of YPD medium (yeast extract 1 w / v%, polypeptone 2 w / v%, glucose 2 w / v%), and 100 ml of SD medium (0.67 w / v% yeast). Nitrogen base, 2w / v% glucose, pH adjusted to 6.5) and SM medium (0.67w / v% yeast nitrogen base, 2w / v% maltitol, pH adjusted to 6.5) 0.5v each / v% inoculated and cultured at 30 ° C. and 225 rpm. The sugar source of SD medium is glucose, and the sugar source of SM medium is maltitol. FIG. 1A shows a graph in which the OD 600 of the culture broth was measured as an indicator of cell growth and the change with time was plotted.
＜比較例１＞
実施例１において、Candida utilisの代わりにSaccharomyces cerevisiae NBRC101557を用いたこと以外は実施例１と同様に行った。その結果を図１Bに示す。
<Comparative Example 1>
In Example 1, it carried out similarly to Example 1 except having used Saccharomyces cerevisiae NBRC101557 instead of Candida utilis. The result is shown in FIG. 1B.
＜比較例２＞
実施例１において、Candida utilisの代わりにPichia pastoris NBRC0948を用いたこと以外は実施例１と同様に行った。その結果を図１Cに示す。
<Comparative Example 2>
In Example 1, it carried out like Example 1 except having used Pichia pastoris NBRC0948 instead of Candida utilis. The result is shown in FIG. 1C.
＜比較例３＞
実施例１において、Candida utilisの代わりにKluyveromyces marxianus NBRC1777を用いたこと以外は実施例１と同様に行った。その結果を図１Dに示す。
<Comparative Example 3>
In Example 1, it carried out like Example 1 except having used Kluyveromyces marxianus NBRC1777 instead of Candida utilis. The result is shown in FIG. 1D.
＜比較例４＞
実施例１において、Candida utilisの代わりにSchizosaccharomyces pombe NBRC0340を用い、前培養にYPD培地の代わりにYE培地（酵母エキス０．５％、グルコース３％）を用いたこと以外は実施例１と同様に行った。その結果を図１Eに示す。
<Comparative example 4>
In Example 1, Schizosaccharomyces pombe NBRC0340 was used instead of Candida utilis, and YE medium (yeast extract 0.5%, glucose 3%) was used instead of YPD medium for pre-culture, as in Example 1. went. The result is shown in FIG. 1E.
実施例１〜比較例４の結果、図１Ａ〜図１Ｅのグラフから明らかなように、 Candida utilisは高いマルチトール資化能力を示した。それに対して、Saccharomyces cerevisiae、Pichia pastoris、Kluyveromyces marxianus、Schizosaccharomyces pombeはいずれもマルチトールを資化することができなかった。 As a result of Example 1 to Comparative Example 4, as can be seen from the graphs of FIGS. 1A to 1E, Candida utilis showed high maltitol utilization ability. In contrast, none of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, and Schizosaccharomyces pombe could assimilate maltitol.
＜マルチトール資化の特異性＞
＜実施例２＞
Candida utilis NBRC0988を５ ｍｌのYPD培地（酵母エキス１％、ポリペプトン２％、グルコース２％）で一晩前培養した培養液を１００ｍｌのSM培地（0.67w/v%イーストニトロゲンベース、2w/v％マルチトール、ｐHを６．５に調整）に０．５％（ｖ／ｖ）植菌し、３０℃、２２５ｒｐｍで培養し、その培養液のOD６００を測定し、経時変化を見た。
<Specificity of maltitol utilization>
<Example 2>
Candida utilis NBRC0988 was pre-cultured overnight in 5 ml YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in 100 ml SM medium (0.67w / v% yeast nitrogen base, 2w / v % Maltitol, pH adjusted to 6.5), 0.5% (v / v) was inoculated, cultured at 30 ° C. and 225 rpm, OD 600 of the culture was measured, and the change with time was observed.
＜比較例５＞
実施例２において、SM培地のマルチトールの代わりに、各々グルコース、フルクトース、スクロース、マルトースを配合した培地を用い、実施例２と同様に試験を行った。
培養開始から４８時間後のOD６００を測定し、その結果を表１に示す。実施例２のマルチトール、比較例５のグルコース、フルクトース、スクロース、マルトースともに増殖は見られたが、糖１ｇあたりのOD６００はマルチトールが一番高く、Candida utilisの糖アルコール資化能力が高いことを示す結果となった。
<Comparative Example 5>
In Example 2, a test was conducted in the same manner as in Example 2 using a medium containing glucose, fructose, sucrose, and maltose instead of maltitol in the SM medium.
The OD 600 after 48 hours from the start of the culture was measured, and the results are shown in Table 1. Maltitol in Example 2 and glucose, fructose, sucrose, and maltose in Comparative Example 5 grew, but OD 600 per gram of sugar was highest in maltitol, and Candida utilis had a high ability to utilize sugar alcohol. The result showed that.
＜多様な糖アルコール資化性＞
＜実施例３＞
Candida utilis NBRC0988を５ ｍｌのYPD培地で一晩前培養した培養液を、１００ｍｌのSM培地（0.67w/v%イーストニトロゲンベース、2w/v％マルチトール、ｐHを６．５に調整）に０．５ｖ/ｖ％植菌し、３０℃、２２５ｒｐｍで培養し、その培養液のOD６００を測定し、経時変化を見た。
<Various sugar alcohol utilization>
<Example 3>
Candida utilis NBRC0988 pre-cultured overnight in 5 ml YPD medium into 100 ml SM medium (0.67 w / v% yeast nitrogen base, 2 w / v% maltitol, pH adjusted to 6.5) 0.5 v / v% was inoculated, cultured at 30 ° C. and 225 rpm, the OD 600 of the culture was measured, and the change with time was observed.
＜比較例６＞
実施例３において、SM培地のマルチトールの代わりに、各々ソルビトール、マンニトールを用いたこと以外は実施例３と同様に試験を行った。
培養開始から４８時間後のOD600を測定し、その結果を表２に示す。糖アルコールの種類によって菌体の増殖に差は見られたものの、いずれの糖アルコールでも増殖は確認できた。
<Comparative Example 6>
In Example 3, the test was performed in the same manner as in Example 3 except that sorbitol and mannitol were used instead of maltitol in the SM medium.
The OD 600 after 48 hours from the start of the culture was measured, and the results are shown in Table 2. Although there was a difference in the growth of bacterial cells depending on the type of sugar alcohol, growth was confirmed with any sugar alcohol.
＜菌株間の増殖速度の差異＞
＜実施例４＞
Candida utilis NBRC0987とCandida utilis NBRC0988を５ ｍｌのYPD培地（酵母エキス１％、ポリペプトン２％、グルコース２％）で一晩前培養した培養液を１００ｍｌのSD培地とSM培地に０．５％（ｖ／ｖ）植菌し、３０℃、２２５ｒｐｍで培養し、その培養液のOD６００を測定し、経時変化を見た。
なお、この２菌株ともCandida utilisのスタンダードな菌株であり、NBRCから入手できる。
<Difference in growth rate between strains>
<Example 4>
Candida utilis NBRC0987 and Candida utilis NBRC0988 were pre-cultured overnight in 5 ml of YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in 100 ml SD medium and SM medium 0.5% (v / V) Inoculated, cultured at 30 ° C. and 225 rpm, OD 600 of the culture was measured, and changes with time were observed.
These two strains are standard strains of Candida utilis and can be obtained from NBRC.
図２AにCandida utilis NBRC0987の増殖の経時変化を、図２BにCandida utilis NBRC0988の増殖の経時変化を示す。糖源がマルチトールの時、NBRC0987はNBRC0988より増殖の立ち上がりが遅れたが、いずれの菌株もマルチトールを資化していることから、キャンディダ・ユーティリス（Candida utilis）に属する菌株は概ねマルチトールの資化性を保有していることが示された。 FIG. 2A shows the time course of the growth of Candida utilis NBRC0987, and FIG. 2B shows the time course of the growth of Candida utilis NBRC0988. When the sugar source is maltitol, the growth start of NBRC0987 was delayed from that of NBRC0988, but since all strains assimilated maltitol, strains belonging to Candida utilis are mostly maltitol. It was shown that it possessed the assimilation property.
以上説明してきたように、本発明によると、キャンディダ・ユーティリスはマルチトールやソルビトールを資化できることから、それらを多く含む糖アルコール工程液を糖源として、培養することができる。
As described above, according to the present invention, Candida utilis can assimilate maltitol and sorbitol, and therefore can be cultured using a sugar alcohol process liquid containing a large amount thereof as a sugar source.
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