JP2008000075A - Method for producing distilled liquor - Google Patents
Method for producing distilled liquor Download PDFInfo
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- JP2008000075A JP2008000075A JP2006172915A JP2006172915A JP2008000075A JP 2008000075 A JP2008000075 A JP 2008000075A JP 2006172915 A JP2006172915 A JP 2006172915A JP 2006172915 A JP2006172915 A JP 2006172915A JP 2008000075 A JP2008000075 A JP 2008000075A
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- lactococcus lactis
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 38
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- Alcoholic Beverages (AREA)
Abstract
Description
本発明は、香味豊かな蒸留酒の製造方法に関し、更に詳細には、泡盛、焼酎等の蒸留酒の製造において、もろみ中に含まれるフェルラ酸の４−ビニルグアヤコール（４−ＶＧ）への転化を促進することによる、香味の豊かな蒸留酒の製造方法に関する。 The present invention relates to a method for producing a flavored distilled liquor, and more specifically, in the production of distilled liquor such as awamori and shochu, conversion of ferulic acid contained in moromi into 4-vinyl guaiacol (4-VG). It is related with the manufacturing method of rich distilled spirits by promoting.
近年、健康志向の高まりから、清酒等のいわゆる醸造酒よりも、泡盛、焼酎等の蒸留酒を愛好する人が多くなっている。この蒸留酒は、長期間の保存が可能であり、長期間保存（熟成）した後の蒸留酒は、製造直後のものよりも香味が高く、古酒として高い評価を得ている。 In recent years, due to an increase in health consciousness, more and more people like distilled sake such as awamori and shochu than brewed sake such as sake. This distilled liquor can be stored for a long period of time, and the distilled liquor after being stored (aged) for a long period of time has a higher flavor than that immediately after production and has been highly evaluated as an old liquor.
ところで、沖縄県には４７泡盛酒造所と１酒造組合があり、沖縄本島内のみならず周辺離島において、蒸留酒である泡盛が広く製造されている。その中でも、最近では特に「泡盛古酒」が注目されており、その芳醇な香りとまろやかな口あたりが人気を集めている。 By the way, there are 47 Awamori Brewery and 1 Brewery Association in Okinawa Prefecture, and Awamori, a distilled liquor, is widely produced not only on the main island of Okinawa but also on the surrounding remote islands. Among them, “Awamori Koshu” has recently attracted attention, and its rich fragrance and mellow taste are gaining popularity.
この泡盛古酒の香り成分は１００種類以上あると言われ、それぞれの含有量やバランスが泡盛の個性を決定しているが、泡盛に含まれる重要な香味成分の一つにバニリンがある。バニリンはバニラに含まれる甘い芳香を持つ物質で、泡盛古酒が有する甘い香りの起因成分の一つとなっている。 It is said that there are over 100 kinds of fragrance components of Awamori Koshu, and each content and balance determines the individuality of Awamori. Vanillin is one of the important flavor components contained in Awamori. Vanillin is a substance with a sweet fragrance contained in vanilla, and is one of the components that cause the sweet scent of Awamori Koshu.
泡盛製造過程において下記式１に示されるようにバニリンは原料米由来のフェルラ酸が遊離し、これがもろみの酸や蒸留過程の熱により４−ＶＧへと変化して泡盛中に移行する。さらに熟成により４−ＶＧがバニリンへと変化することで泡盛中のバニリンが生成すると考えられている（非特許文献１〜３）。
[式１]
[Formula 1]
このような知見に基づき本出願人はもろみ中の４−ＶＧ量と蒸留後の泡盛の４−ＶＧ量との関係を調べたところ、もろみ中の４−ＶＧ量が高い場合には蒸留後の泡盛の４−ＶＧ量も高くなる傾向があることを知った。また、本出願人は通常もろみの発酵に用いられる酵母にはフェルラ酸を４−ＶＧに転化する能力がないので、もろみ中の４−ＶＧ生成に対してはもろみ中の酵母以外の何らかの微生物が関与していると判断した。 Based on such knowledge, the applicant examined the relationship between the amount of 4-VG in the mash and the amount of 4-VG in Awamori after the distillation. I learned that Awamori's 4-VG amount also tends to increase. In addition, since the yeast usually used for fermentation of moromi does not have the ability to convert ferulic acid into 4-VG, any microorganism other than the yeast in the mash is used to produce 4-VG in mash. Judged to be involved.
本発明は、もろみ中でフェルラ酸を４−ＶＧに転化する微生物を明らかにし、その微生物を用い、従来よりも簡便な方法で香味豊かな蒸留酒を製造する方法を提供することをその課題とするものである。 An object of the present invention is to clarify a microorganism that converts ferulic acid into 4-VG in moromi and to provide a method for producing a flavored distilled liquor using the microorganism in a simpler method than before. To do.
本発明者らは、もろみの４−ＶＧ量と、この中に存在する微生物との関係を探索した結果、フェルラ酸を４−ＶＧに転化すると思われる微生物を見出した。そしてもろみにこの微生物を作用させると、もろみ中の４−ＶＧ量が高くなることおよびこのもろみを蒸留した後の蒸留酒中の４−ＶＧ量も高くなることを見出し、本発明を完成した。 As a result of searching for the relationship between the amount of 4-VG in moromi and the microorganisms present therein, the present inventors have found a microorganism that seems to convert ferulic acid into 4-VG. And when this microorganism was made to act on the mash, the amount of 4-VG in the mash was increased, and the amount of 4-VG in the distilled liquor after the mash was distilled was also found, and the present invention was completed.
すなわち本発明は、植物由来の酒造原料を、麹菌および／または酵母で発酵させてもろみとし、これを蒸留する蒸留酒の製造方法において、もろみにラクトコッカス・ラクチス（Lactococcus lactis）を作用させることを特徴とする蒸留酒の製造方法である。 That is, the present invention relates to a method for producing distilled liquor in which a plant-derived brewing raw material is fermented with koji mold and / or yeast, and distilled from the mash, and the mash is allowed to act with Lactococcus lactis. It is the manufacturing method of the distilled spirit characterized by the above.
また本発明は、植物由来の酒造原料を、麹菌および／または酵母で発酵させてもろみとし、このもろみにラクトコッカス・ラクチスを作用させた後、これを蒸留することにより得られる蒸留酒である。 In addition, the present invention is a distilled liquor obtained by distilling a brewing material derived from a plant from mash by fermentation with koji mold and / or yeast, and reacting the mash with lactococcus lactis.
更に本発明は、植物由来の酒造原料を、麹菌および／または酵母で発酵させてもろみとし、これを蒸留する蒸留酒の製造方法において、もろみにラクトコッカス・ラクチスを作用させることを特徴とする蒸留酒に香味を付与する方法である。 Furthermore, the present invention provides a distilled liquor characterized by allowing lactococcus lactis to act on moromi in a method for producing distilled liquor in which a brewing material derived from a plant is fermented with koji mold and / or yeast and distilled. It is a method of imparting flavor to sake.
また更に本発明は、フェルラ酸にラクトコッカス・ラクチスを作用させることを特徴とする４−ＶＧの製造方法である。 Furthermore, the present invention is a method for producing 4-VG, characterized in that Lactococcus lactis acts on ferulic acid.
本発明の蒸留酒の製造方法によれば、もろみの原料に含まれるフェルラ酸の４−ＶＧへの転化が、ラクトコッカス・ラクチスの作用により促進されるため、もろみ中の４−ＶＧ量を高めることができる。その結果、もろみを蒸留後の蒸留酒中においても蒸留酒の香味の一つであるバニリンの前駆体である４−ＶＧ量を有意に高めることができる。 According to the method for producing distilled liquor of the present invention, the conversion of ferulic acid contained in the raw material of moromi into 4-VG is promoted by the action of Lactococcus lactis, so the amount of 4-VG in the mash is increased. be able to. As a result, the amount of 4-VG, which is a precursor of vanillin, which is one of the flavors of distilled spirits, can be significantly increased even in distilled spirits after distillation of moromi.
従って、この製造方法により得られる蒸留酒は、通常の製法で得られる蒸留酒と同期間熟成させた場合であっても、甘い芳香と香味の増強された蒸留酒となる。 Therefore, the distilled liquor obtained by this production method becomes a distilled liquor with enhanced sweet aroma and flavor even when it is aged for the same period as that obtained by a normal production method.
また、本発明の蒸留酒に香味を付与する方法によれば、簡便な方法で蒸留酒に香味を付与することができる。 Further, according to the method for imparting flavor to the distilled liquor of the present invention, flavor can be imparted to distilled spirit by a simple method.
更に、本発明の４−ＶＧの製造方法によれば、フェルラ酸からバニリンの前駆体となる４−ＶＧを効率よく製造することができる。 Furthermore, according to the method for producing 4-VG of the present invention, 4-VG that is a precursor of vanillin can be efficiently produced from ferulic acid.
本発明の蒸留酒の製造方法（以下、「本発明製法」という）は、もろみにラクトコッカス・ラクチスを作用させる以外は、発酵したもろみを蒸留する公知の蒸留酒の製造方法により実施することができる。 The method for producing a distilled liquor of the present invention (hereinafter referred to as “the method of the present invention”) can be carried out by a known method for producing distilled liquor that distills fermented mash, except that Lactococcus lactis is allowed to act on moromi. it can.
本発明製法に用いられるラクトコッカス・ラクチスは、自然界に広く分布するホモ発酵型乳酸菌の１種で、古くからゴーダーチーズ、チェダーチーズ等の半硬質タイプのチーズ、バター、発酵乳等の発酵食品の製造に用いられており、極めて安全な菌株である。このラクトコッカス・ラクチスの生理、生化学性状等は例えば、「乳酸菌の科学と技術」（乳酸菌研究談会編、学会出版センター、１９９６年）等に記載されている。また、ラクトコッカス・ラクチスの１６Ｓ ｒＤＮＡは細胞基準株データベース（http://www.nbrc.nite.go.jp）に登録されている。本発明製法においてはこれら生理、生化学性状および１６Ｓ ｒＤＮＡから、ラクトコッカス・ラクチスに分類される菌株であれば特に制限無く、例えば、ラクトコッカス・ラクチス・サブスピーシーズ・ラクチス、ラクトコッカス・ラクチス・サブスピーシーズ・クレモリス等の何れの亜種も使用することができる。なお、ラクトコッカス・ラクチスにフェルラ酸を４−ＶＧに転化する能力があることはこれまで知られていない。 Lactococcus lactis used in the production method of the present invention is a kind of homo-fermenting lactic acid bacteria widely distributed in nature, and has been producing fermented foods such as gourd cheese, cheddar cheese and other semi-rigid cheeses, butter, fermented milk, etc. It is an extremely safe strain. The physiology, biochemical properties, etc. of Lactococcus lactis are described in, for example, “Science and Technology of Lactic Acid Bacteria” (edited by the Lactic Acid Bacteria Research Conference, Society Publishing Center, 1996). In addition, 16S rDNA of Lactococcus lactis is registered in the cell reference strain database (http://www.nbrc.nite.go.jp). In the production method of the present invention, any strain classified into Lactococcus lactis based on these physiological, biochemical properties and 16S rDNA is not particularly limited. For example, Lactococcus lactis subspices lactis, Lactococcus lactis subs Any subspecies such as species Cremoris can be used. It has not been known so far that Lactococcus lactis has the ability to convert ferulic acid to 4-VG.
本発明製法に用いることのできるラクトコッカス・ラクチスの一例としては、独立行政法人製品評価技術基盤機構（〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８）等から購入可能な以下のものが挙げられる。
・ラクトコッカス・ラクチス・サブスピーシーズ・ラクチス ＮＢＲＣ１２００７
・ラクトコッカス・ラクチス・サブスピーシーズ・ラクチス ＮＢＲＣ１００９３３
・ラクトコッカス・ラクチス・サブスピーシーズ・クレモリス ＮＢＲＣ１００６７６
Examples of Lactococcus lactis that can be used in the production method of the present invention include the following, which can be purchased from the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture), etc. Things.
・ Lactococcus ・ Lactis ・ Subspecies ・ Lactis NBRC12007
・ Lactococcus ・ Lactis ・ Subspecies ・ Lactis NBRC100933
・ Lactococcus ・ Lactis ・ Sub-Species ・ Cremollis NBRC100676
上記ラクトコッカス・ラクチスをもろみに作用させるには、植物由来の酒造原料を、麹菌および／または酵母で発酵させるもろみ調製工程の何れかの段階において、ラクトコッカス・ラクチスを接種するだけでよい。具体的にラクトコッカス・ラクチスを焼酎のもろみに作用させる場合には、一次もろみと二次もろみの二段のもろみ調製工程の何れかまたは両方のもろみ仕込み時あるいはもろみ発酵過程中にラクトコッカス・ラクチスを接種すればよい。また、ラクトコッカス・ラクチスを泡盛のもろみに作用させる場合には、一次もろみのみの一段のもろみ調製工程のもろみ仕込み時あるいはもろみ発酵過程中にラクトコッカス・ラクチスを接種すればよい。また、焼酎または泡盛のもろみを調製する何れの場合であっても、製麹開始時または製麹中の麹にラクトコッカス・ラクチスを接種して付着させ、この麹を用いてもろみを調製することにより、もろみにラクトコッカス・ラクチスを作用させてもよい。本発明製法において、ラクトコッカス・ラクチスのもろみへの接種量は、もろみ１Ｌあたり１×１０３〜１×１０１８ｃｆｕ程度、好ましくは１×１０８〜１×１０１３ｃｆｕ程度である。 In order to make the above Lactococcus lactis act on the mash, it is only necessary to inoculate the Lactococcus lactis at any stage of the mash preparation process in which the brewing raw material derived from the plant is fermented with koji mold and / or yeast. Specifically, when Lactococcus lactis is allowed to act on mash of shochu, Lactococcus lactis is either during the mash preparation or during the mash fermentation process in either or both of the two-stage mash preparation steps of primary mash and secondary mash. You should inoculate. Further, when Lactococcus lactis is allowed to act on Awamori mash, Lactococcus lactis may be inoculated at the time of mash preparation in the first mash preparation process or during the mash fermentation process. Also, in any case of preparing shochu or awamori mash, inoculate and attach Lactococcus lactis to the koji at the start of koji or during koji making, and prepare koji using this koji Thus, Lactococcus lactis may be allowed to act on the moromi. In the production method of the present invention, the amount of Lactococcus lactis inoculated into the moromi is about 1 × 10 3 to 1 × 10 18 cfu, preferably about 1 × 10 8 to 1 × 10 13 cfu per liter of moromi.
本発明製法に用いられるもろみとは、米、麦（麦芽）、トウモロコシ、そば、ひえ、あわ等の穀類、じゃがいも、さつまいも等の芋類、くり、ごま等の植物、黒糖、白ぬか、清酒粕等の植物由来の酒造原料を、アスペルギルス・オリゼー等の黄麹菌、アスペルギルス・カワチ等の白麹菌、アスペルギルス・アワモリ、アスペルギルス・サイトウイ等の黒麹菌等の麹菌および／またはサッカロマイセス・セレビシエ等の焼酎酵母、サッカロマイセス・パストリアヌス等のビール酵母等の酵母で発酵させたものをいう。なお、本発明製法においては、上記酒造原料のデンプン質を酵素等により糖化したものを上記酵母で発酵させたものももろみに含まれる。本発明製法においては、これらもろみの中でも穀類を麹菌および酵母で発酵させたものが好ましく、特に穀類を黒麹菌および焼酎酵母で発酵させたものが好ましい。前記穀類を原料とするもろみはフェルラ酸を多く含有するので本発明製法による効果が特に認められる。 The moromi used in the production method of the present invention is grains such as rice, wheat (malt), corn, buckwheat, barnyard millet, potatoes, potatoes and sweet potatoes, potatoes, sesame and other plants, brown sugar, white bran, sake lees Brewing raw materials such as Aspergillus oryzae, white koji molds such as Aspergillus kawachi, koji molds such as Aspergillus awamori, Aspergillus saitoi, etc. and / or shochu yeast such as Saccharomyces cerevisiae , Fermented with yeast such as Saccharomyces pastorianus beer yeast. In addition, in the manufacturing method of this invention, what fermented with the said yeast what saccharified the starch of the said brewing raw material with an enzyme etc. is contained in mash. In the production method of the present invention, among these mashes, those obtained by fermenting cereals with koji molds and yeasts are preferred, and those obtained by fermenting cereals with black koji molds and shochu yeasts are particularly preferred. The effect of the method of the present invention is particularly recognized because the moromi mash containing cereals contains a large amount of ferulic acid.
上記のようにしてラクトコッカス・ラクチスを作用させたもろみは、次に、公知の条件で蒸留することにより焼酎、泡盛等の蒸留酒が得られる。本発明においてはこれら蒸留酒の中でも泡盛が好ましい。 The mash from which Lactococcus lactis is allowed to act as described above is then distilled under known conditions to obtain distilled liquors such as shochu and awamori. In the present invention, awamori is preferable among these distilled spirits.
本発明製法によれば、もろみにラクトコッカス・ラクチスを作用させることにより、もろみ中のフェルラ酸から４−ＶＧへの転化が促進され、もろみ中の４−ＶＧ量が高くなる。そして、このもろみを蒸留して蒸留酒とすれば、もろみ中の４−ＶＧが蒸留酒中に移行し、蒸留酒中の４−ＶＧ量が高くなる。具体的には、蒸留酒中の４−ＶＧ量は１〜５０μｇ／ｍｌ程度になり、ラクトコッカス・ラクチスを作用させていないもろみを蒸留して得られる蒸留酒と比べて５.０質量％（以下、単に「％」という）以上、好ましくは１０.０〜５０．０％程度高くなる。更に、蒸留酒中に含まれる４−ＶＧは蒸留工程での熱や、その後の熟成等により蒸留酒の香味成分の１つであるバニリンとなるので、本発明製法で得られる蒸留酒は香味豊かなものとなる。 According to the production method of the present invention, by causing Lactococcus lactis to act on the mash, the conversion of ferulic acid in the mash into 4-VG is promoted, and the amount of 4-VG in the mash is increased. And if this mash is distilled into distilled liquor, 4-VG in the mash will move into the distilled liquor and the amount of 4-VG in the distilled liquor will increase. Specifically, the amount of 4-VG in the distilled liquor is about 1 to 50 μg / ml, and is 5.0% by mass as compared with the distilled liquor obtained by distilling the moromi not reacted with Lactococcus lactis ( Hereinafter, it is simply referred to as “%”), preferably about 10.0 to 50.0%. Furthermore, since 4-VG contained in distilled liquor becomes vanillin which is one of the flavor components of distilled liquor due to heat in the distillation process and subsequent aging, the distilled liquor obtained by the method of the present invention is rich in flavor. It will be something.
本発明製法の好ましい一態様として、泡盛の製造方法を示せば次の通りである。
まず、タイ米を洗米後、水に数十分間から一晩漬浸したものを蒸煮して蒸米とする。この蒸米を放冷後、市販黒麹菌を蒸米１ｋｇ当たり１×１０６〜１×１０１０ｃｆｕ程度接種し、これを３０〜４５℃程度で、３８〜４５時間程度、適宜手入れを行いながら製麹する。この麹に、汲み水歩合１５０〜１８０％程度の水を加え、泡盛１０１号等の酵母をもろみ１Ｌ当たり１×１０８〜１×１０１３ｃｆｕ程度およびフェルラ酸を４−ＶＧに変換するラクトコッカス・ラクチスをもろみ１Ｌあたり１×１０６〜１×１０１８ｃｆｕ程度接種してもろみを仕込み、１５〜３０℃程度で仕込みタンク中で発酵する。もろみの発酵時間は７日〜２８日程度でよい。発酵の終了した熟成もろみを従来の方法に従い、蒸留機等を用いて８５〜１００℃程度で蒸留し、蒸留液として泡盛を得る。
As a preferred embodiment of the production method of the present invention, a method for producing Awamori is as follows.
First, Thai rice is washed and then soaked in water for tens of minutes to overnight. After allowing the steamed rice to cool, inoculate about 1 × 10 6 to 1 × 10 10 cfu of commercially available black koji mold per 1 kg of steamed rice, and knead it at about 30 to 45 ° C. for about 38 to 45 hours with appropriate care. To do. Lactococcus that adds about 150-180% of the water ratio of the pumped water to this koji, mashes yeast such as Awamori No. 101, etc. and converts 1 × 10 8 to 1 × 10 13 cfu per liter and ferulic acid into 4-VG.・ Lactus is mashed and the mash is fermented at about 15-30 ° C. and fermented at about 15-30 ° C. even if inoculated at about 1 × 10 6 to 1 × 10 18 cfu per liter. Moromi fermentation time may be about 7 to 28 days. The matured moromi mash after fermentation is distilled at about 85 to 100 ° C. using a distiller or the like according to a conventional method to obtain awamori as a distillate.
また、ラクトコッカス・ラクチスは上記した蒸留酒の製造以外にも、フェルラ酸を原料とした４−ＶＧの製造に用いることもできる。具体的には、市販のフェルラ酸を含有させたＭＲＳ等の培地にラクトコッカス・ラクチスを培地１Ｌあたり１×１０６〜１×１０２０ｃｆｕ程度接種し、２０〜４５℃程度の条件で数時間〜数日程度培養することにより培地中に４−ＶＧが生成される。この培地中に生成された４−ＶＧは、高速液体クロマトグラフィー等の公知の方法により精製することもできる。また、上記のようにして得られる４−ＶＧは熱や熟成、文献（特開２００３−１３５０５１号公報）に記載のオゾンへの接触等によりバニリンにすることもできる。 Lactococcus lactis can also be used for the production of 4-VG using ferulic acid as a raw material, in addition to the above-mentioned production of distilled liquor. Specifically, Lactococcus lactis is inoculated in a medium such as MRS containing commercially available ferulic acid in an amount of about 1 × 10 6 to 1 × 10 20 cfu per liter of the medium, and the condition is about 20 to 45 ° C. for several hours. By culturing for about several days, 4-VG is produced in the medium. 4-VG produced in this medium can also be purified by a known method such as high performance liquid chromatography. Further, 4-VG obtained as described above can be converted into vanillin by heat, aging, contact with ozone described in the literature (Japanese Patent Laid-Open No. 2003-135051), or the like.
以下、実施例を挙げて本発明を詳細に説明するが、本発明はこれらの実施例に何ら制限されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not restrict | limited to these Examples at all.
実 施 例 １
フェルラ酸を４−ＶＧに転化する微生物のスクリーニング：
（１）４−ＶＧを多く含む酒造所の泡盛のもろみ中から分離した多数の菌株をそれぞれＭＲＳ培地にて１０−１〜１０−５まで段階希釈した。次に、それぞれ希釈したものを０.５％の炭酸カルシウムを含有するＭＲＳ寒天培地（１５ｍｌ）に混釈し、それをシャーレに注いだ。更に、シャーレを嫌気条件下、３０℃で２４時間以上維持した後、ハローを形成したコロニーを釣菌した。
Example 1
Screening for microorganisms that convert ferulic acid to 4-VG:
(1) A large number of strains isolated from the mash of Awamori of a brewery containing a large amount of 4-VG were each serially diluted from 10 −1 to 10 −5 in MRS medium. Next, each diluted one was mixed with MRS agar medium (15 ml) containing 0.5% calcium carbonate, and poured into a petri dish. Further, the petri dish was maintained at 30 ° C. for 24 hours or more under anaerobic conditions, and then the colony that formed a halo was fished.
試験管に滅菌したＭＲＳ培地を２〜１０ｍｌ分注し、フェルラ酸を１００μｇ／ｍｌになるよう添加した。その後、上記で釣菌された菌株をＭＲＳ培地で前培養し、その１０〜１００μｌを上記ＭＲＳ培地中に播種し、３０℃で４８時間培養を行った。培養終了後、遠心分離した上清を０.４５μｍのフィルターに通し、培地中のフェルラ酸量および４−ＶＧ量をＨＰＬＣにより測定した。ＨＰＬＣ分析により培地中のフェルラ酸量が減少し、４−ＶＧ量が上昇した菌株としてＳ１株を得た。 2-10 ml of sterilized MRS medium was dispensed into a test tube, and ferulic acid was added to 100 μg / ml. Thereafter, the strains strained above were pre-cultured in MRS medium, 10 to 100 μl thereof was seeded in the MRS medium, and cultured at 30 ° C. for 48 hours. After completion of the culture, the centrifuged supernatant was passed through a 0.45 μm filter, and the amount of ferulic acid and the amount of 4-VG in the medium were measured by HPLC. S1 strain was obtained as a strain in which the amount of ferulic acid in the medium decreased and the amount of 4-VG increased by HPLC analysis.
（２）上記（１）で得たＳ１株について、培地中のフェルラ酸量と４−ＶＧ量の経時変化と菌体量の増加を以下の方法により測定した。まず、５０ｍｌ容量のスクリューキャップ付遠心沈殿管にフェルラ酸を１００μｇ／ｍｌに調整したＭＲＳ培地を３０ｍｌ分注し、別途前培養を行ったＳ１株を０.１ｍｌ（１.０×１０８ｃｆｕ）添加して、３０℃で３０時間培養を行った。この培養中は２時間ごとに１ｍｌのサンプリングを行い、これを０.４５μｍのフィルターに通し、各培養時間におけるフェルラ酸量および４−ＶＧ量をＨＰＬＣにより測定した。また、各培養時間における濁度をバイオフォトメーター（ADVANTEC製）で吸光度６００ｎｍの吸収を測定することにより求めた。これらの結果を図１に示した。 (2) About S1 strain | stump | stock obtained by said (1), the time-dependent change of the ferulic acid amount and 4-VG amount in a culture medium, and the increase in the amount of microbial cells were measured with the following method. First, 30 ml of MRS medium in which ferulic acid was adjusted to 100 μg / ml was dispensed into a 50 ml screw-centrifuged centrifugal sedimentation tube, and 0.1 ml (1.0 × 10 8 cfu) of S1 strain separately pre-cultured. After the addition, the cells were cultured at 30 ° C. for 30 hours. During this culture, 1 ml of sampling was performed every 2 hours, and this was passed through a 0.45 μm filter, and the amount of ferulic acid and the amount of 4-VG in each culture time were measured by HPLC. Moreover, the turbidity in each culture time was calculated | required by measuring absorption with a light absorbency of 600 nm with a biophotometer (made by ADVANTEC). These results are shown in FIG.
（３）上記（１）のようにして得られたＳ１株の１６Ｓ ｒＤＮＡについて、ＢＬＡＳＴを用いた相同性検索を行ったところ、Ｓ１株の１６Ｓ ｒＤＮＡはラクトコッカス・ラクチスの３亜種の１６Ｓ ｒＤＮＡと高い相同性を示した。その中でもラクトコッカス・ラクチス・サブスピーシーズ・ラクチスとは１００％の相同性を示した。また、Ｓ１株の分子系統解析の結果、Ｓ１株はラクトコッカス・ラクチスの３亜種の１６Ｓ ｒＤＮＡが形成するクラスター内に含まれることが判った（図２）。更に、Ｓ１株の生理、生化学性状試験を行ったところ、Ｓ１株はラクトコッカス・ラクチス・サブスピーシーズ・ラクチスの性状と一致した。以上の結果からＳ１株はラクトコッカス・ラクチス・サブスピーシーズ・ラクチスに帰属する菌株と同定された。なお、Ｓ１株については寄託機関に寄託をしていないが、本株は特許法施行規則第２７条の３の条件を満足する分譲の請求があった場合は、出願人において、同条の条件に従って分譲することを保証する。 (3) When the homology search using BLAST was performed for the 16S rDNA of the S1 strain obtained as described in (1) above, the 16S rDNA of the S1 strain was found to be 16S rDNA of 3 subspecies of Lactococcus lactis. And showed high homology. Among them, Lactococcus lactis subspecies lactis showed 100% homology. Further, as a result of molecular phylogenetic analysis of the S1 strain, it was found that the S1 strain was contained in a cluster formed by 16S rDNA of three subspecies of Lactococcus lactis (FIG. 2). Furthermore, when the physiological and biochemical property tests of the S1 strain were performed, the S1 strain was consistent with the properties of Lactococcus lactis, Subspecies lactis. From the above results, the S1 strain was identified as a strain belonging to Lactococcus lactis subspecies lactis. S1 shares have not been deposited with the depositary institution. However, if there is a request for distribution of the shares that satisfies the conditions of Article 27-3 of the Patent Law Enforcement Regulations, the applicant shall Assured to sell according to.
実 施 例 ２
寄託菌株のフェルラ酸−４−ＶＧ転化活性についての検討：
（１）ラクトコッカス・ラクチス・サブスピーシーズ・ラクチスＮＢＲＣ１２００７、ラクトコッカス・ラクチス・サブスピーシーズ・ラクチスＮＢＲＣ１００９３３およびラクトコッカス・ラクチス・サブスピーシーズ・クレモリスＮＢＲＣ１００６７６（いずれも独立行政法人製品評価技術基盤機構より購入）を、実施例１（２）と同様にフェルラ酸を添加した培地中で培養し、培養１８時間後におけるフェルラ酸量および４−ＶＧ量をＨＰＬＣにより測定し、フェルラ酸を４−ＶＧに転化する活性（フェルラ酸−４−ＶＧ転化活性）の有無を調べた。なお活性の有無は以下の評価基準で評価した。
Example 2
Examination of ferulic acid-4-VG conversion activity of the deposited strain:
(1) Lactococcus lactis subspecies lactis NBRC12007, Lactococcus lactis subspecies lactis NBRC100933 and Lactococcus lactis subspecies cremoris NBRC100676 (all purchased from the National Institute for Product Evaluation Technology Base) In the same manner as in Example 1 (2), the cells were cultured in a medium supplemented with ferulic acid, and the ferulic acid amount and 4-VG amount after 18 hours of culture were measured by HPLC to convert ferulic acid into 4-VG. The presence or absence of (ferulic acid-4-VG conversion activity) was examined. The presence or absence of activity was evaluated according to the following evaluation criteria.
＜フェルラ酸−４−ＶＧ転化活性評価基準＞
（評価） （内容）
＋＋ ：培養１８時間でフェルラ酸の９０％以上を４−ＶＧに転化
＋ ：培養１８時間でフェルラ酸の３％以上〜９０％未満を４−ＶＧに転化
− ：培養１８時間でフェルラ酸の３％未満を４−ＶＧに転化
<Evaluation criteria for ferulic acid-4-VG conversion activity>
(Evaluation) (Content)
++: Convert 90% or more of ferulic acid to 4-VG in 18 hours of culture +: Convert 3% or more to less than 90% of ferulic acid into 4-VG in 18 hours of culture −: 3 of ferulic acid in 18 hours of culture % Is converted to 4-VG
フェルラ酸を４−ＶＧに転化する能力はラクトコッカス・ラクチス・サブスピーシーズ・ラクチスおよびサブスピーシーズ・クレモリスにも認められた。以上の結果より、フェルラ酸を４−ＶＧに転化する能力は前記Ｓ１株だけでなく、ラクトコッカス・ラクチス全般に認められることが判った。 The ability to convert ferulic acid to 4-VG was also observed in Lactococcus lactis subspecies lactis and subspecies cremoliths. From the above results, it was found that the ability to convert ferulic acid to 4-VG was observed not only in the S1 strain but also in Lactococcus lactis in general.
実 施 例 ３
ラクトコッカス・ラクチスを用いたもろみの調製（１）：
タイ米を洗米し、水に浸漬した後水切りを行い、５０ｇずつフラスコに分取した。次いで、この洗浄米を１２１℃のオートクレーブで１０分間の蒸煮をして蒸米を得た。麹菌は事前にＰＤＡ培地で培養を行い、胞子懸濁液を調製し、これをフラスコ内の蒸米１ｇに対し４×１０５ｃｆｕ／ｇになるように接種した。培養は、温度３８℃、相対湿度９５％の恒温恒湿機（ＣＲＨ−２１０、タバイエスペック製）で４２時間培養を行った。培養開始から１８時間と２４時間にフラスコ内の米を攪拌して麹を得た。得られた麹５０ｇに水８０ｍｌを添加し、これに前培養した酵母を１.０×１０７ｃｆｕ／ｍｌとなるように加え、更に、前培養を行ったＳ１株（実施例１で得られたもの）を１.０×１０７ｃｆｕ／ｍｌとなるように添加し、２５℃で１４日間発酵させてもろみを得た。発酵開始直後、３日後、６日後、１０日後および１４日後のフェルラ酸量および４−ＶＧ量を実施例１と同様にＨＰＬＣにより測定した。なお、Ｓ１株を添加しない以外は上記と同様にして調製されたもろみを対照とした。これらの結果を図３および図４に示した。
Example 3
Preparation of moromi using Lactococcus lactis (1):
Thai rice was washed, dipped in water, drained, and dispensed into flasks by 50 g. Next, this washed rice was steamed for 10 minutes in an autoclave at 121 ° C. to obtain steamed rice. Aspergillus was cultured in advance in PDA medium to prepare a spore suspension, and this was inoculated to 1 g of steamed rice in the flask at 4 × 10 5 cfu / g. Incubation was performed for 42 hours with a constant temperature and humidity machine (CRH-210, manufactured by Tabay Espec) at a temperature of 38 ° C. and a relative humidity of 95%. The rice in the flask was stirred 18 hours and 24 hours after the start of the culture to obtain a koji. 80 ml of water was added to 50 g of the obtained koji, and the precultured yeast was added to 1.0 × 10 7 cfu / ml, and the precultured strain S1 (obtained in Example 1) was added. Was added to 1.0 × 10 7 cfu / ml and fermented at 25 ° C. for 14 days to obtain mash. Just after the start of fermentation, the amount of ferulic acid and the amount of 4-VG after 3 days, 6 days, 10 days and 14 days were measured by HPLC as in Example 1. In addition, moromi prepared in the same manner as above except that the S1 strain was not added was used as a control. These results are shown in FIG. 3 and FIG.
もろみ中のフェルラ酸量は、Ｓ１株を添加したもろみとＳ１株を添加していないもろみとで、ほとんど変化がなかった。しかし、Ｓ１株を添加したもろみ中の４−ＶＧ量は、Ｓ１株を添加していないもろみよりも約２０％増加していた。 The amount of ferulic acid in the mash was almost unchanged between the mash with the addition of the S1 strain and the mash with no addition of the S1 strain. However, the amount of 4-VG in the mash to which the S1 strain was added was about 20% higher than that of the mash to which the S1 strain was not added.
実 施 例 ４
蒸留酒の製造（１）：
実施例３で得られたＳ１株を添加したもろみ（発酵１４日）について８５〜１００℃程度で蒸留を行い、蒸留酒（泡盛）を製造した。蒸留酒中のフェルラ酸量および４−ＶＧ量を実施例１と同様にＨＰＬＣにより測定した。また、Ｓ１株を添加していないもろみについても同様に蒸留を行い、蒸留酒を製造し、同様の測定をした。
Example 4
Production of distilled liquor (1):
About the mash (
フェルラ酸は、もろみの蒸留により蒸留酒中に移行しないため、Ｓ１株を添加したもろみから蒸留された蒸留酒およびＳ１株を添加していないもろみから蒸留された蒸留酒のいずれにも検出されなかった。しかし、４−ＶＧ量は、Ｓ１株を添加したもろみから蒸留された蒸留酒の方が、Ｓ１株を添加していないもろみから蒸留された蒸留酒よりも約３０％も増加していた。 Since ferulic acid does not migrate into distilled liquor due to distillation of moromi, it is not detected in either distilled liquor distilled from mash with added S1 strain or distilled liquor distilled from mash without added S1 strain. It was. However, the amount of 4-VG was about 30% higher in distilled spirits distilled from the mash to which the S1 strain was added than in distilled spirits distilled from the mash to which the S1 strain was not added.
実 施 例 ５
蒸留酒の製造（２）：
実施例３の方法に従い作製した麹５０ｇに水８０ｍｌを添加し、これに前培養した酵母を１.０×１０７ｃｆｕ／ｍｌとなるように加え、更に、前培養を行ったラクトコッカス・ラクチス・サブスピーシーズ・ラクチスＮＢＲＣ１２００７を１.０×１０７ｃｆｕ／ｍｌとなるように添加し、２５℃で７日間発酵させてもろみを得た。
Example 5
Production of distilled liquor (2):
80 ml of water was added to 50 g of the koji prepared according to the method of Example 3, and yeast that had been pre-cultured was added to 1.0 × 10 7 cfu / ml, and then Lactococcus lactis subjected to pre-culture. -Subspecies Lactis NBRC 12007 was added to 1.0 × 10 7 cfu / ml and fermented at 25 ° C. for 7 days to obtain a mash.
上記で得られたもろみ（発酵７日間）について８５〜１００℃程度で蒸留を行い、蒸留酒（泡盛）を製造した。蒸留酒中の４−ＶＧ量を実施例１と同様にＨＰＬＣにより測定したところ、ラクトコッカス・ラクチス・サブスピーシーズ・ラクチスＮＢＲＣ１２００７株を添加したもろみから蒸留された蒸留酒の方が、ラクトコッカス・ラクチス・サブスピーシーズ・ラクチスＮＢＲＣ１２００７株を添加していないもろみから蒸留された蒸留酒よりも４−ＶＧ量が約３０％も増加していた。 About the mash (fermentation 7 days) obtained above, it distilled at about 85-100 degreeC, and manufactured distilled liquor (Awamori). The amount of 4-VG in the distilled liquor was measured by HPLC in the same way as in Example 1. -The amount of 4-VG was increased by about 30% compared with distilled spirits distilled from mash not added with Subspecies lactis NBRC12007.
以上の結果より、もろみにラクトコッカス・ラクチスを作用させることにより、もろみを蒸留した蒸留酒中に、蒸留酒の香味の一つであるバニリンの前駆体である４−ＶＧ量を有意に増加できることが判った。 From the above results, the amount of 4-VG which is a precursor of vanillin which is one of the flavors of distilled liquor can be significantly increased in distilled spirits obtained by distilling moromi by reacting Lactococcus lactis with mash. I understood.
本発明の蒸留酒の製造方法によれば、ラクトコッカス・ラクチスの作用により、蒸留酒中の４−ＶＧを増加させることができる。この様にして製造される蒸留酒は、通常の製法で得られる蒸留酒と同期間熟成させた場合であっても、甘い芳香と香味の増強された蒸留酒となり、商品価値が高いものとなる。 According to the method for producing distilled liquor of the present invention, 4-VG in distilled liquor can be increased by the action of Lactococcus lactis. Distilled liquor produced in this way becomes distilled liquor with enhanced sweet aroma and flavor even when it is aged for the same period as distilled liquor obtained by a normal production method, and has high commercial value. .
また、ラクトコッカス・ラクチスはフェルラ酸からバニリンの前駆体となる４−ＶＧを製造するのにも利用することができる。 Lactococcus lactis can also be used to produce 4-VG that is a precursor of vanillin from ferulic acid.
Claims (6)
A process for producing 4-vinyl guaiacol, wherein Lactococcus lactis is allowed to act on ferulic acid.
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| JP2010187633A (en) * | 2009-02-20 | 2010-09-02 | Fundokin Shoyu Kk | Method for producing lactic acid fermented amazake |
| WO2012033229A1 (en) * | 2010-09-09 | 2012-03-15 | サントリーホールディングス株式会社 | Process for production of distilled spirit |
| JP2012055235A (en) * | 2010-09-09 | 2012-03-22 | Suntory Holdings Ltd | Method for producing novel shochu |
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| CN104762163B (en) * | 2015-04-08 | 2017-03-01 | 江苏洋河酒厂股份有限公司 | A kind of method preparing biological song |
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