JP4524355B2 - Acquisition of natural yeast and production of liquors using colored rice - Google Patents

Description

  The present invention relates to a method for producing alcoholic beverages such as sake made from natural yeast and colored rice living on the fruit surface.

As for the separation of natural yeast, direct plate separation method and enrichment culture method are known, but none of them can be separated after confirming the presence of rare yeast Saccharomyces cerevisiae. It was. In addition, for the production of alcoholic beverages made from colored rice, it is known to use unpolished colored rice and to extract pigments from colored rice, but it is related to the method of producing alcoholic beverages representing sake. There is no.

In the conventional separation method as described above, it is not easy to effectively separate Saccharomyces cerevisiae yeast. In addition, producing alcoholic beverages such as sake using yeast distributed by the Japan Brewing Association and mutants of the yeast does not give a product that fully exhibits the characteristics of colored rice.

  As a result of repeated earnest studies in view of this point, the inventor has reached the present invention, and effectively and selectively separates and acquires natural yeasts that inhabit the surface of fruits by special electrophoresis, Sake and other alcoholic beverages are produced from this and colored rice. In the examples, in order to produce alcoholic beverages using yeast isolated from Yamagata Prefecture special cherries, purple black rice, which is a purple-red liquor that can image the color of cherries even in colored rice, is used. If the fruit immersed in the medium is La France, a special product of Yamagata Prefecture, it is possible to produce sake unique to Yamagata Prefecture by using green rice, which is a green liquor that can image the color of La France. It becomes.

  By acquiring natural yeasts that inhabit the surface of local specialty fruits, the image of special fruits is created, and by combining the characteristics of colored rice with the characteristics of flavors derived from natural yeasts, unprecedented liquors To manufacture.

(1) About yeast culture The fruit which seems to have adhered yeast is immersed in the aseptic medium containing the nutrients required for growth of yeast. Remove the soaked fruit and leave the medium at 30 ° C. As expected, when yeast is present, the medium becomes turbid and the carbon dioxide is generated. The presence of the bacteria can be confirmed by observing this culture solution under a microscope, but it cannot be clearly distinguished from Saccharomyces cerevisiae which is a yeast suitable for the production of alcoholic beverages. In this example, cherries were used as the fruits. However, since it seems that yeasts are attached to fruits that are cultivated outdoors, the type of fruits is not the essence of the present invention. But the same is true for plums.

(2) About DNA separation In order to determine whether the yeast Saccharomyces cerevisiae suitable for the production of liquor is present in the culture solution obtained in (1), DNA is separated by electrophoresis. DNA is extracted from a part of the culture solution and subjected to electrophoresis. In normal electrophoresis, DNA is separated by molecular weight. However, since the DNA encoding the small subunit ribosomal RNA (hereinafter referred to as SSUrDNA), which is the subject of this study, has almost the same molecular weight, the DNA of Saccharomyces cerevisiae cannot be clearly distinguished. Therefore, DNA was separated by temperature gradient electrophoresis (Temperature gradient Gel Electrophoresis) that can discriminate even a slight difference in gene information written in SSUrDNA. At this time , if DNA of known Saccharomyces cerevisiae is subjected to electrophoresis and the DNA is separated at the same position, it can be determined that Saccharomyces cerevisiae is present in the culture solution. The point of confirming the presence of Saccharomyces cerevisiae by electrophoresis is one of the essence of the present invention. Without this step, the yeast separation in the next step (3) cannot be efficiently achieved.

(3) Separation of yeast In the culture solution obtained in (1), yeast is separated from the culture solution in which the presence of Saccharomyces cerevisiae was confirmed in (2). The culture solution is appropriately diluted 1000 to 10,000 times and applied to a solidified medium. If the dilution is appropriate, colonies derived from one bacterium are formed on the solid medium. The colonies are planted one by one in a new medium and separated. Since the step (2) has been performed, the isolated yeast is almost certainly Saccharomyces cerevisiae . The separated yeast can be subjected to the step (2) again, or the gene information directly written in the SSUrDNA can be read by a technique called DNA sequencing to confirm that it is a Saccharomyces cerevisiae .

(4) About colored rice In this example, in order to produce alcoholic beverages using yeast isolated from cherries, purple-black rice was used as a red-purple liquor so that the color of the cherries could be imaged. Is not to be done. For example, if the first fruit to be immersed in the culture medium is La France, green rice can be used as a green liquor so that the image of La France can be imaged.

Ａｎａｌｙｚｅｒ（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ社）を用いた。得られた塩基配列情報をインターネット上にある遺伝子情報データベース Ｒｉｂｏｓｏｍａｌ Ｄａｔａｂａｓｅ Ｐｒｏｊｅｃｔ ＩＩに送信してホモロジー検索を行った。Experimental Method (1) Strain Used Yeast S1 strain and S4 strain presumed to be Saccharomyces cerevisiae isolated from cherries in Example 1 were used. As a control, Japanese Brewing Association Sake Yeast No. 7 (K7) was used.
(2) SSUrDNA information analysis About 500 bp of the SSUrDNA fragment was amplified by PCR from the total DNA of the isolated yeast strains S1 and S4, and the base sequence was read by cycle sequencing. The primers used are shown in Table 1, and the PCR conditions are shown in Table 2.

Analyzer (PE Biosystems) was used. The obtained base sequence information was transmitted to the gene information database Ribosomal Database Project II on the Internet, and a homology search was performed.

総米１５００ｇで１５℃１２日間の発酵とした。仕込配合は表３のとおりとした

白米による仕込みは、麹米と掛米ともに出羽燦々（精米歩合５０％）を使用した。有色米には紫黒米品種である朝紫を使用した。有色米による仕込みは、麹米に出羽燦々（精米歩合５０％）を使用し、掛米に朝紫（精米歩合９９％）を使用した。発酵期間中は経時的に炭酸ガス減量を測定して発酵経過の指標とした。(3) Small preparation test

The fermentation was performed at 1500 ° C. for 12 days at 15 ° C. The charging composition was as shown in Table 3.

For the preparation with white rice, we used Dewa Rinzu (milled rice ratio 50%) for both rice and kake rice. As the colored rice, Aso Purple, a purple-black rice variety, was used. As for the preparation with colored rice, Dewa Rinzu (milled rice ratio 50%) was used for sticky rice, and Asa purple (milled rice ratio 99%) was used for kake rice. During the fermentation period, the carbon dioxide gas loss was measured over time and used as an index of fermentation progress.

(4) General component analysis The alcohol content was measured with Alcomate AL-2 (Riken Keiki Co., Ltd.), and other general component analyzes were conducted according to the National Tax Agency's prescribed analysis method.

(5) Absorbance measurement A spectrophotometer was used. The degree of coloring indicating the degree of yellow color peculiar to sake was measured by measuring absorbance at 430 nm in a 10 mm optical path length cell according to the National Tax Agency's specific analysis method comment. Similarly, the absorbance at 520 nm was measured as an indicator of anthocyanin which is a pigment component peculiar to purple black rice varieties.

Experimental results (1) SSUrDNA information analysis As a result of analyzing the SSUrDNA information, the yeast strains S1 and S4 isolated from the cherries were almost identical to the standard Saccharomyces cerevisiae, and thus determined to be the same species. This result confirmed the results of 3.3 separation and classification and 3.4 SSUrDNA information analysis in Example 1.

白米の場合、Ｓ１株とＳ４株ともにＫ７に比べて発酵経過が遅れたが（図１▲１▼）、生成酒のアルコール度はＳ１株がＫ７と同じ１２％台となった。有色米の場合、Ｋ７に比べてＳ１株は発酵経過が進み、Ｓ４株は同等であった（図１▲２▼）。有色米の生成酒のアルコール度は、Ｋ７とＳ１株が１５％台で、Ｓ４株は１３％台であった。この結果から、サクランボから分離した酵母Ｓ１株とＳ４株は白米でも十分な発酵力があるが、特に有色米を使用した場合にＫ７と同等以上の発酵力を示すことがわかった。またＳ４株は、白米でも有色米でも、酸度が高い一方でアミノ酸度が低い特徴を示した。このことからＳ４株は酸味があり淡麗な酒質を醸し出すと予想される。(2) Process of fermentation and general components The carbon dioxide gas loss during the fermentation period is shown in FIG. 1, and the analysis results of the general components are shown in Table 4.

In the case of white rice, the fermentation progress was delayed for both S1 and S4 strains compared to K7 (FIG. 1, (1)), but the alcohol content of the produced liquor was in the same 12% range as that of K7. In the case of colored rice, the fermentation progress of S1 strain was similar to that of K7, and S4 strain was equivalent (FIG. 1 (2)). The alcohol content of the colored sake produced by colored rice was about 15% for K7 and S1 and 13% for S4. From this result, it was found that the yeast strains S1 and S4 isolated from cherries have sufficient fermenting power even with white rice, but particularly when colored rice is used, they exhibit fermenting power equivalent to or higher than that of K7. In addition, the S4 strain showed characteristics of high acidity but low amino acidity in both white rice and colored rice. From this, it is expected that the S4 strain has a sour taste and a fresh liquor quality.

(3) Absorbance In the case of white rice, there is no significant difference in absorbance. However, in the case of colored rice, especially the S4 strain shows a larger value of the ratio of 520 nm / 430 nm than K7. This means that the produced liquor has a light yellowish color and a reddish-purple color that stands out, making the color characteristics of purple and black rice stronger.

(4) Aroma component composition and organic acid composition The results of the aroma component analysis are shown in FIG. In the case of white rice, the S1 strain had a very high isobutyl acetate / isobutyl alcohol ratio compared to K7 and exhibited a characteristic aroma component composition. On the other hand, the S4 strain showed a high isobutyl acetate / isobutyl alcohol ratio but also a high isoamyl alcohol ratio. The reason why the aroma component composition is slightly different from that in Example 1 is considered to be due to the properties of the raw rice and the ratio of polished rice. In the case of colored rice, the S1 strain did not show a characteristic aroma component composition when using white rice. On the other hand, the S4 strain showed the characteristics that the ratio of isobutyl acetate / isobutyl alcohol and the ratio of isoamyl acetate / isoamyl alcohol were high when using colored rice as well as when using white rice.
The organic acid composition is shown in FIG. In the case of white rice, the S1 and S4 strains tended to have less succinic acid and more lactic acid than K7, as in Example 1, and malic acid was slightly less. On the other hand, in the case of colored rice, the S1 strain had slightly more succinic acid and more lactic acid. On the other hand, the S4 strain had less succinic acid and moderately higher lactic acid as in the case of white rice, and citric acid and malic acid had almost the same concentration as K7. Since citric acid and malic acid have a refreshing acidity, it was considered that the S4 strain had a refreshing acidity while producing appropriate characteristics.

これらのことからサクランボの果実から分離した一連の酵母は、有色米を原料とした酒類の製造に非常に適していると判断できる。(5) Sensory evaluation In the case of white rice, the results of sensory evaluation were comparable to K7 for S1 and S4 strains. On the other hand, in the case of colored rice, the S1 and S4 strains were evaluated higher than K7, and the S4 strain in particular obtained the best evaluation. (Table 6)

From these facts, it can be judged that a series of yeasts separated from cherries fruits are very suitable for the production of alcoholic beverages using colored rice as a raw material.

(experimental method)
(1) Strain used The yeast S4 strain highly evaluated in Example 2 was used for sake production using colored rice as a raw material. As a control, sake yeast KA strain distributed for production of ginjo sake in Yamagata Prefecture was used for ordinary sake production using white rice as a raw material.

有色米による仕込みは、麹米に出羽燦々（精米歩合５０％）を使用し、掛米に朝紫（精米歩合９９％）を使用した。白米による仕込みは、麹米と掛米ともに山形８６号（精米歩合５０％）を使用し、製麹は、麹室で蓋麹法により行った。酒母および醪の仕込みには、サーマルタンク（新洋技研工業製）を使用した。原料用アルコールは添加せず、製法品質上の純米酒とした。(2) Preparation The preparation composition was as shown in Table 1.

As for the preparation with colored rice, Dewa Rinzu (milled rice ratio 50%) was used for sticky rice, and Asa purple (milled rice ratio 99%) was used for kake rice. For the preparation of white rice, Yamagata No. 86 (50% of polished rice ratio) was used for both rice and kake rice. A thermal tank (manufactured by Shinyo Giken Kogyo Co., Ltd.) was used for the preparation of the sake mother and the lees. The alcohol for the raw material was not added, and pure rice liquor with a high quality was obtained.

(3) General component analysis and absorbance measurement General component analysis was in accordance with the analytical method prescribed by the NTA. Absorbance measurement was as in Example 2.

(4) Aroma component and organic acid analysis The aroma component was analyzed by head space gas chromatography (Shimadzu GC-15A), and the organic acid was analyzed by high performance liquid chromatography (Shimadzu LC-9A).

有色米の清酒は、サクランボをイメージするため、アルコール度は低くして日本酒度を甘くした。また、有色米の清酒は酸度が高い特徴があるが、一方で精米歩合が高いためアミノ酸度も高くなった。しかし実施例２と同程度に、酸度が高いわりにアミノ酸度が低い特徴を示した。紫黒米品種に特有のアントシアニン色素の指標である５２０ｎｍの吸光度などを表３に示す。

実施例２に比べるとやや低い値となったが、目視では十分な赤紫色を呈していた。以上より、白米による通常の清酒と比較して、Ｓ４酵母と有色米を原料とした清酒は概ね目的とした酒質に到達することができた。(Experimental result)
(1) Fermentation process and general components The koji process with colored rice is 12.0 ° C, 16.7 ° C dance, 13.0 ° C mid, 8.0 ° C distillate, 13.5 ° C maximum product temperature, 8 days I put it in my eyes. The temperature of the rice bran was 13.8 ° C, 15.0 ° C dance, 13.0 ° C Naka, 10.3 ° C distillate, and 12.0 ° C maximum product temperature. The analysis results of the general components are shown in Table 2.

The colored rice refined sake has a sweetness of sake by lowering the alcohol level in order to imagine a cherry. In addition, colored rice refined sake has a high acidity, but on the other hand, the percentage of polished rice is high, so the amino acid content is also high. However, as in Example 2, the acidity was high but the amino acidity was low. Table 3 shows the absorbance at 520 nm, which is an index of anthocyanin pigment peculiar to purple black rice varieties.

Although the value was slightly lower than that of Example 2, it was sufficiently reddish by visual observation. From the above, compared with ordinary sake made from white rice, sake made from S4 yeast and colored rice as a raw material was able to reach the intended quality of sake.

(2) Aroma component composition and organic acid composition The results of the aroma component analysis are shown in FIG. As in Example 1, colored rice sake using yeast S4 has a low isoamyl acetate / isoamyl alcohol ratio (iAmyl / Acet Ratio) but a high isobutyl acetate / isobutyl alcohol ratio (iButyl / Acet Ratio). Indicated. Thus, it was confirmed that not only the small preparation but also the pilot scale showed a characteristic aroma component composition of yeast S4. The organic acid composition also showed favorable characteristics such as a large amount of citric acid and malic acid having a refreshing acidity.

(3) Sensory evaluation Although the numerical value of sensory evaluation did not reach that of regular sake made from pure white Daiginjo sake in terms of manufacturing quality, sufficient evaluation was obtained with the goal of commercialization in the future.

Applicability of invention

  As described above, the sake of the present invention is a sake with a characteristic that matches the Yamagata of the cherry kingdom. It can be advertised and sold at special product exhibitions and roadside stations regardless of whether it is in or outside the prefecture, and its usage is large.

  Carbon dioxide loss chart during fermentation   Analysis of aroma components   Composition of organic acid

Claims (2)

  A method for selectively obtaining a strain of Saccharomyces cerevisiae from a mixture of various natural yeasts that inhabit the fruit surface by temperature gradient electrophoresis of gene sites amplified using a primer pair whose base sequences are AGGGTTCGATTCCGGGAGA and ATTACCGCGGCTGCTGGCA.   The rice is refined using purple black rice, which is colored rice, as a raw material, and a strain of Saccharomyces cerevisiae is obtained by the method according to claim 1, and isobutyl alcohol, isobutyl acetate, isoamyl alcohol and isoamyl acetate derived from yeast are obtained using the strain. A method for producing sake having a fruity fragrance.

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