KR101577038B1 - Pichia fabianii AY56 and process for preparing fermented alcoholic drink using same - Google Patents

Abstract

The present invention provides a novel strain of Pichia sp. Having excellent ability to produce ethyl caproate, and also discloses a method for producing a conventional fermented wine using the same, which is Pichia anomala AY56 (Deposit No. KACC93178P) As a result, it is possible to improve and enhance the taste and flavor of traditional rice wine as well as to improve the cooling sensation and to enhance the competitiveness of traditional rice wine by providing the fermented alcoholic yeast having the ability of producing ethyl caproate, There is an expected effect.

Description

Pichia anomala AY56 having high ethyl caproate production ability and a method for producing a fermented beverage using the same,

The present invention relates to a microorganism of the genus Pichia, having high ethyl caproate-producing ability, and a process for producing a traditional fermentation product using the microorganism.

Alcohol is an alcoholic beverage.

Unique brewing methods have been introduced to suit the specific climate and climate of the area suitable for the natural environment. Korean traditional liquor is divided into Takju, Yakju and Soju. It is not as gorgeous as western liquor but it is not only good for health but also contains the wisdom, style and elegance of life because it contains the food customs of our ancestors.

The raw materials of traditional Korean rice are rice, glutinous rice, barley rice, brown rice, corn, sweet potato, etc. Since the main ingredient of cereal is converted into sugar to produce alcohol, an enzyme produced by microorganism is needed and its fermentation source is leaven. Aspergillus kawachii, Aspergillus oryzae and Rhizopus sp. Were used in the yeast and sterilized starch raw materials, which are produced by breeding many kinds of microorganisms such as fungi, yeast and fungi in the natural state according to the manufacturing method . Which is made by inoculation of pure cultured bacteria. Amino acids, organic acids, and volatile flavor components produced by fermentation of alcohol by yeast are harmonized with color due to various enzymatic actions by these microorganisms. Therefore, enzyme activity, organic acid productivity, and alcohol fermentation ability vary depending on the type of yeast, and therefore, the volatile flavor component, taste and color quality characteristics of the fermented beverage greatly influence the characteristics.

On the other hand, in the process of brewing conventional traditional liquor, yeast has been conventionally used as an fermentation agent. However, the liquor using the yeast fermentation agent thus produced has no change in taste and aroma and the quality may be different depending on the fermentation conditions. There was difficulty in homogenization. Especially, in order to overcome the above problems, various attempts have been made to make new yeast. However, since the production of Yakju and Takju is carried out by small and medium brewers, improvement and quality control of the new strain are poor, It is difficult to expect a variety of flavors and aromas because it can not produce fermented fermented beverages.

Therefore, in order to promote the continuous consumption of traditional Korean rice, it is necessary to precede research on the seed culture such as search for excellent strains and improvement of strains. Research on the optimum fermentation condition of raw materials, glycation condition, sensory improvement, It is strongly desired to provide an appropriate fermentation agent for consumers' preference.

In recent years, a trend to add new flavor to traditional liquor has been developed, and methods for improving taste and flavor in traditional liquor have been steadily developed.

In the prior art, Prior Art 1 (KR 1998-049302 B1) relates to a novel saccharomyces cerevisiae strain TD03 and a method for producing a saccharomyces cerevisiae strain using the same, The Saccharomyces cerevisiae DR yeast was mutagenized with ethyl methanesulfonate (EMS) and separated and improved on a selective medium to produce a large amount of amylase and isoamyl alcohol, A method for easily producing the above-mentioned method is disclosed.

Background Art [0002] Prior Art 2 (KR 10-2008-053629 A) relates to a method for preparing a safflower using Saccharomyces cerevisiae T084 and a plant medicinal ingredient, which increases the aroma component of a mainstream, And then adding phytopharmaceuticals such as Cordyceps and Red Sulfuric Acid to the fermentation broth to produce a fragrant-rich fermented beverage, as well as containing phytopharmaceuticals such as Cordyceps and Red Seaweed, A method for producing a functional sugar such as a recovery has been disclosed.

However, as described above, in order to increase the aroma component of the traditional wine, it is possible to improve the flavor of the traditional fermented wine by adding various fragrance yeasts and components. However, the report on the genus Saccharomyces cerevisiae, There are many cases of developing fragrance yeast as a new strain.

In addition, it is inadequate to produce a mainstream containing a large amount of a perfume ingredient, which is a major factor of mainstream quality.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

KR 1998-049302 B1 KR 10-2008-053629 A

The present invention is to provide a novel strain having excellent ability to produce ethyl caproate.

The present invention also provides a method for producing a conventional fermentation broth using a new strain having excellent ability to produce ethyl caproate.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present invention provides Pichia anomala AY56 (KACC93178P), which has excellent ability to produce ethyl caproate.

In addition, the present invention provides a method for producing a traditional fermentation broth using Pichia anomala AY56 (KACC93178P), which has excellent ability to produce ethyl caproate.

The present invention provides a piquant alcoholic yeast having an ability to produce ethyl caproate isolated from a traditional yeast, thereby improving and enhancing various flavors and flavors of the traditional alcoholic beverages as well as enhancing the cooling sensation, thereby enhancing the competitiveness of traditional alcoholic beverages There is an expected effect.

The present invention provides a new type of Pichia enteric yeast having high ethyl caproate-producing ability.

Since the novel Pycnogeny yeast of the present invention has a high ethyl caproate-producing ability, it is possible to provide a high-quality alcoholic beverage because it contains a large amount of desirable aroma components when it is applied to the manufacture of traditional fermented liquors such as Chinese liquor. Ethyl caproate is derived from a middle chain fatty acid produced as a metabolite in yeast cells and is synthesized from ethyl alcohol and caproyl coA by alcohol acyltransferase , And is synthesized from ethyl alcohol and caproic acid by esterase.

In the present invention, in order to discover a yeast capable of producing a large amount of aroma components in the preparation of traditional fermentation liquor, cerulenin, which contains 25-50 uM of cerulenin which separates various microorganisms from the yeast that has been conventionally used and inhibits yeast fatty acid synthesis YPD (yeast extract 1.0%, peptone 2%, dextrose 2%, agar 2.0%) The strains were cultured at 30 ° C for 3 days on a plate medium. The primary screened strains were cultivated again on a new plate medium in the same environment, and olfactory sensory tests were performed. Nine strains were screened secondarily. As a result of gas chromatography analysis, three strains of high - ethyl caproate were identified. Each of these strains was identified phylogenetically to belong to Pichia anomala or Pichia fabianii . These strains were confirmed to be novel strains that did not exist in the past. Thus, the novel strains isolated and identified in the present invention were deposited with the National Center for Agricultural Genetic Resources (KACC) as Pichia anomala AY56 and assigned accession number KACC93178P " Pichia anomala AY56 (Accession No. KACC93178P) ".

The above-mentioned " Pichia anomala AY56 (Accession No. KACC93178P)" which has been separated and identified from the conventional yeast causes effective alcohol fermentation at 15 to 18 DEG C, and under the usual fermentation conditions, Octylcaproate having an ethyl acetate productivity of up to 180 ppm%.

The present invention also provides a method for preparing a traditional fermented juice using the novel " Pichia anomala AY56 (Accession No. KACC93178P)" yeast.

Traditional nuruk, which has been used as a fermentation source in the production of traditional fermented beverages, contains various microorganisms and has a disadvantage in that it can not produce a uniform flavor due to a large change in taste and flavor. Also, the fragrance component is present in a small amount, and the presence or absence of the fragrance component in the alcoholic beverage is an important factor for determining the quality. In the case of using the novel " Pichia anomala AY56 (Accession No. KACC93178P)" yeast of the present invention in place of the traditional yeast in producing the conventional fermentation liquor, the mainstream of a large amount of the ethyl caproate Can be produced.

Although there is no limitation in the method of producing the traditional juice using the Pycnogeny yeast of the present invention, for example, 1 kg of rice is thoroughly washed, the juice is drained, mixed with 1.2 L of boiling water, When cooled, 40 g of wheat flour, 4 g of enzyme (potency 15,000 sp), 2 mL (10 ⁶ / mL) of yeast culture solution in which the new pickling oil is cultured, and yeast are added and fermented at 15 to 18 ° C for 10 to 14 days. It is of course possible to further include a two-stage immersion or an enzyme.

In the above and the following description, the term "traditional wine" or "traditional fermented wine" includes all the alcoholic beverages such as Takju, fruit wine, distilled wine, liqueur, It should be understood.

The conventional wine obtained using the novel " Pichia anomala AY56 (Accession No. KACC93178P)" yeast of the present invention is conventionally used as a conventional yeast used for the manufacture of Chinese cabbage or as a strain of the genus Saccharomyces Saccharomyces sp. ), For example, Saccharomyces cerevisiae is used, and thus it has a high quality by containing a large amount of a fragrant ethyl caproate.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example 1 Isolation and Identification of Pycnidia sp. Strain having ability to produce high ethyl caproate

(1-1) Isolation of strain

The present inventors selected and identified strains having excellent alcohol content and flavor-producing ability from conventional yeast.

Specifically, 1 g of the conventional yeast was suspended in 1,000 mL of sterilized water, and then spread on YPD (yeast extract 1.0%, peptone 2%, dextrose 2%, agar 2.0%) medium and the independent colonies were separated and cultured on YPD medium . These strains were cultured in YPD plate medium containing 25 ~ 50uM of cerulenin, which inhibited the fatty acid synthesis of yeast, respectively, and cultured at 30 ℃ for 3 days. The surviving strains were firstly screened. The primary screened strains were cultivated again on a new plate medium in the same environment, and olfactory sensory tests were performed. Nine strains were screened secondarily. As a result of gas chromatography analysis, three strains of high - ethyl caproate were identified.

(1-2) Identification of selected strains

Three yeast strains producing high ethyl caproate were PCR-amplified using the ITS 1,4 primer [ITS 1 (5'-TCCGTAGGTGAACCTGCGG-3 ') and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3')] in 18S ribosomal DNA region Conduct. The resulting PCR product was assigned to Solgent Corporation and the result of identification was taken. The homology and taxa of the three yeasts were identified by searching the National Center for Biotechnology Information (NCBI; Basic Local Alignment Search Tool (BLAST)). The three species were Pichia anomala , Pichia fabianii) . In addition, it was confirmed that the three strains were new strains that did not exist in the past , and on March 14, 2013, the National Institute of Genetic Resources (KACC) KACC93178P, KACC93176P, and KACC93177P.

In the present invention, the above-identified new strain is named Pichia anomala AY56 (Accession No. KACC93178P), Pichia fabianii AY42 (Accession No. KACC93176P) and Pichia fabianii AY47 Accession No. KACC93177P).

<Example 2> Preparation of fermented juice

In order to investigate alcohol fermentation ability and perfume component production ability of the new strain Pichia anomala AY56 (Accession No. KACC93178P) according to the present invention, a new strain and commercial strain according to the present invention were used to prepare a fermentation liquor. The yeast are commercially available as Saccharomyces cerevisiae, are each experimental convenience, AY36, AY38, AY39 was given a pseudonym in actual, Saccharomyces cerevisiae (Aromatic, Bioferm Inc. , Beverlo, Belgium), Saccharomyces cerevisiae (Fermivin (No. 7013), DSM Food Specialties, Lille, France), and Saccharomyces cerevisiae (La Parisienne (S. l.) Lesaffre Inc., Lesaffre, France).

Fermentation was carried out with one stage of immersion, and the composition of the raw materials in each stage is shown in Table 1 below.

Immersion times Primary soaking Raw material name (a) rice (rice balls) (b) Sterile water  (c) Enzymes (d) Yeast Raw material amount 1,000 g 1,200 mL 4g
(15,000 SP) 10 &lt; ⁶ &gt; / mL, 2 mL

(1) Preparation of Yeast Culture Medium: Yeast cultures were obtained by aseptically culturing selected strains in YPD medium (yeast extract 1.0%, peptone 2.0%, dextrose 2.0%) at room temperature for about 48 hours at 25 ° C.

(2) The number of yeasts in the culture was adjusted to 10 ⁶ / mL, and 2 mL of the mixture was inoculated into a mixture of high-fat rice, water (sterilized water), and enzyme according to the compounding amount shown in Table 1 above.

(3) The mixture of the above (2) was fermented at 18 DEG C for 7 days (168 hours) to obtain a fermentation product.

(4) The finally obtained fermented product was filtered using a filter paper to prepare a cauliflower.

Test Example 1: Analysis of flavor of fermented beverage

The results of analyzing the fragrance of the yeast and the fermented product thus prepared are shown in Table 2 below.

Fragrance analysis was performed using Gas Chromatography (GC 2010, Shimadzu Co., Kyoto, Japan). 1 μL of each calf was injected into the GC injector. The column used for GC was a HP-INNOWAX capillary column (60 m x 0.25 mm id x 0.25 μM film thickness, J & W Scientific, Folsom, CA, USA). Nitrogen gas was used as the mobile phase gas and the flow rate was 22.0 cm / sec (linear velocity). The oven temperature of the column was maintained at an initial 45 ° C for 5 minutes, then raised to 100 ° C at a rate of 5 ° C per minute and held for 5 minutes. Thereafter, the temperature was raised to 200 DEG C at a rate of 10 DEG C per minute and held for 10 minutes. The split ratio was adjusted to 50: 1. The injector and detector (FID) temperatures were maintained at 250 ° C.

Yeast No. Acet
aldehyde EtoAc Iso
butanol Isoamyl
alcohol Ethyl
caprylate Acetic
acid Fufral Linalool Butyrate Ethyl
caproate 2-Phenyl
alcohol AY36
(Commercially available) 1281.2 + - 82.9 ^bcde - - - 76.5 ± 10.4 ^bc 3180.4 + - 124.8 ^cd 40.5 ± 4.6 ^d 30.7 ± 2.4 ^cd 7.3 ± 0.9 ^de 12.3 ± 1.1 ^d 4.9 ± 0.4 ^de AY38
(Commercially available) 1541.9 ± 146.1 ^abc - - 15.8 ± 2.3 ^ab 25.1 ± 2.54 ^de 2722.7 ± 212.8 ^de 37.2 ± 3.8 ^d 22.5 ± 2.2 ^d 7.2 ± 0.8 ^de 11.4 ± 0.6 ^d 39.4 ± 6.9 ^a AY39
(Commercially available) 1257.1 + - 157.8 ^cde 3.5 ± 1.2 ^d 5.3 ± 0.3 ^a 11.2 ± 2.9 ^bcd - 2351.5 ± 132.0 ^ef - 2.2 ± 0.2 ^a 24.3 ± 2.2 ^b 19.1 ± 3.4 ^d 6.4 ± 0.3 ^cde AY42
( P. fabianii ) 1504.8 ±
182.1 ^abc 49.9 ± 8.6 ^c - 7.7 ± 1.7 ^cd 65.3 ± 9.7 ^bc 3026.6 ± 373.4 ^cde 149.3 ± 19.5 ^c 67.5 ± 17.4 ^c 17.0 + - 7.8 ^bcd 99.4 + - 16.5 ^b 5.6 ± 0.7 ^de AY47
( P. fabianii ) 1386.6 ± 97.4 ^abcd 82.5 ± 6.6 ^c - 6.7 ± 1.7 ^d 55.4 ± 3.1 ^cd 3452.1 ± 192.8 ^bcd 62.0 ± 19.4 ^d 152.6 ± 25.1 ^a 7.6 ± 2.4 ^de 176.6 ± 17.4 ^a 6.9 ± 1.7 ^cde AY56
( P. anomala ) 1398.7 ± 38.4 ^abcd - - 8.1 ± 1.5 ^cd 1.5 ± 0.3 ^e 1851.1 ± 173.2 ^fg 68.7 ± 12.0 ^d 125.1 ± 17.8 ^ab 53.4 ± 5.1 ^a 170.1 ± 11.6 ^a 3.4 ± 0.5 ^e

It was confirmed that Saccharomyces cerevisiae, which is a control, has an ability to produce ethyl caproate at 11.4 to 19.1 ppm.

In contrast, the production of ethyl caproate was superior to that of the other treatments using the new strains of the present invention, Pichia fabianii AY42, Pichia fabianii AY47 and Pichia anomala AY56.

Test Example 2: Sensory evaluation of the fermented beverage

The sensory evaluation and acceptability of the fermented product prepared in Example 2 were evaluated. First, 20 trained inspectors are selected, and a 9-point scale (1-not good, 3-bad, 5-average, 7-good, And ANOVA analysis were used for the statistical analysis. Mean significance (P <0.05) was determined by Duncan's multiple range test.

The results are shown in Table 3 below.

Likelihood Sample color incense flavor Overall
Likelihood AY36
(Commercially available) 7.4 ± 0.9 ^a 5.3 ± 1.9 ^abc 4.5 ± 2.1 ^bcd 4.5 ± 2.2 ^bcde AY38
(Commercially available) 6.7 ± 1.4 ^ab 5.7 ± 1.8 ^ab 4.7 ± 2.1 ^abcd 4.8 ± 2.2 ^bcd AY39
(Commercially available) 6.7 ± 1.1 ^ab 5.1 ± 1.8 ^abc 4.0 ± 1.9 ^cd 4.0 ± 1.8 ^cde AY41
( P.anomala ) 5.9 ± 2.2 ^bc 5.4 ± 1.4 ^ab 5.3 ± 1.6 ^abc 5.4 ± 1.6 ^ab AY42
( P.fabianii ) 5.5 ± 2.0 ^bcd 6.4 ± 1.4 ^a 5.9 ± 1.5 ^a 6.1 ± 1.5 ^a AY47
( P. fabianii ) 4.2 ± 1.3 ^e 4.4 ± 2.1 ^bc 5.1 ± 1.8 ^abcd 4.7 ± 1.9 ^bcde AY56
( P. anomala ) 5.3 ± 1.8 ^cde 5.3 ± 1.7 ^abc 5.4 ± 1.4 ^ab 5.5 ± 1.2 ^ab

As a result of the test, the flavor yeast Pichia anomala AY56 (Accession No. KACC93178P), Pichia fabianii AY42 (Accession No. KACC93176P), Pichia anomala AY56 The preference of Pichia fabianii AY47 (Accession No. KACC93177P) was generally high, and in particular, Pichia anomala AY56 (Accession No. KACC93178P), Pichia fabianii AY42 (Accession No. KACC93176P) I got a good score on scent.

From these results, it can be confirmed that the quality of the liquor is influenced by the degree of harmony between the ethyl caproate content and the other components.

From this, it can be seen that the flavor yeast of the present invention can improve and enhance the taste and aroma in the manufacture of Chinese cabbage and takju, and can provide a traditional Chinese cabbag with a high quality and a refreshing sensation.

National Institute of Agricultural Science KACC93178P 20130415

Claims (2)

Pichia anomala AY56 (Accession No. KACC93178P) which has excellent ability to produce ethyl caproate.
A method for producing a conventional fermentation broth using Pichia anomala AY56 (Accession No. KACC93178P) having excellent ability to produce ethyl caproate.