KR20190132814A - Optimal manufacturing methods of Hanseniaspora uvarum starters which produce high level of aromatic compounds using air-blast drying technology - Google Patents

Abstract

The present invention relates to an optimal production method for a Hanseniaspora uvarum starter producing a high level of aromatic compounds using a low-temperature air-blast drying technology, and more specifically, to a production method for a Hanseniaspora uvarum starter with an increased survival rate, and to a method of increasing the storability of Hanseniaspora uvarum starter. The production method for a Hanseniaspora uvarum starter with an increased survival rate and the method of increasing the storability of a Hanseniaspora uvarum starter can prevent damage due to heat, compared to the conventional hot air drying technology, and compared to the conventional freeze-drying technology, can produce the starter with significantly less energy and costs. Furthermore, the present invention can enhance the survival rate and storability of the Hanseniaspora uvarum starter produced under optimal conditions for Hanseniaspora uvarum strain, and thus can contribute to increasing the quality of domestic fruit wine as well as to the development and commercialization of indigenous yeast starters, and also can be used in the wine industry for various purposes.

Description

Optimal manufacturing methods of Hansenia spora uvarum yeast spawn using low temperature air drying technique {{Optimal manufacturing methods of Hansenia spora uvarum starters which produce high level of aromatic compounds using air-blast drying technology}

The present invention relates to a method for the preparation of home-grown Hansenia spora ubarum yeast spawn using cold air drying technique, and more particularly, to a method for preparing Hansenia spora ubarum yeast spawn with improved survival rate and Hansenia spore. It relates to a method of increasing the shelf life of the Lauvarum yeast seed.

Wine is the oldest alcoholic drink in human history, and wine produced for domestic consumption in Europe has gradually become known around the world, expanding its production and consumption markets. Domestic wine consumption has grown by double digits every year for the past 10 years until 2008, and for the first time in 2015, wine imports surpassed those of Western liquors such as whiskey and brandy. Although interest in wine has increased, imported wine accounts for a large part of the domestic wine market. In the domestic wine industry, sales volume and number of companies are increasing in appearance, but they are not implementing various products to strengthen their competitiveness. In order to improve the competitiveness of domestic wines, we manufacture wines from various fruits such as grapes, maroons, persimmons, apples, etc. Is going on.

Wine is the product of complex interactions between fruit and yeast, mold and bacteria. These microorganisms metabolize glucose and other constituents into a number of secondary end products during fermentation, enhancing the character and personality of wine. The aroma of wine consists of compounds with various aromatic properties, and more than 400 of the more than 1000 volatile compounds in the wine are produced by yeast during fermentation. Since the nature and concentration of these compounds are determined by the species of yeast that ferment, it can be said that the aroma content of wine depends on the species of yeast used for fermentation. Recently, in addition to Saccharomyces cerevisiae , a yeast commonly used for wine fermentation, non- Saccharomyces species known to contribute to the improvement of flavor and quality of wine have been studied. These non- Saccharomyces yeasts are capable of anaerobic and aerobic growth, and can affect wine bouts by producing secondary metabolites by competing with Saccharomyces yeasts during fermentation.

On the other hand, most of the wine yeast is distributed in the form of powder dried by lyophilization. Lyophilization is widely used for microbial preservation because of its long-term viability and ease of distribution. However, such lyophilization is difficult for commercial use because of a long drying time and high consumption of energy and cost.

The survival rate of microbial cells during drying includes initial microbial concentrations, protective additives, and rehydration conditions. Among them, the protective additives include sugars, amino acids, vitamins, and the like, and the degree of protection by the additives varies depending on the microbial species. In addition, the dried microorganisms are recovered through the rehydration process, and it is important to establish the optimum conditions according to the microbial species since the most suitable rehydration conditions differ according to the microbial species.

Accordingly, the present inventors selected non- Saccharomyces yeast with excellent fragrance among native yeast separated from persimmon grown in Korea and apple and Aaronia separated for persimmonization of native yeast and development of domestic wine with excellent fragrance. . In addition, Hansenia sp. Improved the survival rate using the low-temperature air drying method while studying the conditions for increasing the survival rate of each dry yeast by using the low-temperature air drying method, drying protector, rehydration solution and antioxidant for powdering of the separated yeast. The present invention was completed by developing an optimal method for producing seedlings of lauvarum yeast.

Accordingly, it is an object of the present invention to provide a method for producing Hansenia spora ubarum yeast seed with improved survival rate and a method for increasing the shelf life of Hansenia spora ubarum yeast seed.

In order to achieve the above object, the present invention comprises the steps of (S1) Hansenia spora uvarum ( Hansenia spora uvarum ) cells and a dry protective composition for preparing a seed solution; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution. Provides a method of preparing a Hansenia spora ubarum yeast spawn with improved survival rate.

In addition, the present invention (S1) Hansenia spora uvarum SJ69 ( Hanseniaspora uvarum SJ69) (Microorganism Accession No .: KACC93298P) step of preparing a seed solution by mixing the cells and the composition for dry protection; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution; wherein the dry protective composition comprises 5 to 15% (w / v) skim milk; And 5 to 15% (w / v) of one or more sugars selected from the group consisting of fructose, maltose, sucrose and trehalose, wherein the dry protective composition comprises 2 to 4 mM vitamins. And C or 4 to 7 mM glutathione, wherein the rehydration solution is at least one selected from the group consisting of distilled water, PBS buffer, NaCl solution, and peptone water. Provided is a method for preparing a seedling of Uvarum yeast.

In addition, the present invention (S1) Hansenia spora uvarum ( Hansenia spora uvarum ) step of preparing a spawn solution by mixing the cells and the dry protective composition; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution. Provides a method of increasing shelf life of the Hansenia spora ubarum yeast spawn.

According to the method for producing or improving the shelf life of the Hansenia spora ubarum yeast seed with improved survival rate according to the present invention, it is possible to prevent damage by heat as compared to conventional hot air drying and to produce seed with much less energy and cost than conventional freeze drying. can do. In addition, it is possible to increase the survival rate and shelf life of Hansenia spora uvarum yeast spawn produced under the optimum conditions according to Hansenia spora uvarum yeast, which can contribute to the high quality of domestic fruit wine as well as the localization and commercialization of native yeast. It can be used in various ways in the wine industry.

는 S. cerevisiae를,

는 P. anomala를,

는 H. uvarum를,

는 P. kluyveri를,

는 C. zemplinina 균주를 나타낸다. 상기 설명은 이하 도 4 내지 도 9에서도 동일하게 해당된다.
도 4는 분리 효모의 알코올 내성을 분석하기 위해, 8 %(v/v) 알코올 함량에서의 생육도를 측정한 결과를 나타낸 도이다.
도 5는 분리 효모의 알코올 내성을 분석하기 위해, 12 %(v/v) 알코올 함량에서의 생육도를 측정한 결과를 나타낸 도이다.
도 6은 분리 효모의 내당성을 분석하기 위해, 20 %(w/v) 글루코오스 농도에서의 생육도를 측정한 결과를 나타낸 도이다.
도 7은 분리 효모의 내당성을 분석하기 위해, 50 %(w/v) 글루코오스 농도에서의 생육도를 측정한 결과를 나타낸 도이다.
도 8은 분리 효모의 내산성을 분석하기 위해, pH 2.0일 때의 생육도를 측정한 결과를 나타낸 도이다.
도 9는 분리 효모의 내산성을 분석하기 위해, pH 4.0일 때의 생육도를 측정한 결과를 나타낸 도이다.
도 10은 본 발명에서 향기 생성능이 우수한 균주로 선정한 상주 20번(P. anomala SJ20), 상주 69번(H. uvarum SJ69), 청도 34번(P. kluyveri CD34), 청도 80번(C. zemplinina CD80), 양평 1번(P. caribbica YP1), 청송 7-16번(P. anomala CS7-16) 균주의 계통수(phylogenetic tree)를 나타낸 도이다.
도 11은 본 발명에서 실시한 저온송풍건조 방법을 도식화하여 나타낸 도이다.
도 12는 본 발명의 저온송풍건조 방법으로 제조된 P. anomala SJ20 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다. 위쪽 그래프는 저온송풍건조 후의 결과를, 아래쪽 그래프는 저온송풍건조 전의 결과를 나타내며, ■는 생존율, □는 수분함량을 나타낸다. 통계분석 결과(^**P<0.01, ^*P<0.05)는 당을 첨가하지 않고 증류수로 재수화 시킨 샘플과 비교하여 나타낸 결과이다. 상기 설명은 이하 도 13 내지 17에서도 동일하게 해당된다.
도 13은 본 발명의 저온송풍건조 방법으로 제조된 H. uvarum SJ69 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다.
도 14는 본 발명의 저온송풍건조 방법으로 제조된 P. kluyveri CD34 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다.
도 15는 본 발명의 저온송풍건조 방법으로 제조된 C. zemplinina CD80 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다.
도 16은 본 발명의 저온송풍건조 방법으로 제조된 P. caribbica YP1 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다.
도 17은 본 발명의 저온송풍건조 방법으로 제조된 P. anomala CS7-16 종균의, 여러 가지 당 및 재수화 용액에 따른 생존율, 수분함량 및 생균수를 측정하여 나타낸 도이다.
도 18은 본 발명의 저온송풍건조 방법으로 제조된 P. anomala SJ20 및 H. uvarum SJ69 종균의, 여러 가지 농도의 비타민 C(V) 및 글루타티온(G) 첨가에 따른 생존율을 알아보기 위해 4℃에서 8주간 보관하며 생균수를 측정하여 나타낸 도이다.
도 19는 본 발명의 저온송풍건조 방법으로 제조된 P. caribbica YP1 및 P. kluyveri CD34 종균의, 여러 가지 농도의 비타민 C(V) 및 글루타티온(G) 첨가에 따른 생존율을 알아보기 위해 4℃에서 8주간 보관하며 생균수를 측정하여 나타낸 도이다.
도 20은 본 발명의 저온송풍건조 방법으로 제조된 C. zemplinina CD80 및 P. anomala CS7-16 종균의, 여러 가지 농도의 비타민 C(V) 및 글루타티온(G) 첨가에 따른 생존율을 알아보기 위해 4℃에서 8주간 보관하며 생균수를 측정하여 나타낸 도이다.
도 21은 본 발명 건조 종균의 저장 온도 및 항산화제(비타민 C(V) 및 글루타티온(G)) 첨가에 따른 저장성을 확인하기 위해, 4℃에서 8주간 보관하며 외관을 촬영하여 나타낸 도이다.
도 22는 본 발명 건조 종균의 형태학적 관찰을 위해 SEM으로 4,000배에서 촬영하여 나타낸 도이다. 가장 왼쪽 위의 결과부터 오른쪽으로 세 개의 결과는 각각 SJ20(P. anomala SJ20), SJ69(H. uvarum SJ69), YP1(P. caribbica YP1)를 나타내며, 가장 왼쪽 아래의 결과부터 오른쪽으로 세 개의 결과는 각각 CS7-16(P. anomala CS7-16), CD80(Candida zemplinina CD80), CD34(P. kluyveri CD34)의 결과를 나타낸다.1 is a diagram showing a yeast DNA separation process according to the present invention.
Figure 2 is a diagram showing the results of PCR-RFLP pattern analysis of the 5.8S-ITS region using Hinf I and Hae III through the isolated yeast DNA. Figure 2a shows the results of the resident (SJ) persimmon wine, Figure 2b shows the results of the Qingdao (CD) persimmon wine.
Figure 3 is a diagram showing the results of measuring the sulfuric acid (500ppm) resistance of isolated yeast in a YPD medium using a spectrophotometer. SJ is the result of resident persimmon wine, and CD is the result of Qingdao persimmon wine.

S. cerevisiae ,

P. anomala ,

H. uvarum ,

P. Kluyveri ,

Represents a strain of C. zemplinina . The above description also applies to FIGS. 4 to 9 below.
Figure 4 is a view showing the results of measuring the growth at 8% (v / v) alcohol content in order to analyze the alcohol resistance of isolated yeast.
5 is a view showing the results of measuring the growth at 12% (v / v) alcohol content in order to analyze the alcohol resistance of isolated yeast.
6 is a view showing the results of measuring the growth at 20% (w / v) glucose concentration in order to analyze the glucose tolerance of the isolated yeast.
7 is a view showing the results of measuring the growth at 50% (w / v) glucose concentration in order to analyze the glucose tolerance of the isolated yeast.
8 is a view showing the results of measuring the growth at pH 2.0 to analyze the acid resistance of the isolated yeast.
9 is a view showing the results of measuring the growth at pH 4.0 in order to analyze the acid resistance of the isolated yeast.
10 is selected as a strain having excellent fragrance generating ability in the present invention 20 ( P. anomala SJ20) , resident 69 ( H. uvarum SJ69) , Qingdao 34 ( P. kluyveri CD34), Qingdao 80 ( C. zemplinina CD80), Yangpyeong 1 ( P. caribbica YP1), Cheongsong 7-16 ( P. anomala CS7-16) is a diagram showing the phylogenetic tree (phylogenetic tree).
11 is a diagram schematically showing a low temperature air blowing drying method performed in the present invention.
12 is a view showing the survival rate, water content and viable cell number of P. anomala SJ20 seedlings prepared by various methods of sugar and rehydration of the present invention prepared by the low temperature air drying method. The upper graph shows the results after the low-temperature blow drying, the lower graph shows the results before the low-temperature blow drying, and ■ indicates the survival rate and □ indicates the water content. The statistical analysis results ( ^** P <0.01, ^* P <0.05) are compared with the sample rehydrated with distilled water without adding sugar. The above description also applies to FIGS. 13 to 17 below.
13 is a view showing the survival rate, water content and viable cell number of H. uvarum SJ69 spawn prepared by various methods of sugar and rehydration solution prepared by the low temperature air drying method of the present invention.
14 is a view showing the survival rate, water content and viable cell number of P. kluyveri CD34 spawn prepared by various methods of sugar and rehydration of the P. kluyveri CD34 spawn prepared by the method of the present invention.
15 is a view showing the survival rate, water content and viable cell number of C. zemplinina CD80 spawn prepared by various methods of sugar and rehydration solution prepared by the low temperature air drying method of the present invention.
16 is a view showing the survival rate, water content and viable cell number of P. caribbica YP1 spawn prepared by the cold-air drying method of the present invention according to various sugars and rehydration solutions.
17 is a view showing the survival rate, water content and viable cell number of P. anomala CS7-16 spawn prepared by the cold-air drying method of the present invention according to various sugars and rehydration solutions.
Figure 18 is a P. anomala SJ20 and H. uvarum SJ69 spawn prepared by the cold-air drying method of the present invention, at 4 ℃ to determine the survival rate according to the addition of various concentrations of vitamin C (V) and glutathione (G) This figure shows the number of viable cells stored for 8 weeks.
Figure 19 is a P. caribbica YP1 and P. kluyveri CD34 spawn prepared by the cold-air drying method of the present invention, at 4 ℃ to determine the survival rate according to the addition of various concentrations of vitamin C (V) and glutathione (G) This figure shows the number of viable cells stored for 8 weeks.
20 is a view showing the survival rate according to the addition of various concentrations of vitamin C (V) and glutathione (G) of C. zemplinina CD80 and P. anomala CS7-16 spawn prepared by the cold-air drying method of the present invention 4 It is a figure showing the measurement of the number of viable cells stored for 8 weeks at ℃.
Figure 21 is a view showing the storage temperature of the dried seed of the present invention and storage of the antioxidant (vitamin C (V) and glutathione (G)) addition, stored for 8 weeks at 4 ℃ and photographed the appearance.
Figure 22 is a photograph taken at 4,000 times by SEM for the morphological observation of the dry seed of the present invention. The three results from the top left to the right represent SJ20 ( P. anomala SJ20), SJ69 ( H. uvarum SJ69), and YP1 ( P. caribbica YP1), respectively. Shows the results of CS7-16 ( P. anomala CS7-16), CD80 ( Candida zemplinina CD80), CD34 ( P. kluyveri CD34), respectively.

The present invention comprises the steps of preparing a seed solution by mixing (S1) Hansenia spora uvarum cells and the dry protective composition; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution. Provides a method of preparing a Hansenia spora ubarum yeast spawn with improved survival rate.

In addition, the method of preparing the Hansenia spora ubarum yeast spawn improved survival rate of the present invention may further comprise the step of preparing the Hansenia spora ubarum bacteria having excellent flavor before the step (S1). In the present invention, the "preparation" may be a screening and using the Hansenia spora ubarum bacteria having excellent flavor producing ability, the advantage of the wine can be further increased when the wine is produced using the prepared spawn have.

In the present invention, the Hansenia spora uvarum cell is a non- Saccharomyces yeast, preferably Hansenia spora uvarum SJ69 (microbial accession number: KACC93298P). In addition, the term "cell" of the present invention may be used interchangeably with the terms microorganism, yeast, strain.

Hansenia spora Ubarum SJ69 cells of the present invention (microbial accession number: KACC93298P) is a strain selected as a strain having excellent fragrance generating ability through the 52 strains. Therefore, there is an advantage that the flavor of the wine can be further increased when the wine is produced using the spawned Hansenia spora Ubarum SJ69 cells of the present invention.

The dry protective composition is 5 to 15% (w / v), preferably 7 to 13% (w / v), more preferably 9 to 11% (w / v), most preferably 10% (w / v) 5-15% (w / v), preferably 7-13%, of skimmed milk and at least one sugar selected from the group consisting of fructose, glucose, maltose, raffinose, sucrose and trehalose; w / v), more preferably 9 to 11% (w / v), most preferably 10% (w / v). The skim milk protects the drying of the microorganism, and the sugar replaces the structural water flowing out of the cell membrane of the cell during drying of the microorganism, and prevents loosening and aggregation of the polar protein group that is hydrogen-bonded in the cell.

In addition, in the step (S1), the cells may be in the form of pellets, and the seed solution may further include an excipient after mixing of the dry protective composition. The excipient may be lactomil and the addition ratio of the cell pellet and lactomil may be 1: 4 (w / w), preferably 1: 3.5 (w / w). Using an excipient lactomil can turn the cells into powder.

In the step (S1), the seed solution may further include an antioxidant, and the antioxidant may be vitamin C or glutathione. When the antioxidant is included, the oxidation process that occurs during long-term storage of dry yeast prepared by blowing air can be suppressed as much as possible, thereby further improving the shelf life. The vitamin C may comprise 2 to 4 mM, preferably 2 to 3 mM, more preferably 3 mM. In addition, the glutathione may comprise 4 to 7 mM, preferably 4 to 6 mM, more preferably 5 mM. When the dose includes vitamin C or glutathione, the shelf life of the Hansenia spora ubarum yeast spawn is further increased to effectively prevent the reduction of viable cell count even during storage for 4 to 8 weeks.

Further, in the step (S2), the seed solution may be prepared by low temperature air drying at 30 to 45 ° C., preferably at 35 to 40 ° C., more preferably at 37 ° C. When the low-temperature air drying in the above temperature range, it is possible to prevent the damage caused by heat compared to the conventional hot air drying and can be dried at much less energy and cost than conventional freeze drying. In addition, the cold air drying may be performed for 20 to 60 minutes, preferably 30 to 40 minutes, but is not limited thereto.

The rehydration solution may be one or more selected from the group consisting of distilled water, Phosphate buffered saline (PBS) buffer, NaCl solution, and peptone water, but is not limited thereto. In addition, the PBS buffer is 1X to 2X, preferably 1XPBS buffer, NaCl solution is 0.5 to 1%, preferably 0.7 to 0.9%, more preferably 0.85% NaCl solution, peptone number is 0.5 to 1.5%, preferably May be 0.8 to 1.3%, more preferably 1% peptone water. The use of the rehydration solution can recover the dried microorganisms through the rehydration process, and more effectively the use of the rehydration solution at this capacity.

The Hansenia spora uvarum cell may be Hansenia spora uvarum SJ69 (microbial accession number: KACC93298P), wherein the dry protective composition is 5 to 15% (w / v), preferably Preferably 7 to 13% (w / v), more preferably 9 to 11% (w / v), most preferably 10% (w / v) skim milk; And 5 to 15% (w / v), preferably 7 to 13% (w / v), more preferably 9, at least one sugar selected from the group consisting of fructose, maltose, sucrose and trehalose. To 11% (w / v), most preferably 10% (w / v). When the Hansenia spora Uvarum SJ69 cells are mixed with at least one sugar selected from the group consisting of skim milk and fructose, glucose, maltose, sucrose and trehalose in the same ratio, Hansenia compared to the unmixed control group The survival rate of Spora ubarum SJ69 yeast spawn can be significantly increased.

In addition, when the Hansenia spora uvarum cell is Hansenia spora uvarum SJ69 (Microbial Accession No .: KACC93298P), the dry protective composition may further include vitamin C or glutathione. If the vitamin C or glutathione is further included, the storage capacity of the Hansenia spora uvrum SJ69 yeast spawn prepared by blowing air may be increased to prevent a decrease in the number of viable cells according to the storage period.

In addition, the vitamin C may be included 2 to 4 mM, preferably 2 to 3 mM, more preferably 3 mM. In addition, the glutathione may be included 4 to 7 mM, preferably 4 to 6 mM, more preferably 5 mM. The inclusion of vitamin C or glutathione in the dose increases the shelf life of the Hansenia spora uvrum SJ69 yeast spawn, thereby effectively preventing the reduction of viable cell count even during storage for 4 to 8 weeks.

In addition, the Hansenia spora uvarum cell may be Hansenia spora uvarum SJ69 (Microbial Accession No .: KACC93298P), wherein the rehydration solution is distilled water, PBS buffer, NaCl solution and peptone water. It may be one or more selected from the group consisting of. The use of one or more rehydration solutions selected from the group consisting of distilled water, PBS buffer, NaCl solution and peptone water can increase the survival rate of the dried Hansenia spora Uvarum SJ69 yeast spawn, further enhancing the usability of the spawn. Can be. In addition, the PBS buffer is 1X to 2X, preferably 1XPBS buffer, NaCl solution is 0.5 to 1%, preferably 0.7 to 0.9%, more preferably 0.85% NaCl solution, peptone number is 0.5 to 1.5%, preferably May be 0.8 to 1.3%, more preferably 1% peptone water. By using the rehydration solution at the above capacity, it is possible to more effectively recover the dried microorganisms and increase the survival rate of the dried Hansenia spora uvrum SJ69 yeast seed.

According to a preferred embodiment of the present invention, the present invention, (S1) Hansenia spora uvarum SJ69 ( Hanseniaspora uvarum SJ69) (Microorganism Accession No .: KACC93298P) mixing the cells and the composition for dry protection to prepare a seed solution; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution; wherein the dry protective composition comprises 5 to 15% (w / v) skim milk; And 5 to 15% (w / v) of one or more sugars selected from the group consisting of fructose, maltose, sucrose and trehalose, wherein the dry protective composition comprises 2 to 4 mM vitamins. And C or 4 to 7 mM glutathione, wherein the rehydration solution is at least one selected from the group consisting of distilled water, PBS buffer, NaCl solution, and peptone water. Provided is a method for preparing a seedling of Uvarum yeast. According to the above production method, the survival rate of Hansenia spora uvrum SJ69 yeast spawn can be most effectively increased.

The present invention also comprises the steps of (S1) Hansenia spora uvarum ( Hansenia spora uvarum ) cells and a dry protective composition to prepare a seed solution; (S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And (S3) treating the spawn powder with a rehydration solution. Provides a method of increasing shelf life of the Hansenia spora ubarum yeast spawn.

In addition, after the step (S2), the seed powder is stored at 0 to 10 ℃, preferably 2 to 7 ℃, more preferably 3 to 5 ℃, most preferably 4 ℃; further comprising a Can be.

When the spawn powder prepared by the low-temperature air drying is prepared and stored in the same manner as above, the survival rate of the Hansenia spora uvrum yeast spawn is improved to increase the shelf life of the spawn and to change the color of the spawn powder. It is possible to prevent the growth of other microorganisms can effectively increase the shelf life of Hansenia spora Uvarum yeast seed.

Another description of the storage method of the Hansenia spora ubarum yeast spawn of the present invention is omitted to avoid the overlap with the description of the method for producing the Hansenia spora ubarum yeast spawn improved survival rate.

Therefore, according to the method of manufacturing or increasing the shelf life of the Hansenia spora ubarum yeast seed with improved survival rate of the present invention, it is possible to prevent damage by heat as compared to conventional hot air drying and to produce the seed with much less energy and cost than conventional freeze drying. can do. In addition, it is possible to increase the survival rate and shelf life of Hansenia spora uvarum yeast spawn produced under optimal conditions according to Hansenia spora uvarum yeast, which can contribute to the quality of domestic fruit wine along with the localization and commercialization of indigenous yeast.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated more concretely based on an Example. The examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1 Persimmon Wine Preparation and Yeast Separation

Example 1-1. Persimmon wine manufacturing

Hongsi used to make persimmon wine for the separation of yeast was made by softening young persimmons purchased in the two provinces of Sangju and Qingdao in late November 2016. After crushing the hongsi of each of the two areas by 3 kg, 200 ppm of potassium metabisulfite (K ₂ S ₂ O ₅ ) was added to prevent contamination of various bacteria. The shredded hongsi were put into 10 L fermentation vessel and spontaneously fermented at 20 ° C. Persimmon wine was prepared by fermentation for 6 days in the case of resident Hongsi and 12 days in the case of Qingdao Hongsi.

Example 1-2. Separation of yeast

YPD solid medium (1.0% yeast extract, 2.0% peptone, 2.0% glucose, 2.0% agar) was used for strain isolation from the prepared persimmon wine. Samples were taken at intervals of two days from the start of fermentation of persimmon wine to the end of fermentation, plated on medium, and incubated at 30 ° C. for 48 hours. Strains were purely separated in YPD solid medium based on the type of colonies generated among the first isolates.

As a result, 191 strains of yeast were separated, and 99 strains of resident persimmon wine and 92 strains of Qingdao persimmon wine were separated.

Example 2. PCR-RFLP Analysis

To determine the genetic diversity pattern of yeast isolated during fermentation of Sangju and Qingdao Persimmon wine, PCR-RFLP analysis and sequencing were performed in the internal transcribed spacer (ITS) region of the isolated strains by the following method. did.

Example 2-1. Yeast DNA Isolation and Manipulation

191 strains isolated in Example 1 were inoculated in a YPD liquid medium and incubated with a shaker set at 150 ° C. at 30 ° C. for 24 hours, followed by centrifugation (13,000 rpm, 10 min) to recover the cells. 0.4 mL of 5% chelex was added to the recovered cells, vortexed to allow constant mixing of the cells and the cells, and then boiled at 90-95 ° C for 10 minutes and left for 10 minutes. The same procedure was repeated one more time and centrifuged (13,000 rpm, 10 min) to separate the supernatant, which was used for PCR analysis. The procedure is illustrated in FIG.

Example 2-2. Polymerase chain reaction (PCR)

The primers used for PCR were purchased from Bioneer (Daejeon, Korea) and synthesized in both directions of universal primers ITS1 and ITS4 in the yeast internal transcribed spacer (ITS) region. PCR was performed using Tpersnal 48 (Biometra Co., Gottingen, Germany) equipment, and the type and sequence of primers used are shown in Table 1. In addition, the amount of the reaction solution used for PCR was adjusted to a total of 50μL, the composition of the reaction solution is shown in Table 2, the reaction conditions are shown in Table 3.

Table 1. PCR primer types and sequences primer
(Primer) Sequence  ITS1 5`-TCC GTA GGT GAA CCT GCG G-3`  ITS4 5`-TCC TCC GCT TAT TGA TAT GC-3`

Table 2. PCR reaction solution composition Composition Amount, μL 10X Taq reaction buffer  5.00 Template DNA  2.00 Forward primer (10 pmol / μl)  2.00 Reverse primer (10 pmol / μl)  2.00 dNTP mixture (10 mM)  1.00 Taq polymerase (5 U / μl)  0.25 Distilled water 37.75 Total volume 50.00

Table 3. PCR reaction conditions cycle
(Cycles) denaturalization
(Denaturation) Combination
(Annealing) extension
(Extension) Frequency
(Frequency) One 94 ℃ / 3 min 50 ℃ / 1 min 72 ℃ / 1 min One 2 94 ℃ / 30 s 50 ℃ / 1 min 72 ℃ / 1 min 32 3 72 ℃ / 10 min One

Example 2-3. Restriction Enzyme Treatment

In order to obtain a DAN fragment, restriction enzymes were used as Hinf I, Hae III, and reacted for 1 hour at 37 ° C with the composition shown in Table 4.

Table 4. Compositions for restriction enzyme treatment of amplified yeast DNA Composition Amount, μL PCR product  5.00 Enzyme ( Hinf I, Hae III)  0.5 Enzyme buffer (× 10)  0.75 Distilled water  8.75 Total volume 15

Example 2-4. DNA electrophoresis

The obtained DNA fragments were confirmed by electrophoresis in the following manner. DNA electrophoresis was performed at 100V voltage and 1.5% agarose gel (Molecular Biology grade, Korea) and 0.5 × TBE buffer were used. After the electrophoresis gel was developed with 0.5 mg / mL EtBr (Ethdium Bromide) solution for 10 minutes, washed in distilled water for 10 minutes and then UV transilluminator (Vilber Lour mat.TFX-20M, B.0.66 TORCY-Z, I SUD , Paris, France). As a marker for measuring the size of DNA fragments by strain, 100 bp Plus DNA ladder (Biofact, Lot No, M54G338IG, Korea) was used.

The results of electrophoresis are shown in FIG. 2, and FIG. 2A shows the results of the Sangju (SJ) persimmon wine, and FIG. 2B shows the results of the Qingdao (CD) persimmon wine. In addition, in the results of the following drawings, SJ means resident, and CD means Qingdao Persimmon wine.

As a result of RFLP pattern analysis using Hinf I and Hae III through Example 2, the pattern of the strain isolated from the resident persimmon wine as shown in FIG. Isolated strains showed a pattern of relatively diverse shapes. This is because yeasts in different climate conditions such as temperature, humidity, and wind show different species diversity.

Example 3 Identification of Isolated Yeast

By comparing the pattern shape of the strain isolated in Example 2, a pattern determined to be different in shape was selected, and the homology was investigated after analyzing the nucleotide sequence of the ITS1-5.8S rDNA-ITS2 region by the following method. It was.

Specifically, based on the nucleotide sequence of the ITS site of 5.8S rDNA of the isolated strain was identified through phylogenetic analysis. DNA sequencing was performed using the sequencing service of Solgent (Daejeon, Korea). Homology of sequencing was performed using BLAST of the National Center for Biotechnology Information (NCBI). Multiple Sequence Alignment for systematic analysis was performed using ClustalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI). Used. Phylogenetic trees were prepared and analyzed using Molecular Evolutionary Genetics Analysis (MEGA7).

A total of seven strains of Sangju 6, Changju 7, Changju 53, Qingdao 3, Qingdao 4, Qingdao 19, and Qingdao 81 were selected through homology analysis. The results are shown in Table 5 below.

Table 5. BLAST results of isolates Isolates Species and strain designation Identity (%) SJ-6 Saccharomyces cerevisiae strain ATCC 208606 100 SJ-7 Pichia anomala strain MTCC 237 100 SJ-53 Hanseniaspora uvarum strain UFLA CWFY3 99 CD-3 Pichia kluyveri strain CBS 188 100 CD-4 Hanseniaspora uvarum strain JBBB-1 100 CD-19 Pichia anomala strain MTCC 3815 100 CD-81 Candida zemplinina isolate MCR9 99 SJ; SangJu persimmon wine, CD; CheongDo persimmon wine

As shown in Table 6, Sangju No. 6 showed 100% homology with Saccharomyces cerevisiae , which is commonly used for wine production, and Changju No. 7 and Qingdao No. 19 showed 100% homology with Pichia anomala . Resident 53 and Qingdao No. 4 and Hanseniaspora uvarum and respectively 99% and 100%, Qingdao three times P. kluyveri, Qingdao 81 times was found to have a homology of Candida zemplinina and 100%. Therefore, six strains except Sangju 6 were identified as non- Saccharomyces species.

In addition, the isolates were distinguished by judging the group having the same pattern as the pattern of the strains that had been subjected to homology to the same species.

Table 6. Number of Yeasts by Strain Pattern Isolated from Permanent and Qingdao Persimmon Wines Strain Isolated yeast can all Resident Qingdao Saccharomyces cerevisiae 7 * ND 7 Pichia anomala 51 20 71 Hanseniaspora uvarum 41 60 101 Pichia kluyveri ND 10 10 Candida zemplinina ND 2 2 all 99 92 191 * ND = not detected.

As shown in Table 6, the most isolates of P. anomala were 51 isolates from Persimmon persimmon wine, and the most were H. uvarum (60) in Qingdao. It was also confirmed that P. kluyveri and C. zemplinina, which are non- Saccharomyces strains, were not found in Persimmon wine.

Example 4 Analysis of Environmental Tolerance and Selection of Yeasts According to Environmental Tolerance

Example 4-1. Sulfurous acid resistance assay

Sulfite is widely used in food storage because of its ability to inhibit oxidation and inhibit the growth of bacteria, and is a food store that plays a role in improving the appearance and quality of food and wine. In order to investigate the sulfurous acid (K ₂ S ₂ O ₅ ) resistance of the isolated strain was added sulfuric acid of 500ppm (w / v) concentration to YPD liquid medium and 5 mL each was dispensed into the test tube. Inoculate 5% (v / v) of the preculture of the isolated strain to each strain and incubate for 48 hours at 30 rpm at 150 rpm, followed by 600 nm using a spectrophotometer (Shimazdu Co. UV-1601, Japan). Absorbance was measured at to measure the growth of bacteria, and the results are shown in FIG. 3.

As shown in Figure 3, it was confirmed that the strains isolated in two regions of Changju and Qingdao showed similar growth levels.

Example 4-2. Alcohol Tolerance Assay

One of the most common stresses yeasts undergo during fermentation is their high ethanol content. Ethanol increases the fluidity of the plasma membrane and destroys normal membrane structure. As the fermentation of the wine increases the alcohol content, it is necessary to study the alcohol resistance of the isolate strain for smooth wine production.

In order to investigate the alcohol resistance of the isolated strain, the alcohol content of the YPD liquid medium was adjusted to 8% (v / v) and 12% (v / v), respectively, and 5 mL was dispensed into a test tube. Inoculated with 5% (v / v) of the pre-culture of the isolated strain to each strain and incubated for 48 hours at 30 rpm at 150 ℃, and then measured the absorbance at 600 nm with a spectrophotometer to measure the growth of the bacteria . Among them, the results at 8% (v / v) alcohol content are shown in FIG. 4, and the results at 12% (v / v) alcohol content are shown in FIG. 5.

As shown in FIG. 4, it was confirmed that the S. cerevisiae strain and the P. anomala strain showed higher alcohol resistance than other strains at an alcohol content of 8%.

In addition, as shown in Figure 5 in the alcohol content of 12% strains 3, 4, 6, 17, 23, 26, 27 S. cerevisiae strain showed the best resistance, the other strains showed that the growth is significantly inhibited Confirmed.

Therefore, as a result of alcohol resistance analysis, it was confirmed that P. anomala strain had the best alcohol resistance among non- Saccharomyces yeast.

Example 4-3. Tolerance Analysis

In wine production or in certain other circumstances, yeast must cope with osmotic stress caused by high sugar concentrations. In order to investigate the glucose tolerance of the isolated strain, the glucose concentration of the YPD liquid medium was adjusted to 20% (w / v) and 50% (w / v), respectively, and 5 mL was dispensed into the test tube. Inoculated with 5% (v / v) of the pre-cultivation of the isolated strain to each strain and incubated for 48 hours at 30 rpm at 150 ℃, and then measured the absorbance at 600 nm with a spectrophotometer to measure the growth of the bacteria . The results at 20% (w / v) glucose concentration are shown in FIG. 6 and the results at 50% (w / v) glucose concentration are shown in FIG. 7.

As shown in FIG. 6, when the 20% glucose was added, it was confirmed that the strain initially isolated from the resident persimmon wine exhibited relatively high growth.

In addition, as shown in FIG. 7, when the 50% glucose was added, the strain showing the highest growth was S. cerevisiae , the strain 23, 26, and 27 resident. Also, among the same non- Saccharomyces yeast, the growth rate of P. anomala was higher than that of H. uvarum .

Example 4-4. Acid Resistance Analysis

In order to investigate the acid resistance of the isolated strain, the pH of the YPD liquid medium was adjusted to pH 2.0 and pH 4.0, and 5 mL were dispensed into the test tube. Inoculated with 5% (v / v) of the pre-cultivation of the isolated strain to each strain and incubated for 48 hours at 30 rpm at 150 ℃, and then measured the absorbance at 600 nm with a spectrophotometer to measure the growth of the bacteria . Among them, the results at pH 2.0 are shown in FIG. 8, and the results at pH 4.0 are shown in FIG. 9.

As shown in FIG. 8, P. anomala excluding Permanent No. 18 showed relatively lower value than the growth of H. uvarum in Persimmon Persimmon wine at pH 2.0. As a result, it was confirmed that P. anomala hardly grow under the pH 2.0 environmental conditions. On the other hand, the strains isolated from Qingdao Persimmon wine did not show characteristic growth.

In addition, as shown in FIG. 9, it was confirmed that at pH 4.0, all strains isolated from the resident and the Qingdao Persimmon wine exhibited no significant difference.

Example 4-5. First Selection of Yeast According to Environmental Tolerance

In general, alcohol content of wine is known to be the best when it is 12%. Fruits such as grapes, apples, and persimmons grown domestically have low initial sugars, and sugar is added to adjust the sugar to about 24 Brix after fermentation. It is used. When sugar is added to increase the sugar, the osmotic pressure in the juice increases, so there is a possibility that some strains may inhibit growth. In addition, when the alcohol content is increased during the fermentation process, in case of strains having low alcohol resistance, growth may be inhibited and killed. Therefore, prior to the selection of fermented yeast having excellent fragrance generating ability, it is necessary to first select a yeast showing excellent growth in the environment of general wine, and in the present invention, the environmental resistance analysis results of Examples 4-1 to 4-4 are described. On the basis of the following, fermented yeast with excellent growth was selected as follows.

In the case of sulfurous acid resistance, it was determined that the yeast isolated in the present invention does not have a great effect. In addition, in case of acid resistance, considering that the pH range of general fruit wine is between 3.6 and 4.0, most of them showed excellent growth in the pH 4 environment and were excluded from consideration. In the case of alcohol tolerance, the superior yeast was selected by comparing the growth in 8% alcohol environment considering that non- Saccharomyces yeast affects the initial aroma production. In addition, even in the case of glucose tolerance, excellent yeasts were selected for each species by comparing the growth degree in a 20% glucose environment similar to the general wine fermentation environment.

As a result, in P. anomala resident 1, 2, 7, 11, 15, 16, 18, 19, 20, 24, 37, 47 showed higher alcohol and glucose tolerance than other P. anomala , H In uvarum , resident 53, 61, 68, 69, 77, 81, 88 and Qingdao 8, 14, 35, 39, 77, 78, 82 showed relatively high growth rate. P. kluyveri and C. zemplinina were separated into a small number of 10 and 2, respectively, were included in the primary selection target, and a total of 38 strains were selected first.

Example 5 Secondary Selection of Yeast According to Sniffing Test

Sniffing test (sniffing test) is an experiment that smells through the nose, it is an experiment that can determine whether the flavor is produced easily and quickly. In order to separate non- Saccharomyces native fermented yeast with excellent fragrance-generating ability through this experiment, 38 strains selected first and apple isolates previously isolated and stored in this laboratory A total of 52 strains were used to isolate Aaronia isolates 14, 16, Yangpyeong 1, 5, 9, 24, 30, 37, 38, 39, 40, and Danyang 32. Information on the 52 strains used in the experiments is shown in Table 7.

Table 7. Isolated Yeasts Used for Sniffing Tests No. Yeasts Strains Environmental resistance Origin Ref. In glucose 20% In alcohol 8% One SJ-1 P. anomala 23.960 ± 0.56 4.983 ± 0.05 feeling In this sudy 2 SJ-2 P. anomala 21.960 ± 0.16 7.233 ± 0.04 feeling In this sudy 3 SJ-7 P. anomala 22.220 ± 0.14 6.057 ± 0.06 feeling In this sudy 4 SJ-11 P. anomala 23.027 ± 0.48 6.167 ± 0.019 feeling In this sudy 5 SJ-15 P. anomala 21.613 ± 0.18 6.470 ± 0.08 feeling In this sudy 6 SJ-16 P. anomala 22.133 ± 0.98 4.393 ± 0.01 feeling In this sudy 7 SJ-18 P. anomala 22.067 ± 0.30 6.120 ± 0.07 feeling In this sudy 8 SJ-19 P. anomala 22.947 ± .023 6.390 ± 0.06 feeling In this sudy 9 SJ-20 P. anomala 21.587 ± 0.06 6.060 ± 0.05 feeling In this sudy 10 SJ-24 P. anomala 13.747 ± 0.06 7.443 ± 0.02 feeling In this sudy 11 SJ-37 P. anomala 22.547 ± 0.19 4.290 ± 0.10 feeling In this sudy 12 SJ-47 P. anomala 10.787 ± 0.04 6.667 ± 0.02 feeling In this sudy 13 SJ-53 H. uvarum 12.040 ± 0.04 0.884 ± 0.004 feeling In this sudy 14 SJ-61 H. uvarum 6.875 ± 0.03 1.935 ± 0.01 feeling In this sudy 15 SJ-68 H. uvarum 9.851 ± 0.03 1.045 ± 0.01 feeling In this sudy 16 SJ-69 H. uvarum 7.749 ± 0.12 1.039 ± 0.02 feeling In this sudy 17 SJ-77 H. uvarum 9.632 ± 0.02 1.207 ± 0.01 feeling In this sudy 18 SJ-81 H. uvarum 9.045 ± 0.05 1.163 ± 0.01 feeling In this sudy 19 SJ-88 H. uvarum 8.912 ± 0.21 1.196 ± 0.0 feeling In this sudy 20 CD-1 P. kluyveri 7.451 ± 0.06 1.434 ± 0.06 feeling In this sudy 21 CD-2 P. kluyveri 10.240 ± 0.15 1.204 ± 0.01 feeling In this sudy 22 CD-3 P. kluyveri 10.656 ± 0.11 0.363 ± 0.01 feeling In this sudy 23 CD-8 H. uvarum 12.592 ± 0.29 0.980 ± 0.01 feeling In this sudy 24 CD-14 H. uvarum 13.003 ± 0.22 1.037 ± 0.01 feeling In this sudy 25 CD-23 P. kluyveri 12.683 ± 0.25 0.406 ± 0.02 feeling In this sudy 26 CD-25 P. kluyveri 8.960 ± 0.09 1.011 ± 0.01 feeling In this sudy 27 CD-34 P. kluyveri 7.109 ± 0.07 1.037 ± 0.02 feeling In this sudy 28 CD-35 H. uvarum 7.109 ± 0.06 0.979 ± 0.02 feeling In this sudy 29 CD-39 H. uvarum 9.477 ± 0.04 1.055 ± 0.02 feeling In this sudy 30 CD-72 P. kluyveri 6.389 ± 0.12 0.700 ± 0.01 feeling In this sudy 31 CD-75 P. kluyveri 7.707 ± 0.02 1.029 ± 0.01 feeling In this sudy 32 CD-76 P. kluyveri 7.643 ± 0.10 0.866 ± 0.03 feeling In this sudy 33 CD-77 H. uvarum 9.765 ± 0.12 1.019 ± 0.02 feeling In this sudy 34 CD-78 H. uvarum 7.323 ± 0.03 1.221 ± 0.01 feeling In this sudy 35 CD-80 C. zemplinina 9.840 ± 0.07 1.079 ± 0.01 feeling In this sudy 36 CD-81 C. zemplinina 9.813 ± 0.10 1.166 ± 0.01 feeling In this sudy 37 CD-82 H. uvarum 9.813 ± 0.07 1.423 ± 0.02 feeling In this sudy 38 CD-83 P. kluyveri 8.347 ± 0.17 1.206 ± 0.01 feeling In this sudy 39 GJ-14 P. anomala 27.400 ± 0.12 4.553 ± 0.01 Aaronia (Cha, 2017) 40 GJ-16 P. anomala 24.340 ± 0.02 4.973 ± 0.02 Aaronia (Cha, 2017) 41 YP-1 P. caribbica 16.580 ± 0.06 1.505 ± 0.01 Aaronia (Cha, 2017) 42 YP-5 P. anomala 27.400 ± 0.24 5.713 ± 0.41 Aaronia (Cha, 2017) 43 YP-9 P. anomala 25.100 ± 0.03 6.157 ± 0.21 Aaronia (Cha, 2017) 44 YP-24 P. anomala 26.980 ± 0.21 5.327 ± 0.14 Aaronia (Cha, 2017) 45 YP-30 P. anomala 27.440 ± 0.14 5.887 ± 0.21 Aaronia (Cha, 2017) 46 YP-37 P. anomala 29.620 ± 0.21 5.253 ± 0.15 Aaronia (Cha, 2017) 47 YP-38 P. anomala 29.260 ± 0.41 5.480 ± 0.04 Aaronia (Cha, 2017) 48 YP-39 P. anomala 29.750 ± 0.12 4.977 ± 0.13 Aaronia (Cha, 2017) 49 YP-40 P. anomala 28.340 ± 0.21 5.723 ± 0.19 Aaronia (Cha, 2017) 50 DY-32 P. caribbica 21.330 ± 0.03 1.570 ± 0.04 Aaronia (Cha, 2017) 51 CS 7-16 P. anomala 30.480 ± 0.21 4.330 ± 0.12 Apple * In lab 52 CS 13-15 P. anomala 31.240 ± 0.32 0.600 ± 0.21 Apple In lab SJ; SangJu persimmon wine, CD; Cheong Do persimmon wine, GJ; Gang Jin Aronia, YP; Yang Pyeong Aronia, DY; Dan Yang Aronia, CS; CheongSong Apple wine
All the data were expressed as meas ± SD (n-3).
* In lab, those strains were previously isolated and stored in our laboratory.

In order to select a strain having excellent fragrance generating ability against the 52 strains of Table 7, the sniffing test was conducted in the following manner. Apples purchased from Hongsi and Cheongsong prepared by softening the persimmons purchased from Qingdao were washed, crushed and filtered to obtain juice. Inoculate 5% (v / v) of the preculture of the strain isolated on sterilized juice, ferment for 2 weeks at 20 ℃, select 10 general sensory personnel consisting of college students and graduate students to smell the five-point scale method The test was carried out. From 1 point (fragrance is very bad, not unique) to 5 points (fragrance is very good, unique), the results of the sniffing test performed by the 10 sensory personnel as shown above are shown in Table 8 below. .

Table 8. Sniffing test results of isolated yeast Yeast strains Flavor Yeast strains Flavor P. anomala feeling Apple H. uvarum feeling Apple SJ-1 + ++ SJ-53 +++ ++ SJ-2 +++ + SJ-61 +++ + SJ-7 + + SJ-68 +++ ++ SJ-11 + + SJ-69 ++++ ++ SJ-15 + + SJ-77 + ++ SJ-16 + ++ SJ-81 ++ ++ SJ-18 +++ ++ SJ-88 ++ ++ SJ-19 +++ ++ CD-8 ++ + SJ-20 +++++ ++ CD-14 + ++ SJ-24 + + CD-35 + + SJ-37 + + CD-39 + ++ SJ-47 + + CD-77 + ++ GJ-14 +++ ++ CD-78 + + GJ-16 +++ ++ CD-82 +++ ++ YP-5 +++ + P. kluyveri YP-9 + + CD-1 ++ + YP-24 + + CD-2 + ++ YP-30 + + CD-3 + ++ YP-37 + + CD-23 ++ + YP-38 + ++ CD-25 ++ ++ YP-39 + + CD-34 ++++ ++ YP-40 + ++ CD-72 ++ ++ CS 7-16 ++ ++++ CD-75 + ++ CS 13-15 ++ ++ CD-76 + + CD-83 ++ + C. zemplinina P. caribbica CD-80 ++++ +++ DY-32 + + CD-81 ++ ++ YP-1 ++++ ++ SJ; SangJu persimmon wine, CD; Cheong Do persimmon wine, GJ; Gang Jin Aronia, YP; Yang Pyeong Aronia, DY; Dan Yang Aronia, CS; CheongSong Apple wine.
Approval number: 2017-0125

As shown in Table 8, the strains that received the highest score in the juice were 20, Sangju 69, Qingdao 34, Cheongdo 80, and Yangpyeong 1, and the strains with the highest score in apple juice were Cheongsong 7-16 and Qingdao 80. Burn-in was confirmed. Qingdao 80 scored high in both juice and apple juice.

According to the results of the sniffing test, resident 20 ( Pichia anomala SJ20, P. anomala SJ20 ) , resident 69 ( Hanseniaspora uvarum SJ69, H. uvarum SJ69), Qingdao 34 ( Pichia kluyveri CD34, P. kluyveri CD34), Total 6 of Qingdao No. 80 ( Candida zemplinina CD80, C. zemplinina CD80), Yangpyeong No. 1 ( Pichia caribbica YP1, P. caribbica YP1), Cheongsong No. 7-16 ( Pichia anomala CS7-16, P. anomala CS7-16) The dog strain was determined to produce a good fragrance and was finally selected. The phylogenetic tree of the selected six strains is shown in FIG. 10.

The six selected strains of hometown flavor were deposited with the National Academy of Agricultural Sciences, and each of No. 20 strain ( P. anomala SJ20) was deposited on Feb. 22, 2018, Accession No. KACC93297P, No. 69 ( H. uvarum). SJ69 ) strain was deposited on February 22, 2018, accession number KACC93298P, Qingdao 34 ( P. kluyveri CD34) strain was deposited on October 18, 2017, accession number KACC93292P, Qingdao 80 ( C. zemplinina CD80) For the strain, deposited on February 22, 2018, accession number KACC93299P, Yangpyeong 1 ( P. caribbica YP1) strain, deposited on October 18, 2017, accession number KACC93291P, Cheongsong 7-16 ( P anomala CS7-16) was deposited on October 18, 2017 and was assigned accession number KACC93290P.

Example 6 Low Temperature Blow Drying and Survival Measurement of Final Selected Yeast

Example 6-1. Strain and Culture Conditions

Sangju 20 ( P. anomala SJ20) , Sangju 69 ( H. uvarum SJ69) , Qingdao 34 ( P. kluyveri CD34), Qingdao 80 ( C zemplinina CD80), Yangpyeong 1 ( P. caribbica YP1), Cheongsong 7-16 ( P. anomala CS7-16) 6 strains were used in the experiment. Inoculate 5% (v / v) of the preculture solution of the strain in 300 mL of the YPD liquid medium was used after incubating for 16 hours at 30 ℃.

Example 6-2. Powder spawn production through low temperature air blowing

Low-temperature air drying was performed using a dust-free dryer (HanBeak, HB-509C, Korea) that can be dried in a dust-free, sterile environment and the low temperature air drying method shown in the present invention is shown schematically in FIG.

As shown in FIG. 11, the protective agent and the excipient were treated during the low temperature air drying of the present invention, and the composition and concentration of the protective agent and the excipient used in the low temperature air drying are shown in Table 9 below.

Table 9. Composition and Concentrations of Protectors and Excipients Protection Excipient Skim milk
(Skim milk) Party
(Sugars) Pellets: Lactomil
(Pellet: Lactomil) 10% (w / v) Fructose
(Fructose) 10% (w / v) 1: 3.5 (w / w) Glucose
(Glucose) maltose
(Maltose) Raffinose
(Raffinose) Sucrose
(Sucrose) Trehalos
(Trehalose)

As shown in Table 9, 10% (w / v) of skim milk as a dry protective agent was used, and additionally 10% (w / v) of 6 sugars (fructose, glucose, maltose, raffinose, sucrose, trehalose) was used. . In addition, the excipient for making the cells into powder form was used lactomil (lactose 89%, maltodextrin 11%) and the ratio of the cell pellet and lactomil was added to 1: 3.5 (w / w).

Thereafter, the powder was spawned by drying for 30 to 40 minutes until the content was 8% to 12%. The moisture content of all treatments was adjusted to 10 ± 2%, which was determined to have the highest survival rate of microorganisms after drying. Moisture content was measured by weighing the sample and dried in a 105 ℃ dry oven for 3 to 4 hours, then allowed to cool for 20 minutes in a desiccator and weighed until the weight was reached.

Example 6-3. Rehydration Solution and Treatment

Distilled water (D.W.), 1x PBS (Phosphate buffered saline) buffer, 0.85% NaCl, and 1% peptone water were used to investigate the effect of rehydration conditions on cryogenic dry yeast. After the low temperature air drying, the sample was put in a rehydration solution, restored to an initial volume, and rehydrated at 25 ° C. for 1 hour. Subsequently, step dilution with sterile water as follows to measure the survival rate in the YPD solid medium.

Example 6-4. Survival Measurement of Strains

. 이에 대한 결과를 도 12 내지 도 17에 나타내었다.The initial bacterial count of the strain was incubated at 30 ° C. for 16 hours in YPD liquid medium, 1 mL of the sample was diluted prior to cryogenic drying, and the colonies grown after incubation at 30 ° C. for 48 hours in YPD solid medium were counted. Measured. Survival rate of the strains by treatment conditions was restored by the initial volume of the air-dried strain using a rehydration solution, and then incubated in the same conditions as the initial number of bacteria and expressed through the following equation. Survival rate:

. The results are shown in FIGS. 12 to 17.

As shown in FIG. 12, in the case of P. anomala SJ20, when the sugar was added as compared to the sugar-free and DW rehydration conditions used as a control, it was confirmed that the survival rate increased in all the test zones, 10% raffinose All of the sugars except fructose, fructose, glucose, maltose, sucrose and trehalose showed a three times higher survival rate than the control. Among them, especially when 10% trehalose was added and rehydrated with 1% peptone water for 1 hour, the highest survival rate was 103.76%. Survival surpasses 100% due to yeast growth in the proper environment during the rehydration process.

In addition, as shown in Figure 13, H. uvarum SJ69 showed a particularly high survival rate when rehydrated with 0.85% NaCl solution in fructose, maltose, sucrose, trehalose addition compared to the control, In some sugars it was confirmed that 1 × PBS solution was also an excellent rehydration condition. In particular, when the 10% sucrose addition group was rehydrated with 0.85% NaCl solution for 1 hour, the survival rate was 144.33%.

In addition, in the case of P. kluyveri CD34, maltose, raffinose, and sucrose were added to the rehydration solution, the survival rate was lower than that of the control group, except for some conditions. It did not increase. However, the survival rate of 148.92% was increased by 148.92% when the 1% peptone number was used in the glucose addition group, and the best survival condition was 182.10 when rehydrated with 0.85% NaCl solution in the fructose addition group. Very high survival rate in%.

In addition, in the case of C. zemplinina CD80 as shown in Figure 15, when rehydrated with 0.85% NaCl solution in the addition of sucrose, the best survival rate was 164.61%.

In addition, as shown in FIG. 16, P. caribbica YP1 showed a high survival rate of more than 100% even when rehydrated in 1 × PBS solution and 1% peptone water for 1 hour without adding sugar, but maltose and 1% peptone water. Survival of 120.66% using, 123.22% using raffinose and DW, and 147.76% using sucrose and 0.85% NaCl showed very good survival rates. In addition, the condition showing the highest survival rate was 160.69% when trehalose and 1 × PBS solution were used. In the fructose and glucose addition groups, the effect of survival rate was not found for each rehydration solution.

In addition, in the case of P. anomala CS7-16, as shown in Figure 17, the initial survival rate of the control group showed a relatively low survival rate of 40.28% compared to other strains, but when using a 0.85% NaCl solution in maltose added 130.74% of Survival rate was very high with 128.65% survival rate when 1 × PBS solution was used in the trehalose solution, and the highest survival rate was 152.46% when 0.85% NaCl solution was used in the trehalose solution. Indicated.

As described above, the drying protection agent and the rehydration solution conditions which can maximize the survival rate during the air drying of the six types of fermented yeasts finally selected in the present invention are shown in Table 10 below.

Strain Low temperature blowing condition Survival rate (%) % Increase Skim milk Per 10% Rehydration solution Test group Control P. anomala
SJ20 10% Trehalos 1% Peptone water 103.76 21.55 ▲ 82.21 H. uvarum
SJ69 Sucrose 0.85% NaCl 144.33 84.07 ▲ 60.26 P. caribbica
YP1 Trehalos 1 × PBS 160.69 55.20 ▲ 105.49 P. kluyveri
CD34 Fructose 0.85% NaCl 182.10 74.26 ▲ 107.84 C. zemplinina CD80 Sucrose 0.85% NaCl 164.61 84.73 ▲ 79.88 P. anomala
CS7-16 Trehalos 0.85% NaCl 152.46 40.28 ▲ 112.18

As shown in Table 10, it was confirmed that the types of sugars that can protect the cells during drying were different for each yeast, and the types of rehydration solutions that may affect the improvement of cell viability due to environmental changes during rehydration were different. Confirmed. Also, sugar-free and D.W. Compared with the rehydration solution treatment group, the survival rate was increased from the lowest 60.26% to the maximum 112.18%. Therefore, the optimal conditions for seeding set forth in the present invention are expected to help the production of prototypes having a high survival rate in the future industrialization stage of the strain.

Example 7 Measurement of Survival Change of Dry Yeast by Addition of Antioxidant

For the successful commercialization of dry yeast, it is very important to ensure the maximum shelf life of the dry yeast prototype. In the case of commonly used lyophilization, the frozen cells are dried by sublimation to stably evaporate moisture, which has the advantage of being stored for a long time. On the other hand, in the case of air drying, the manufacturing cost is nearly 10 times cheaper, but it has a disadvantage of being easily polluted and deteriorated by relatively high moisture content than freeze drying.

  In the present invention, vitamin C and glutathione (γ-L-Glutamyl-L-cysteinylglycine), which are antioxidants, are used to suppress the oxidative process occurring during long-term storage of dry yeast in order to compensate for these disadvantages of air drying and to improve shelf life. , GSH) were added to 1 mM, 3 mM and 5 mM, respectively, to prepare dry cells and to measure the survival rate of dry yeast during storage. Dry cells were stored at 4 ° C., and survival change was measured for 8 weeks at 4 week intervals. The protective agent and the rehydration solution were used for the optimum conditions obtained in the previous experiment, and the dry cell without antioxidant was used as a control. The results are shown in FIGS. 18 to 20.

As shown in FIG. 18, in the case of P. anomala SJ20, there was no significant difference in all the test groups after 4 weeks, but after 8 weeks, it was confirmed that the lowest viable cell count was added to the 3 mM glutathione-added cells. However, even in the control group to which the antioxidant was not added, the viable cell count decreased only about 1 log CFU / mL, and it was confirmed that the P. anomala SJ20 did not show the effect of improving the shelf life according to the addition of the antioxidant.

In addition, in the case of H. uvarum SJ69 as shown in Figure 18, the experimental group added 3 mM vitamin C or 1, 3, 5 mM glutathione after 4 weeks, the number of viable cells was less than the control group, 5 mM after 8 weeks In the experimental group added glutathione, the reduction of viable cell count was the least. Compared with the control group, the number of viable cells was decreased by 0.31 log CFU / mL, indicating that the H. uvarum SJ69 had an increase in shelf life due to the addition of antioxidants.

In addition, in the case of P. caribbica YP1 as shown in Fig. 19, all antioxidants treated group showed better shelf life than the control group after 4 and 8 weeks, among which 5 mM vitamin C was added. The decrease in viable cell count was the lowest in the bulb.

In addition, in the case of P. kluyveri CD34 as shown in FIG. 19, the number of viable cells was decreased in the 1 mM glutathione-added group compared to the control group, but after 8 weeks, the control group, which was not added to the antioxidant group, showed the least reduction in viable cell count. No effect was found with the addition of antioxidants.

In addition, as shown in Figure 20, C. zemplinina CD80, the control group showed the smallest decrease in viable cell count after 4 weeks, but after 8 weeks, the decrease in viable cell count of the control group increased significantly, resulting in a significantly lower shelf life than the antioxidant added groups. Confirmed. On the other hand, the addition of 5 mM vitamin C in the experimental group added antioxidants reduced the number of viable cells by 0.83 log CFU / mL less than the control group was confirmed to show the best storage effect.

In addition, as shown in FIG. 20, in the case of P. anomala CS7-16, after 4 weeks, the number of viable cells of the control group and vitamin C-added groups was similar, but the number of viable cells of the control group was significantly reduced after 8 weeks. On the other hand, except for 1 mM of vitamin C, the viable cell counts of the antioxidants, vitamin C and glutathione added groups were reduced, and in particular, the decrease in viable cell counts in glutathione added groups significantly improved the shelf life. In addition, in the case of glutathione, the shelf life was further increased according to the addition ratio, and when 5 mM glutathione was added, it was confirmed that the number of viable cells decreased by 2.03 log CFU / mL less than the control group, which showed the best storage effect.

Example 8 Confirmation of Shelf-life of Dry Yeast with Storage Temperature and Antioxidant Addition

As in Example 7, it was confirmed that the addition of antioxidant inhibited the reduction of viable cell count during the storage period. In general, the storage temperature of dry yeast is recommended to 4 ℃, there is a possibility of contamination or breeding of germs caused by alteration of excipients or dry protective agent above 20 ℃. Therefore, in the present embodiment, in order to investigate how the storage temperature when the antioxidant is added to the dry yeast during the storage period, each of the dry yeasts were stored at 4, 20, 30 ° C. for 8 weeks and the viable cell count was measured. The result is shown in FIG. 21.

As shown in Figure 21, in the case of dry yeast stored at 4 ℃, even after 8 weeks there was no change in color was confirmed a single yeast. On the other hand, in the case of 20 ℃ and 30 ℃, it was confirmed that the color of the dry yeast is changed from 4 weeks passed, and more than two microorganisms are proliferated, so accurate counting is difficult. Therefore, the storage temperature of the low temperature air drying yeast developed in the present invention was determined to be suitable 4 ℃.

Example 9 Morphological Observation of Low Temperature Blow Dry Yeast Spawn Using Scanning Electron Microscopy (SEM)

In order to investigate the morphological characteristics of each of the six yeasts blown with the addition of a protective agent and skim milk as in the above example, the surface of the dried yeast was observed using a scanning electron microscope (SEM) in the following manner. For surface observation, the dried yeast powder was evaporated in a spin vacuum dryer (VC-36R, Taitec, Saitama-ken, Japan) for 30 minutes at 37 ° C, and then stubed using double-sided metal tape. Fixed on. Then, after coating with gold using a sputter coater (WI-RES-Coater-001) was observed at 4,000 times by applying a 5 kV voltage by SEM (SU8220, Hitachi, Tokyo, Japan). The results are shown in FIG. 22. In FIG. 22, the three results from the leftmost top to the right represent SJ20 ( P. anomala SJ20), SJ69 ( H. uvarum SJ69), and YP1 ( P. caribbica YP1), respectively. The three results represent the results of CS7-16 ( P. anomala CS7-16), CD80 ( C. zemplinina CD80) and CD34 ( P. kluyveri CD34), respectively.

As a result of microscopic observation, as shown in Figure 22, each yeast was present in a dense form surrounded by sugars of very large grains and very small crystal structure presumed skim milk (skim milk). In addition, H. uvarum SJ69, etc., was not smooth because the surface of the yeast was coated on the protective agent solution, while P. anomala SJ20 and P. anomala CS7-16 maintained the surface covered with skim milk and sugars. This is believed to be due to the structure of cell walls of various yeasts. In addition, some yeasts such as P. anomala SJ20, C. zemplinina CD80, and P. anomala CS7-16 were found, which caused resuscitation of the one-hour rehydration solution to increase survival. It was judged that.

In all experiments, the mean and standard deviation of the data measured three times or more were determined, and the significance was verified by using a paired Student's t-test (p≤0.05 or p≤0.01).

National Academy of Agricultural Science KACC93297P 20180222 National Academy of Agricultural Science KACC93298P 20180222 National Academy of Agricultural Science KACC93292P 20171018 National Academy of Agricultural Science KACC93299P 20180222 National Academy of Agricultural Science KACC93291P 20171018 National Academy of Agricultural Science KACC93290P 20171018

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Optimal manufacturing methods of Hansenia spora uvarum starters          which produce high level of aromatic compounds using air-blast          drying technology <130> KNU1.312P <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> 223 ITS4 primer <400> 2 tcctccgctt attgatatgc 20

Claims (10)

(S1) preparing a seed solution by mixing Hansenia spora uvarum cells with a dry protective composition;
(S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And
(S3) comprising the step of treating the rehydration solution to the seed powder; method of producing a Hansenia spora ubarum yeast spawn with improved survival rate.
The method of claim 1,
The Hansenia spora ubarum cell is characterized in that the Hansenia spora ubarum SJ69 ( Hanseniaspora uvarum SJ69) (microbial accession number: KACC93298P), improved survival rate Hansenia spora ubarum yeast spawn method.
The method of claim 1,
The Hansenia spora uvarum cell is Hansenia spora uvarum SJ69 (microbial accession number: KACC93298P),
The dry protective composition is 5 to 15% (w / v) skim milk; And 5 to 15% (w / v) of at least one sugar selected from the group consisting of fructose, maltose, sucrose and trehalose; Hansenia spora ubarum yeast with improved survival rate Preparation of spawn.
The method of claim 3,
The dry protective composition is characterized in that it further comprises vitamin C or glutathione, method of producing a Hansenia spora ubarum yeast spawn improved survival rate.
The method of claim 4, wherein
The vitamin C is characterized in that it comprises 2 to 4 mM, the method of producing a Hansenia spora ubarum yeast spawn with improved survival rate.
The method of claim 4, wherein
The glutathione is characterized in that it comprises 4 to 7 mM, improved survival rate Hansenia spora ubarum yeast spawn production method.
The method of claim 1,
The Hansenia spora uvarum cell is Hansenia spora uvarum SJ69 (microbial accession number: KACC93298P),
The rehydration solution is distilled water, PBS buffer, NaCl solution and peptone water, characterized in that at least one selected from the group consisting of, improved survival rate Hansenia spora ubarum yeast spawn method.
(S1) Hansenia spora uvarum SJ69 ( Hanseniaspora uvarum SJ69) (Microorganism Accession No .: KACC93298P) mixing the cells and a dry protective composition to prepare a seed solution;
(S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And
(S3) treating the seed powder with a rehydration solution; and
The dry protective composition is 5 to 15% (w / v) skim milk; And 5 to 15% (w / v) of at least one sugar selected from the group consisting of fructose, maltose, sucrose and trehalose;
The dry protective composition further comprises 2 to 4 mM vitamin C or 4 to 7 mM glutathione,
The rehydration solution is distilled water, PBS buffer, NaCl solution and peptone water, characterized in that at least one selected from the group consisting of, improved survival rate Hansenia spora ubarum yeast spawn method.
(S1) preparing a seed solution by mixing Hansenia spora uvarum cells with a dry protective composition;
(S2) preparing a seed powder by cold-driing the seed solution at 30 to 45 ° C .; And
(S3) a step of treating the rehydration solution to the seed powder; containing, Hansenia spora Ubarum yeast spawn storage method of increasing.
The method of claim 9,
After the step (S2), the step of storing the seed powder at 0 to 10 ℃; characterized in that it further comprises, Hansenia spora Ubarum yeast spawn growth method.

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