JPWO2009020008A1 - Yeast culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquor mother having sufficient fermentability and capable of providing a fermented drink with an excellent balance of aroma components when the fermented drink is produced with the obtained liquor mother. The present invention relates to a method for culturing yeast, and more particularly, to a method for obtaining a liquor mother capable of producing a fermented beverage having excellent flavor characteristics by efficiently growing yeast in a short period of time. More specifically, the present invention cultivates yeast under culture conditions in which the occurrence rate of abnormal yeast having a cell minor axis: diameter ratio of 1: 3 or more is 20% or less of the whole yeast at the end of the culture. Provide a method.

Description

  The present invention relates to a yeast culture method. In particular, the present invention relates to a method for obtaining a liquor mother that can efficiently proliferate yeast in a short period of time and produce a fermented beverage having excellent flavor characteristics.

In the production of alcoholic beverages, for example, malt fermented beverages, so-called “ream” may be performed in which yeast produced during the production is collected at the end of fermentation and reused as a liquor mother for the next fermentation. However, there is a limit to the number of times that yeast can be reused, and after several consecutive brews, yeast that can produce liquor on the scale of several kiloliters is newly expanded through small-scale expansion from a small scale such as a test tube. It is necessary to ensure. In recent years, the need for low-volume, multi-variety production has been increasing in order to meet a wide range of consumer preferences. In these cases, yeasts suitable for each variety must be cultured sequentially to prepare for fermentation. Don't be.
In such a background, it is very important to shorten the production lead time and improve the flexibility of the production site. In the cultivation process, yeast per unit time can be obtained by culturing while supplying oxygen or air. Generally, the amount of growth (growth efficiency) is increased.

  However, as one of the phenomena observed in yeast rapidly cultured under such aeration conditions, a generally spherical cell may change to an elongated ellipse extending in a more polar direction. In such abnormal yeast, the growth rate and fermentation rate often decrease, and in fermented beverages obtained by fermentation using such yeast, the aroma component balance is deteriorated and the quality of the liquor is not necessarily good. It has been known. For this reason, it is necessary to repeat the adaptive fermentation several times until the ratio of the abnormal yeast is sufficiently lowered. After all, it takes a lot of time until the main fermentation.

On the other hand, as a yeast modification method, for example, in Japanese Patent No. 2537361 (Patent Document 1), yeast is soaked in a treatment solution to which high concentration of zinc is added to absorb the zinc, thereby fermenting the yeast. It is said that durability can be improved. In addition, in Japanese Patent Publication No. 5-442268 (Patent Document 2), in culturing yeast used for the production of brewed foods such as soy sauce and miso, fermentation of the yeast obtained by limiting the amount of dissolved oxygen in the culture It is said that it can improve sex. However, these documents do not disclose a method for producing a large amount of yeast for producing a fermented beverage from a small-scale culture without undergoing adaptive fermentation. In JP 2006-212024 A (Patent Document 3), adding a small amount of mineral to the fermentation stock solution, limiting the supply of oxygen or air, reducing the dissolved carbon dioxide concentration in the culture solution, or these It is said that a practical liquor mother can be efficiently obtained in a short time by the combination of
Japanese Patent No. 2537361 Japanese Patent Publication No. 5-442268 JP 2006-212024

  In view of the above situation, when producing fermented beverages with sufficient fermentability and the obtained liquor mother, the aeration conditions are limited for liquor mothers who can provide fermented beverages with excellent balance of aroma components. Therefore, it is desired to develop a culture method that can be obtained quickly without doing so.

The inventors of the present invention have completed the present invention as a result of various investigations of culture conditions under which a desired liquor can be obtained based on the idea that it is important to maintain a normal yeast form. That is, the present invention provides the following yeast culturing method, liquor obtained by such culturing method, fermented beverage obtained using the liquor, and the like.
(1) A method of culturing yeast under culture conditions in which the incidence of abnormal yeast having a cell minor axis: diameter ratio of 1: 3 or more is 20% or less, preferably 10% or less of the whole yeast at the end of the culture. .
(2) The method according to (1) above, wherein the culturing is performed under the condition that the components related to the ergosterol biosynthesis system are increased in the culture solution.
(2a) A yeast culture method for improving the quality of the liquor (for example, improving the flavor) by culturing under conditions where the components related to the ergosterol biosynthesis system are increased in the culture solution.
(3) The method according to (2) or (2a) above, wherein the component is ergosterol and / or a precursor of ergosterol.
(4) The method according to (2) or (2a) above, wherein the component is an enzyme and / or a coenzyme that catalyzes an intermediate reaction in ergosterol biosynthesis.

(5) The method according to (2) or (2a) above, wherein the component is mevalonic acid.
(6) The method according to (2) or (2a) above, wherein the component is pantothenic acid.
(7) The method according to any one of (1) to (6), wherein the yeast is a liquor for producing a fermented beverage.
(8) The method according to (7) above, wherein the fermented beverage is beer or a beer-taste fermented beverage.
(9) A liquor obtained by the method according to any one of (1) to (8) above.
(10) Alcoholic beverages produced using the liquor mother described in (9) above.

According to the method of the present invention, 1) maintaining the aeration conditions (not limiting) can increase the growth efficiency of yeast and shorten the culture period, and 2) the time required for adaptive fermentation due to the good quality of the mother's liquor. Can be shortened. As a result, a sufficient amount of sake mother having excellent brewing characteristics can be rapidly obtained from small-scale cultured yeast. In addition, even when a small amount of various varieties are produced and different yeasts are required for each product, the time required to make a sake mother can be greatly reduced.
The liquor produced by the method of the present invention usually has such characteristics as being capable of producing a fermented beverage having excellent fermentability and excellent flavor balance.

1. Summary of the Invention It is important for the present inventors to maintain a normal yeast form, and by suppressing the occurrence of abnormally shaped yeast, the present invention provides a fermented beverage with excellent fermentability and excellent aroma component balance. The idea was that it would be possible to produce a liquor mother who could do it with high efficiency. Based on this idea, as a result of intensive studies on the influence of culture conditions and medium components on yeast, the fermentation stock solution using cereal raw materials such as malt was added to components related to the yeast ergosterol biosynthetic system, specifically mevalonic acid, for example. Alternatively, it was found that the addition of pantothenic acid or the like can suppress the occurrence of abnormal morphological yeasts even under aeration conditions. As a result, it has been found that a sufficient amount of a liquor mother that can provide a fermented beverage with excellent fermentability and excellent aroma component balance can be produced with high efficiency.

2. First, according to the present invention, the occurrence rate of abnormal yeast having a short axis: diameter ratio of cells of 1: 3 or more is 20% or less of the whole yeast (preferably 10% or less, More preferably, the present invention relates to a method for culturing yeast under culture conditions of 6% or less. The incidence of abnormal yeast can be measured by using a microscope and a hemocytometer. More specifically, the present invention relates to a yeast culturing method in which culturing is performed under the condition that the components related to the ergosterol biosynthesis system are increased in a culture solution.
(yeast)
The yeast used in the present invention is not particularly limited as long as it is any yeast that can be used for brewing. For example, brewing yeasts such as beer, wine and sake can be mentioned. Specific examples include yeasts of the genus Saccharomyces. In the present invention, beer yeasts such as Saccharomyces pastorianus W34 / 70, Saccharomyces carlsbergensis NCYC453, NCYC456, etc. Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954, etc. can be used. Furthermore, whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as Association Wines No. 1, 3, and 4 and sake sakes such as Association Yeast Nos. 7 and 9 can be used. It is not limited to. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used.

(Sake mother)
In the present invention, a liquor mother is produced using the above yeast culture method.
In the present specification, “sake mother” includes yeast that is in a state used for fermentation of fermented beverage production and yeast grown for the purpose of use. The liquor may be grown from a small-scale culture solution in a test tube scale through several stages of scale-up, or may be yeast recovered after production of the fermented beverage. When a liquor is obtained by scaling up from a small culture solution, a liquor with good fermentability can be obtained if the method of the present invention is carried out in the final culturing process. The yeast recovered after the production of the fermented beverage can usually be used as a liquor as it is, but it is preferable to carry out the method of the present invention at least once in order to enhance the fermentability as a liquor. Therefore, in the present specification, “yeast used as a raw material for a liquor” includes all starting yeasts for culturing at least once by the method of the present invention to produce a liquor having good fermentability.

(Culture medium)
In the method of the present invention, as a medium for producing a liquor, the same fermentation stock solution for producing a fermented beverage, for example, wort similar to beer production can be used. Happoshu and beer-taste beverages have a low malt use ratio or use raw materials other than malt, but the fermentation stock solution can also be used for the production of a sake mother. However, for example, in the case where the nutrient source necessary for the growth of the yeast is insufficient such that the amino acid content is low in the low-malt-foamed sake wort, it may be appropriately supplemented. These typical manufacturing conditions, e.g. W. Kunze, Technology Brewing and Malting, 3 rd completely updated Ed. (2004), Miyaji, beer brewing technology, reference may be made to the food industry newspaper (1999) .

(Ingredients related to ergosterol biosynthesis system)
The method of the present invention is characterized in that the concentration in the culture solution of components relating to the ergosterol biosynthesis system of yeast is increased. Ergosterol is one of the components of the yeast cell membrane, and its functions are known to vary. For example, there is a domain called raft rich in ergosterol on the cell membrane, which plays a major role in the function of membrane proteins represented by transporters for taking up nutrient components in the medium.
Yergosterol biosynthesis in yeast consists of more than 20 reactions starting from acetyl-CoA and passing through mevalonic acid, squalene, lanosterol, and the like. The main components involved in this biosynthetic system include ergosterol itself and enzymes that catalyze the respective reactions of the ergosterol biosynthetic system, such as hydroxymethylglutaryl CoA synthase, hydroxymethyl CoA reductase, and squalene synthase. Other than these, there are pantothenic acid, which is a coenzyme, such as mevalonic acid and squalene, which are precursors of ergosterol (intermediate metabolites). In view of the solubility of the acid, mevalonic acid, pantothenic acid or a mixture thereof is preferable. In the present invention, it is particularly preferable to use pantothenic acid. In addition, a preferable additive component may change with kinds of yeast to be used. Therefore, a preferable additive component can be appropriately selected by performing a culture test and a brewing test for each yeast to be used.
The method for increasing the concentration of these substances in the fermentation stock solution may be performed by selecting a method for producing the fermentation stock solution, for example, a malt production method or a wort production method, or may be added in the form of a free form or a salt. It may be done by doing. The concentration of the above components in the culture solution is not particularly limited, and for example, it is adjusted to a concentration that enhances the ability to improve the quality of the yeast obtained. For example, in the state of being dissolved in a fermentation stock solution such as wort, the intermediate metabolite is 1 to 1000 ppm, preferably about 1 to 100 ppm, and the coenzyme is about 0.01 to 100 ppm, preferably about 0.01 to 10 ppm. What is necessary is just to add. Moreover, it is preferable to add such an additive to the culture stock solution from the start of culture until the yeast enters the logarithmic growth phase.

(Ventilation conditions)
The supply of oxygen speeds up yeast growth. Supply of oxygen necessary for yeast growth can be performed by supplying a gas (for example, oxygen or air) containing oxygen to the culture solution (see, for example, JP-A-2006-212024). What is necessary is just to set oxygen supply amount suitably according to various culture conditions, target yeast concentration, or a strain. For example, when culturing at a yeast concentration of 20 x 10 ⁶ cells / mL using W he ih enstephan-3 4 strain and growing 6 to 8 times in 24 hours, a gas containing oxygen at the start of culture as a standard The culture is started at an aeration rate of about 0.2 to 2.0 mL per minute as oxygen per liter of culture solution, preferably about 1.2 mL per minute as oxygen per liter of culture solution. For example, in the case of air, the culture is started with an aeration rate of 1 to 10 mL per minute, preferably about 6 mL.
The supply of oxygen or air may be continuous or intermittent. The supply method is not particularly limited, and can be selected as appropriate, for example, by installing a pipe to the culture tank. The oxygen supply amount can be adjusted by changing the length of time for supplying a constant flow rate of oxygen, or can be controlled by adjusting the flow rate with a constant supply time.

(Yeast concentration)
In the method of the present invention, the yeast concentration at the start of culture is not particularly limited, but is usually 1 × 10 ^{6 to} 5 × 10 ⁸ cells / mL, more preferably 1 × 10 ⁷ to 1 × 10 ⁸ cells / mL. is there. The yeast concentration at the end of the culture is preferably 5 × 10 ⁷ to 1 × 10 ⁹ cells / mL.

3. Alcohol obtained using the liquor of the present invention In the present invention, a fermented beverage is produced using the liquor obtained as described above. Examples of fermented beverages include beer, happoshu, miscellaneous sake, liqueurs, spirits, low alcohol malt fermented beverages (eg, malt fermented beverages having an alcohol content of less than 1%), and the like. According to a preferred embodiment of the present invention, beer and beer-taste beverages are produced. Here, the beer-taste beverage refers to a beverage fermented with yeast using a saccharide raw material such as malt or sugar liquid, hops, etc., and having a flavor like beer.
A fermented beverage (particularly beer or beer-taste beverage) obtained by culturing a liquor obtained by the method of the present invention has a feature of excellent flavor balance.

Hereinafter, the present invention will be described based on examples, but the present invention is not limited thereto.
(Evaluation items and evaluation methods)
Growth amount: Sampling was carried out over time in the culture or fermentation process, and the yeast cell concentration was measured by a direct counting method using a microscope and a hemocytometer.
The ratio of the abnormal morphological yeast: the ratio of the minor axis of the cell to the major axis of 1: 3 or more was counted as the abnormal morphological yeast, and the ratio of the ratio to the whole was calculated.
Fragrance: The sensory evaluation of the happo sake brewed using cultured yeast was conducted by a blind method by five trained panelists, and the target fragrance thickness (good fragrance quality and quantity) was evaluated. A comprehensive evaluation including off-flavor and taste was also conducted.

Example 1: Verification of effect of additive Lower surface beer brewing yeast (BH604) was pre-cultured in a medium containing 5% yeast extract and 2% glucose under aerobic conditions, followed by foaming with a malt use ratio of 24% It used for the main culture which used sake wort as a culture medium. For the additive, a stock solution having a concentration 1000 times the set concentration was prepared, and 1/1000 of the culture solution was added. Table 1 shows the experimental levels.

(Other culture conditions)
Culture temperature: 20 ℃
Operating capacity: 4L
Aeration rate (sterile air): 24mL / min
Stirring speed: 30rpm
Initial yeast concentration: 60 × 10 ⁶ cells / mL
Incubation period: 29h

  The concentration of yeast cells at the end of the culture is shown in FIG. 1, and the ratio of abnormally shaped yeast is shown in FIG. The use of each additive increased the amount of growth and the final yeast concentration increased by 28% and 22%, respectively, relative to the control. In addition, at the level where each additive was used, the appearance frequency of abnormal yeast was very low throughout the culture period, and the proportion of abnormal morphological yeast at the end of the culture was significantly reduced to 20% and 12% of the control, respectively. As described above, it was found that by using an additive having an extremely low concentration, it is possible to suppress the occurrence of abnormally shaped yeast while increasing the growth efficiency of the yeast under aeration conditions.

Example 2: Using a yeast obtained by culturing using a happoshu test brewing additive as a liquor, a happoshu (malt use ratio 24%) was brewed as it was without adapting fermentation. The brewing conditions were the same except for the culture conditions (with or without additives), and the influence of the quality of the sake mother on the quality of the sake was verified.
Fermentation temperature: 15 ℃
Fermentation capacity: 4L
Initial yeast concentration: 20 × 10 ⁶ cells / mL
Fermentation period: 7 days

  As shown in Fig. 3, the brewed product fermented using yeast with or without mevalonic acid or pantothenic acid obtained a higher score in scent thickness than the sample with control (no added) cultured yeast. It was. In general, it is said that the use of aeration-cultured yeast reduces both the quality and quantity of fragrance, but the use of these additives seems to have improved the flavor, possibly through improving the quality of yeast. Moreover, as shown in FIG. 4, the sample brewed product whose fragrance thickness was improved by using cultured yeast added with mevalonic acid or pantothenic acid showed a high score in the overall evaluation.

Example 3 Verification of Effect of Additives A culture test was carried out in the same manner as in Example 1 except that S. pastorianus Weihenstephan Nr. 34 (Nr. 34) was used as the bottom brewer's yeast. That is, using Nr.34, pre-cultured in a medium containing 5% yeast extract and 2% glucose under aerobic conditions, followed by main culture using a mash wort with a malt use ratio of 24% as the medium. Provided. For the additive, a stock solution having a concentration 1000 times the set concentration was prepared, and 1/1000 of the culture solution was added. The addition concentrations of mevalonic acid and pantothenic acid and other culture conditions are the same as in Example 1.

  FIG. 5 shows the yeast cell concentration at the end of the culture, and FIG. 6 shows the proportion of abnormally shaped yeast. At the level where each additive was used, the frequency of appearance of abnormal yeast was very low throughout the culture period, and the proportion of abnormal morphological yeast at the end of the culture was significantly reduced relative to the control. As described above, it was found that by using an additive having an extremely low concentration, it is possible to suppress the occurrence of abnormally shaped yeast while increasing the growth efficiency of the yeast under aeration conditions.

Example 4: Happoshu test brewing A bottom quality test was conducted in the same manner as in Example 2 except that Nr. 34 was used as the bottom beer brewing yeast and nine panelists were used. That is, the yeast obtained by culturing using the additive was used as a liquor, and the sake brewing (malt use ratio 24%) was brewed as it was without performing adaptive fermentation. The brewing conditions were the same except for the culture conditions (with or without additives), and the influence of the quality of the sake mother on the quality of the sake was verified.

As shown in FIG. 7, the brewed product fermented with yeast by pantothenic acid-added culture gave a higher score in the scent thickness than the brewed product with control (non-added) cultured yeast. In general, it is said that the use of aeration-cultured yeast reduces both the quality and quantity of fragrance, but the use of these additives seems to have improved the flavor, possibly through improving the quality of yeast. Moreover, as shown in FIG. 8, the brewed product whose fragrance thickness was improved by using pantothenic acid-added cultured yeast showed a high score in the overall evaluation.
In the case of Nr.34, the result of the sensory evaluation of mevalonic acid was not good, so the compatibility of Nr.34 and mevalonic acid may not necessarily be good.

  As described above, according to the present invention, a sufficient amount of a sake mother having excellent brewing characteristics can be quickly obtained from a small amount of seed yeast. Thereby, the lead time until manufacture of fermented drinks can be reduced significantly, and the industrial utility value is extremely high.

It is the figure which showed the yeast cell density | concentration at the time of completion | finish of culture | cultivation in Example 1. FIG. It shows that the yeast growth efficiency was improved by the addition of mevalonic acid or pantothenic acid. It is the figure which showed the ratio of the abnormal form yeast at the time of completion | finish of culture | cultivation in Example 1. FIG. It shows that the ratio was significantly reduced by the addition of mevalonic acid or pantothenic acid. It is the figure which showed the sensory evaluation result of the happoshu brewed in Example 2. FIG. This also shows that the thickness of the scent can be improved by adding mevalonic acid or pantothenic acid even when aeration is maintained in the culture process. It is the figure which showed the sensory evaluation result of the happoshu brewed in Example 2. FIG. The brewed product whose fragrance thickness is improved by using cultured yeast added with mevalonic acid or pantothenic acid shows a high score in the overall evaluation. FIG. 3 is a graph showing the yeast cell concentration at the end of culture in Example 3. It shows that the yeast growth efficiency was improved by the addition of each additive. FIG. 3 is a graph showing the proportion of abnormally shaped yeast at the end of culture in Example 3. It shows that the ratio was significantly reduced by the addition of each additive. FIG. 6 is a diagram showing sensory evaluation results of the happoshu brewed in Example 4. This also shows that the aroma thickness can be improved by adding pantothenic acid even when aeration is maintained in the culture process. FIG. 6 is a diagram showing sensory evaluation results of the happoshu brewed in Example 4. The brewed product whose fragrance thickness is improved by using pantothenic acid-added cultured yeast shows a high score in the overall evaluation.

Claims (10)

  A method of cultivating yeast under culture conditions in which the occurrence rate of abnormal yeast having a minor axis: diameter ratio of 1: 3 or more is 20% or less of the whole yeast at the end of the culture.   The method according to claim 1, wherein the culture is performed under a condition in which components related to the ergosterol biosynthesis system are increased in the culture solution.   The method of claim 2, wherein the component is ergosterol and / or a precursor of ergosterol.   The method according to claim 2, wherein the component is an enzyme and / or a coenzyme that catalyzes an intermediate reaction in ergosterol biosynthesis.   The method of claim 2, wherein the component is mevalonic acid.   The method of claim 2, wherein the component is pantothenic acid.   The method according to claim 1, wherein the yeast is a liquor for producing a fermented beverage.   The method according to claim 7, wherein the fermented beverage is beer or a beer-taste fermented beverage.   A liquor obtained by the method according to any one of claims 1 to 8.   Alcoholic beverages produced using the liquor mother according to claim 9.