WO2012067106A1

WO2012067106A1 - Method for producing yeast extract

Info

Publication number: WO2012067106A1
Application number: PCT/JP2011/076281
Authority: WO
Inventors: 由香伊藤; 一郎澁谷; 哲司小谷
Original assignee: アサヒグループホールディングス株式会社
Priority date: 2010-11-15
Filing date: 2011-11-15
Publication date: 2012-05-24
Also published as: JPWO2012067106A1

Abstract

Provided are a method for producing a succinic acid-rich yeast which contains succinic acid at a higher concentration than the conventional yeasts, a succinic acid-rich yeast, and a succinic acid-rich yeast extract. The present invention relates to: a method for producing a yeast extract, said method being characterized by comprising extracting a yeast extract, via autolysis, from a yeast which has been cultured under such conditions as controlling the volumetric oxygen transfer coefficient (KLa) to 0.9-195 hr^-1; a method for producing a yeast extract as described above, wherein said yeast has been grown under such conditions as controlling the respiration quotient to 1.3 or greater; a method for producing a yeast extract as described in any of the above, wherein the autolysis is conducted under such conditions as controlling the autolysis solution temperature to 30-55^oC; and a yeast extract which is produced by a method as described in any of the above and has a succinic acid content of 3.0-30.0 wt% on a dry weight basis.

Description

Method for producing yeast extract

The present invention relates to a yeast extract having a high succinic acid content and a method for producing the same.
This application claims the priority based on Japanese Patent Application No. 2010-255396 for which it applied to Japan on November 15, 2010, and uses the content here.

Saccharomyces yeasts such as brewer's yeast and baker's yeast contain natural vitamin B groups, amino acids, minerals, etc. in a well-balanced manner, and are effectively used in addition to being used for producing beer and bread. . For example, dry yeast has been used in Japan for many years as a pharmaceutical, food material, seasoning, etc., and is recognized as a material with high nutritional value and safety. In recent years, it has also been widely used as a raw material yeast for yeast extract.

Yeast extract is prepared from a yeast culture and contains abundant amino acids and has been used as a food additive such as a seasoning for imparting umami and richness. In particular, due to the recent increase in natural orientation, the demand for yeast extract as a seasoning is increasing. Yeast extract prepared from yeast containing abundant taste components can be expected to be used as a better seasoning, and therefore, development of yeasts containing more taste components has been actively conducted.

For example, Patent Document 1 describes that in the preparation of a yeast extract, the properties of the obtained yeast extract vary greatly depending on conditions such as temperature, pH, and reaction time. For example, when preparing a yeast extract by the autolysis method, if the reaction temperature is set to 45-55 ° C, there is no possibility of propagation of various bacteria, but it is not the optimum reaction temperature for the main enzymes contained in yeast. Tend to be less.

In addition to amino acids such as glutamic acid, yeast extract contains various taste-tasting substances, and succinic acid is one type. Succinic acid is an umami component that is abundantly contained in shellfish and the like, and a yeast extract having a high succinic acid content is expected to be useful as a seasoning for seafood. Moreover, for example, Patent Document 2 describes that by increasing the succinic acid content per solid content of the yeast extract, the yeast extract has a further enhanced taste-enhancing effect. Here, in Patent Document 2, a method for preparing an extract from a succinic acid high-producing mutant obtained by ultraviolet irradiation treatment, or by adding succinic acid to a yeast extract prepared from a wild-type yeast, A method for preparing a yeast extract with a high acid content is disclosed.

Succinic acid is an important taste component of sake along with other organic acids such as malic acid, and for this reason, in the field of alcoholic beverages, yeasts that produce these organic acids at high production levels have been developed. For example, Patent Document 3 discloses that a mutant strain resistant to 2-oxoglutaric acid obtained by a mutation treatment with ethyl methanesulfonate (EMS) is higher in various organic acids including succinic acid than the parent strain. Production is described.

Japanese Patent Laid-Open No. 10-179084 JP 2005-102549 A Japanese Patent No. 4402779

The succinic acid content in yeast is very small, and it is difficult to prepare a yeast extract with a high succinic acid content. For example, in Patent Document 2, the yeast extract prepared from the wild strain is only 0.32% in the solid content, and the yeast extract prepared from the succinic acid-rich mutant is 1.32%. Even the yeast extract having a high succinic acid content disclosed in Non-Patent Document 1 has a succinic acid content of only 1.8% in the yeast extract. In addition, the succinic acid content of yeast extracts currently on the market is often less than 1% of the solid content, and even if the content is high, it is only about 2% at most. A yeast extract contained in the concentration is desired.

For example, by producing a mutant having a higher succinic acid content, a yeast extract having a higher succinic acid content may be obtained. However, it is difficult to drastically improve the content of organic acids that originally exist only in trace amounts only by genetic modification. Even if such genetic modification is made, it is very difficult to remarkably increase the succinic acid content in the cells because excess organic acid is easily released outside the cells.

Further, as in Patent Document 2, for example, a yeast extract having a desired succinic acid content can be prepared by externally adding purified succinic acid. However, it is not preferable to add succinic acid separately from the viewpoint of production cost.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a yeast extract containing succinic acid at a higher concentration than before and a method for producing the same.

As a result of intensive studies to achieve the above object, the present inventors have determined that yeast extract from yeast cultured under conditions where KLa (oxygen transfer capacity coefficient) is 0.9 to 195 hr ⁻¹ is obtained by autolysis. As a result, it was found that a yeast extract having a high succinic acid content could be produced, and the present invention was completed. That is, the present invention adopts the following configuration.

(1) A method for producing a yeast extract, comprising extracting yeast extract by self-digestion from yeast cultured under conditions where KLa (oxygen transfer capacity coefficient) is 0.9 to 195 hr ⁻¹ .
(2) The method for producing a yeast extract according to (1), wherein the yeast is a yeast grown under a condition where the respiratory quotient is 1.3 or more.
(3) The method for producing a yeast extract according to (1) or (2), wherein self-digestion is carried out under the condition that the temperature of the self-digestion solution is 30 to 55 ° C.
(4) The amount of succinic acid produced by self-digestion is 1.7 to 6.5% by weight per dry yeast cell weight before self-digestion, according to any one of (1) to (3) above Of producing yeast extract.
(5) The method for producing a yeast extract according to any one of (1) to (4), wherein the yeast is a genus Saccharomyces or a genus Candida.
(6) The production of the yeast extract according to any one of (1) to (5), wherein the succinic acid content of the produced yeast extract is 3.0 to 30.0% by weight per dry yeast extract weight. Method.
(7) Manufactured by the method for producing a yeast extract according to any one of the above (1) to (6), and the succinic acid content is 3.0 to 30.0% by weight per dry yeast extract weight. Yeast extract.
(8) A yeast extract having a succinic acid content produced from yeast of 3.0 to 30.0% by weight per dry yeast extract weight.
(9) A method for producing a seasoning composition comprising the yeast extract according to (7) or (8).
(10) A method for producing a food or drink, comprising the yeast extract according to (7) or (8).

According to the method for producing a yeast extract of the present invention, a yeast extract in which the succinic acid content is remarkably increased without external addition of succinic acid by extraction from yeast cultured under specific culture conditions by autolysis. Can be easily produced.

In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight of fed molasses (Opt1) at the time of culture | cultivation at the stirring speed of 100 rpm. In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight of fed molasses (Opt1) at the time of culture | cultivation at the stirring speed of 200 rpm. In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight of fed molasses (Opt1) at the time of culture | cultivation at the stirring speed of 300 rpm. In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight (Opt1) of the molasses fed over time when culture | cultivating with the stirring speed of 400 rpm. In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight of fed molasses (Opt1) at the time of culture | cultivation at the stirring speed of 500 rpm. In Example 1, it is a figure which shows the result of having measured the respiratory quotient (RQ) and the weight of fed molasses (Opt1) over time when it culture | cultivates with the stirring speed of 600 rpm. In Example 1, the relationship between the amount of succinic acid produced (% by weight) per dry yeast cell of cultured yeast and the content (% by weight) of succinic acid per dry weight of yeast extract, and the stirring speed during culture FIG. In Example 6, it is the figure which showed the result of the comparative sensory test of the clam chowder of samples 1-4. In Example 7, it is the figure which showed the result of the comparative sensory test of the clam chowder of samples 1-3.

The method for producing a yeast extract of the present invention is characterized in that the yeast extract is extracted by autolysis from yeast cultured under a condition that KLa is 0.9 to 195 hr ⁻¹ . Usually, when culturing yeast, it is carried out under conditions that achieve high yield such as aerobic conditions (for example, KLa is 380 hr ⁻¹ ). On the other hand, in the present invention, by using yeast grown under conditions where KLa is 0.9 to 195 hr ⁻¹ , more succinic acid can be produced by subsequent autolysis treatment. A yeast extract having a high succinic acid content can be produced.
Hereinafter, embodiments of the present invention will be described in detail.

KLa shows the ability to move oxygen from the gas phase to the liquid phase and generate dissolved oxygen per unit time during aeration and agitation culture, and is based on oxygen supplied from the gas phase and oxygen consumed by microorganisms. It can be determined by the oxygen balance equation (1). In the balance equation (1), C is the dissolved oxygen concentration (DO) (ppm) in the culture solution, C ^* is the saturated dissolved oxygen concentration (DO) (ppm) at the culture temperature, and X is the cell concentration ( g / L), and Q _O2 is the specific respiration rate (mgO ₂ / (g · cell · h)).
dC / dt = KLa · (C ^* −C) −Q _O2 · X (1)

As a method for reducing the dissolved oxygen concentration, there is a gas replacement method in which nitrogen gas is vented to expel oxygen. In this case, it is only necessary to exclude the term [Q _O2 · X] in the balance equation (1).
Formula (1) is integrated and expressed as Formula (2).
(C ^* −C) = exp (−KLa · t) (2)
Taking the logarithm of both sides of Equation (2), it is expressed as Equation (3).
Ln (C ^* −C) = − KLa · t (3)
That is, by plotting the logarithm of the value of [C ^* -C] (the value obtained by subtracting the measured dissolved oxygen concentration (DO) from the saturated dissolved oxygen concentration (DO)) against the time of aeration, a straight line is obtained. The relationship is obtained and the slope is [−KLa].

The dissolved oxygen concentration and the respiratory quotient in the culture solution are affected by the stirring speed of the culture solution, the shaking speed during the shaking culture, the aeration amount to the culture solution, and the like. For this reason, KLa can be adjusted within a desired range by appropriately adjusting the aeration condition and the stirring condition to the culture solution. Usually, when the stirring speed and the shaking speed are too fast, the dissolved oxygen concentration in the culture medium tends to be high, and the respiration rate tends to be fast. Therefore, in order to culture yeast under conditions where KLa is 0.9 to 195 hr ⁻¹ , for example, when culturing using a 5 L jar fermenter, it is preferable to culture at 500 rpm or less, and 100 to 500 rpm It is more preferable to culture in
KLa when the yeast used in the autolysis culture method of manufacturing the yeast extract of the present invention include, but are not particularly limited as long as it is within the range of 0.9 ~ 195hr ^-1, is 5 ~ 150hr ^-1 Preferably, 9 to 100 hr ⁻¹ is more preferable, and 9.4 to 95 hr ⁻¹ is more preferable.
In addition, yeast used for autolysis is preferably cultured in a slightly anaerobic environment rather than a completely aerobic environment, but it is not preferable to culture in a completely anaerobic environment. For example, the respiratory quotient at the time of culturing yeast used for autolysis is preferably 1.3 or more, more preferably 1.3 to 10.

In the present invention, the yeast cultured in a specific range of the KLa value may be any unicellular fungus. Specifically, Saccharomyces spp., Shizosaccharomyces spp., Pichia spp., Candida spp., Kluyveromyces spp., Williopsis spp. Debaryomyces, Galactomyces, Torulaspora, Rhodotorula, Yarrowia, Zrochos, etc.
Among these, Candida tropicalis, Candida lipolytica, Candida utilis, Candida sake, and Saccharomyces cerevisiae are edible. (Saccharomyces cerevisiae) and the like are preferable, and Saccharomyces cerevisiae and Candida utilis that are widely used are more preferable.

In the present invention, the yeast cultured in a specific range of KLa value may be a natural yeast (a yeast whose gene has not been artificially modified) or a mutant strain. In the present invention, a yeast extract having a high succinic acid content can be prepared from yeast without genetically modifying the succinic acid metabolism / accumulation pathway originally possessed by the yeast. For this reason, succinic acid content can be obtained from a natural yeast by producing a yeast extract by the method for producing a yeast extract of the present invention without performing a genetic modification treatment that may reduce the palatability as a food or drink. High yeast extract can be produced. Even when a mutant strain is used, a yeast extract having a high succinic acid content can be produced by the method for producing a yeast extract of the present invention.

In the present invention and the specification of the present application, the “wild strain” means a yeast that originally existed in nature, that is, a yeast that has not been subjected to artificial mutation treatment. In contrast, “mutant strain” means a yeast obtained by subjecting a gene to artificial mutation treatment. In the present invention, the mutation treatment is not particularly limited as long as it is a treatment capable of mutating a part of a gene possessed by an organism such as yeast, and when producing a mutant strain of a microorganism such as yeast. Any commonly used technique may be used. For example, the yeast can be subjected to a mutation treatment by treating the yeast with ultraviolet rays, ionizing radiation, nitrous acid, nitrosoguanidine, EMS or the like as a mutagen.

The culture format is not particularly limited, and can be appropriately determined in consideration of the culture scale, the intended use of the obtained culture, and the like. For example, batch culture, fed-batch culture, continuous culture and the like can be mentioned, but industrially fed-batch culture or continuous culture is adopted.

In the present invention, the composition of the culture solution used for yeast culture is not particularly limited as long as the yeast can grow, and those used in a conventional method can be used. For example, as the carbon source, one or more selected from the group consisting of glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, and the like, which are used for normal microorganism culture, are used. The nitrogen source is selected from the group consisting of inorganic salts such as urea, ammonia, ammonium sulfate, ammonium chloride or ammonium phosphate, and nitrogen-containing organic substances such as corn steep liquor (CSL), casein, yeast extract or peptone. One type or two or more types are used. Furthermore, phosphoric acid component, potassium component, and magnesium component may be added to the medium. These include normal industrial products such as lime superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium hydrochloride. The raw material can be used. Moreover, you may use inorganic salts, such as zinc, copper, manganese, and an iron ion. In addition, vitamins and nucleic acid-related substances may be added.

In general, the growth rate of yeast when cultivated using a liquid medium containing sufficient nitrogen, phosphorus, minerals, vitamins, etc., contains a relatively small amount of some or all of the above components. It becomes larger than when cultured using a liquid medium. And as shown in the balance equation (1), KLa is also affected by the growth rate of yeast. For this reason, KLa can be adjusted within a desired range also by adjusting the composition of the liquid medium.

Note that the culture conditions of the yeast used as the raw material for the yeast extract may be any conditions as long as the KLa is 0.9 to 195 hr ^−1, and other culture conditions such as the pH and temperature of the culture solution, the culture time, etc. What is necessary is just to follow general yeast culture conditions. For example, the temperature is 20 to 40 ° C., preferably 25 to 35 ° C., and the pH is 3.5 to 7.5, particularly 4.0 to 7.0. Further, the culture may be performed under a condition where the pH of the culture solution is controlled, or may be cultured under a condition where the pH is not controlled.

In the method for producing a yeast extract of the present invention, a yeast extract is prepared by self-digesting a yeast cultured under conditions where KLa is 0.9 to 195 hr ⁻¹ . In general, methods for preparing yeast extract include enzymatic degradation, acid degradation, alkali extraction, and hot water extraction. Compared to these methods, autolysis methods have a relatively high extract yield. This is an extraction method that can be enhanced. That is, by preparing by the self-digestion method, the succinic acid content per solid content of the yeast extract can be increased. On the other hand, the yield of yeast is lower in the culture conditions where KLa is 0.9 to 195 hr ⁻¹ than in the aerobic culture conditions. The present invention is a method that combines a culture method with a low yield and a self-digestion method with a high extract yield. By such a combination, the succinic acid content in the yeast extract can be increased. For the first time.

Yeast self-digestion can be performed by conventional methods. For example, a yeast suspension obtained by suspending yeast collected after culturing in water or a buffer is used as a self-digestion solution, and the self-digestion solution is maintained at 30 to 55 ° C. Yeast is degraded and extracted by the enzyme originally present in In the method for producing a yeast extract of the present invention, the temperature of the autolysate is preferably 35 to 55 ° C. Since the temperature range is a temperature range in which the activity of the enzyme originally contained in the yeast is high, the production efficiency of succinic acid is further increased. After completion of self-digestion, a yeast extract can be obtained by collecting the liquid from the self-digestion solution by centrifugation or the like. The yeast extract can be concentrated, adjusted for pH, etc. by conventional methods.

The self-digestion may be performed under a condition in which the pH of the yeast suspension is controlled, or may be performed under an uncontrolled condition. When performing under pH control, self-digestion can be performed by controlling the pH of the yeast suspension to an arbitrary value within the range of 4-7.

The succinic acid content per solid content in the yeast extract increases with the progress of the autolysis reaction time and eventually reaches a plateau. Therefore, the succinic acid content in the self-digestion solution can be controlled to a desired level by appropriately adjusting the reaction time of self-digestion. In the method for producing a yeast extract of the present invention, for example, when Saccharomyces cerevisiae is used, it is 1.7 to 6.5% by weight, preferably 2.5 to 6% per dry yeast cell before autolysis. 0.5% by weight, more preferably 3.0 to 6.5% by weight, still more preferably 3.5 to 6.5% by weight, particularly preferably 4.0 to 6.5% by weight of succinic acid Can be produced from yeast. In addition, by intermittently measuring the succinic acid content in the autolysate, the production of succinic acid per dry yeast cell and the succinic acid content of the resulting yeast extract can be controlled more easily. be able to.

Thus, according to the method for producing a yeast extract of the present invention, a yeast extract having a high succinic acid content can be prepared without adding succinic acid after autolysis. For example, when a Candida spp. Having a relatively low succinic acid content, such as Candida utilis, is used by the method for producing a yeast extract of the present invention, 3% by weight or more of succinic acid per dry weight in the yeast extract is used. Can be produced. When Saccharomyces cerevisiae is used, succinic acid in the yeast extract is 3 to 30% by weight, preferably 4.5 to 30.0% by weight, more preferably 7.5 to 30.0% by dry weight. % By weight, more preferably 9.5-30.0% by weight, even more preferably 10.5-30.0% by weight, still more preferably 11.0-30.0% by weight, particularly preferably 15-30%. A yeast extract containing 0.0% by weight, most preferably 20.0-30.0% by weight can be obtained.
For this reason, especially the yeast extract obtained by the manufacturing method of this invention yeast extract has high taste of seafood flavor, and when it uses for food-drinks etc., it has a deep taste and can manufacture rich food-drinks.

In the present invention, “succinic acid production amount per dry yeast cell” means the ratio of the amount of succinic acid produced by self-digestion to the solid content obtained by drying yeast cells used as a raw material for self-digestion ( % By weight). The “succinic acid content per dry weight of yeast extract” means the ratio (% by weight) of succinic acid contained in the solid content obtained by drying the yeast extract (that is, succinic acid per dry yeast extract weight). Acid content).

The amount of succinic acid produced per dry yeast cell and the content of succinic acid in the yeast extract are measured using, for example, 1 mL of yeast extract after self-digestion filtered through a filter with a pore size of 0.45 μm as a sample solution for measurement. It can be measured by analyzing the sample solution using an analyzer such as an HPLC organic acid analysis system (device name: Prominence, manufactured by Shimadzu Corporation).

The yeast extract obtained by the method for producing a yeast extract of the present invention is dried and powdered to obtain a yeast extract powder containing a high amount of succinic acid. As a method for preparing the yeast extract powder, any method can be used as long as it is a usual method, but industrially, freeze-drying method, spray-drying method, drum-drying method and the like are adopted. .

Moreover, the obtained yeast extract and the yeast extract powder may be used as a seasoning composition. The seasoning composition may be composed only of the yeast extract of the present invention and contains other components such as a stabilizer and a preservative in addition to the yeast extract of the present invention. May be. Specifically, a seasoning composition can be produced by adding and mixing other components to the yeast extract or the yeast extract powder as necessary. The said seasoning composition can be suitably used for various food-drinks similarly to other seasoning compositions.

Furthermore, the obtained yeast extract and the yeast extract powder can be directly contained in food and drink as raw materials. Specifically, in the production process of food and drink, the succinic acid-rich yeast, the dried yeast cells of the yeast, the yeast extract prepared from the yeast, the yeast extract powder, and the like are the same as other raw materials. By adding, a food or drink containing succinic acid at a high concentration can be produced.

These foods and drinks may be any foods and drinks that can normally be added with dry yeast, yeast extract, and seasoning compositions containing these, for example, alcoholic beverages, soft drinks, fermented foods, seasonings, soups. , Breads and confectionery. In addition, the culture and the like can be consumed as a supplement or the like by processing into soft capsules, hard capsules, tableted tablets or the like.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Reference Example 1]
First, yeast extracts were prepared by a hot water extraction method and an autolysis method, and succinic acid contents in the obtained yeast extracts were compared. Three types of Saccharomyces cerevisiae (AB9170 strain, AB95 strain, and AB933 strain), which are natural yeasts, were used.

<1> Preculture 350 mL of each medium having the following composition was prepared in two Erlenmeyer flasks with 2 L baffles.
(Medium composition)
Molasses 8%
Urea 0.6%
(NH ₄ ) ₂ SO ₄ 0.16% (ammonium sulfate)
(NH ₄ ) ₂ HPO ₄ 0.08% (diammonium hydrogen phosphate)

(Production method)
(1) After 167 mL of molasses (sugar content 36%) was made up to 750 mL with MilliQ water, 350 mL each was dispensed into a 2 L baffled Erlenmeyer flask.
(2) The molasses medium prepared in (1) was autoclaved (121 ° C., 15 minutes).
(3) At the time of use, 1/50 amount of nitrogen component mixture (x100) was aseptically added (7 mL each) to the molasses medium after autoclaving.

(Culture conditions)
Incubation temperature 30 ℃
Shaking 160rpm (rotary)
Incubation time 24 hours

<2> Main culture The main culture was performed by setting the volume at the start of culture to 2 L and the volume at the end of fed-batch to 3 L. First, 2 L each of media having the following composition was prepared in two 5 L jar fermenters (manufactured by Maruhishi Bioengineer).
(Medium composition)
Ammonium chloride 0.18% (3L conversion) 5.3g
(NH ₄ ) ₂ HPO ₄ 0.04% (3 L equivalent) 1.2 g

Subsequently, 300 mL of the preculture solution was inoculated into the prepared medium and cultured for 24 hours under the following conditions.
(Culture conditions)
Incubation temperature 30 ℃
Aeration 3L / min stirring 600rpm
pH control Lower limit control pH 5.0 (with 10% ammonia water), no upper limit control Antifoam Adecanate stock solution fed medium Molasses (sugar content 36%), capacity 700mL (1L medium bottle, final 8%)

2 mL of each main culture solution was collected in an aluminum dish (diameter 5 cm) weighed in advance and dried at 105 ° C. for 4 hours.
The weight after drying (weight after drying of yeast) was measured, and the weight of solid content (weight of dry yeast cells, unit g / L) was calculated by the following formula (4).
(Weight after yeast drying-weight of aluminum dish) x 500 = Weight of dried yeast cells (g / L) (4)

Among the two main culture solutions, yeast extract was prepared by autolysis method and the remaining one by hot water extraction method.
Specifically, in the autolysis method, yeast cells are collected from 3 L of the obtained main culture solution, and the weight of dry yeast in the yeast suspension is 180 g / L in the collected yeast cells. Water was added, and the yeast extract was extracted by maintaining at 52 ° C. under uncontrolled pH for 18 hours.
On the other hand, in the hot water extraction method, specifically, yeast cells are recovered from 3 L of the obtained main culture solution, and the weight of dry yeast in the yeast suspension is 180 g / L. Water was added so that 1 mL of each yeast suspension was fractionated into an aluminum dish (diameter 5 cm) weighed in advance and dried at 105 ° C. for 4 hours. The weight after drying (weight after drying of the yeast suspension) was measured, and the weight of the solid content (dry yeast weight, unit g / L) was calculated by the following formula (5).
(Weight after drying of yeast suspension-weight of aluminum dish) x 1000 = weight of dried yeast (g / L) (5)

The supernatant obtained by heating the yeast suspension at 100 ° C. for 10 minutes and centrifuging the obtained heated extract was used as a yeast extract.
1 mL of each yeast extract solution was collected in an aluminum pan (diameter 5 cm) weighed in advance and dried at 105 ° C. for 4 hours. The weight after drying (weight after drying yeast extract) was measured, and the weight of solid content (weight of dried yeast extract, unit g / L) was calculated by the following formula (6).
(Weight after drying yeast extract-Aluminum dish weight) x 1000 = Weight of dried yeast extract (g / L) (6)

For each yeast extract, the amount of succinic acid produced per dry yeast cell and the content of succinic acid in the yeast extract were measured. Specifically, 1 mL of yeast extract filtered through a filter having a pore size of 0.45 μm is used as a measurement sample solution, and the measurement sample solution is HPLC organic acid analysis system (device name: Prominence, manufactured by Shimadzu Corporation) Was used to measure the succinic acid content in the yeast extract. By dividing the obtained measured value (g / L) by the dry yeast extract weight (g / L), the amount of succinic acid produced per dry yeast extract was calculated. By dividing the obtained measured value by the dry yeast weight (g / L), the amount of succinic acid produced per dry yeast cell was calculated. Table 1 shows the results of succinic acid production per dry yeast cell and succinic acid content in the yeast extract. As a result, regardless of which strain is used, in the hot water extraction method, the amount of succinic acid produced per dry yeast cell is 0.3% by weight or less, and the succinic acid content in the yeast extract is 1. It was a very small amount of 3% by weight or less. In contrast, in the self-digestion method, the amount of succinic acid produced per dry yeast cell was 1.1% by weight or more, which was more than three times that obtained by the hot water extraction method.

[Example 1]
A yeast extract was prepared using Saccharomyces cerevisiae AB933 strain, which is a natural yeast, as a raw material, and the influence of the stirring speed during yeast culture on the succinic acid content of the yeast extract was examined.
First, in the same manner as in Reference Example 1, a preculture solution of AB933 strain was prepared. Subsequently, the main culture is performed in the same manner as in Reference Example 1 except that the stirring speed is 100, 200, 300, 400, 500, 600, 700, or 900 rpm, and the culture time is 24 to 42 hours under non-pH control. Went. During the culture, the temperature, pH, dissolved oxygen concentration (DO), and respiratory quotient (RQ) of the culture solution were measured over time. The measurement results of the respiratory quotient (“RQ” in the figure) and the weight of fed molasses (“Opt1” in the figure) at each stirring speed of 100 to 600 rpm are shown in FIGS. 1 to 6, respectively.

The KLa of each culture was calculated by a static method in the electrode method. Specifically, KLa was measured as follows.
First, 2.5 L of water was put on a 5 L jar fermenter (manufactured by Maruhishi Bioengineer), a DO electrode whose DO value was adjusted to zero with 10% sodium sulfite was inserted, and the water temperature was 30 ° C. The operation was started with the air flow set to 1 vvm and the number of stirrings set arbitrarily. Saturated DO value (C ^* , unit: mmol / L) was set after the air was sufficiently aerated and stirred to stabilize the DO value. Thereafter, the aeration was stopped, nitrogen gas was aerated to expel oxygen, and after the DO value was lowered to 1 to 2 ppm, the aeration was resumed. The DO value (C, unit: mmol / L) after resuming aeration was measured, and aeration was performed using the following formula (7) at the time when the increase in DO value per unit time began to be linear. The logarithm of the value of [saturated dissolved oxygen concentration (DO) −measured dissolved oxygen concentration (DO)] (mmol / L) versus time (t, unit: sec) was plotted.
^{Ln (C * -C) = -KLa} · t / 3600 ··· (7)
A value obtained by multiplying the slope of the obtained straight line by 3600 is −KLa (hr ⁻¹ ). The above operation was performed at a stirring number of 250, 300, 350, 400, 500, and 600 rpm, and a correlation equation between the stirring number rpm and KLa (hr ⁻¹ ) was obtained. From the obtained correlation equation, KLa (hr ⁻¹ ) at a stirring speed of 100 to 900 rpm was determined. The dissolved oxygen concentration (C) was set to O ₂ mmol / L by dividing the DO electrode measured value (ppm) by the molecular weight of oxygen 32.

Table 2 shows KLa and culture time of each culture solution. As a result, KLa correlates with the stirring speed, and in this example, KLa was 0.9 to 195 hr ⁻¹ when the stirring speed was 100 to 500 rpm.

Moreover, water was added to the yeast cells recovered from each 3 L of the culture solution so that the dry yeast weight in the yeast suspension was 180 g / L, and maintained at 40 ° C. for 18 hours under uncontrolled pH. Yeast extract was extracted. Solid-liquid separation was performed on the extracted yeast extract to obtain a yeast extract.
About each yeast extract, it carried out similarly to the reference example 1, and measured the succinic-acid production amount (weight%) per dry yeast cell, and the succinic-acid content (weight%) in a yeast extract. The measurement results are shown in Table 2 and FIG. FIG. 7A shows the succinic acid production amount (% by weight) per dry yeast cell, and FIG. 7B shows the succinic acid content (% by weight) in the yeast extract.

As a result, the succinic acid content per dry weight of the yeast extract is higher than that when KLa is 380 hr ⁻¹ or more when KLa is 0.9 to 195 hr ⁻¹ (when the number of stirring is 100 to 500 rpm). There were also many. That is, it was found that, by appropriately adjusting the stirring speed among the yeast culture conditions, KLa was adjusted to a range of 0.9 to 195 hr ^{−1, and} a yeast extract having a high succinic acid content could be prepared.

As shown in FIGS. 1 to 6, when the stirring speed was as high as 600 rpm or more, the respiratory quotient during the growth phase was as small as about 1.0 to 1.2. This is presumably because they are growing by breathing in the presence of a sufficient amount of oxygen. On the other hand, when the stirring speed was 100 to 500 rpm, the respiratory quotient was as large as 1.3 or more. This is considered because yeast is fermenting in a low oxygen environment. When the stirring speed is 100 to 500 rpm, the yeast growth phase is from the time when the respiratory quotient becomes 1.3 or more after the start of sugar feeding until the end of sugar feeding. For example, in the case of 300 rpm in this example, as shown in FIG. 3, the time is from 3 hours after the start of culture to 22 hours later.

[Example 2]
The effect of autolysis time on the succinic acid content of the yeast extract was examined.
First, the supernatant was removed from the preculture liquid obtained by the same method as the preculture in Reference Example 1, and the precultured yeast suspension concentrated so that the dry yeast weight in the yeast suspension was 180 g / L was obtained. The main culture of AB933 strain was prepared in the same manner as in Reference Example 1 except that 140 mL of the prepared precultured yeast suspension was inoculated and that the stirring speed was 300 rpm and the culture time was 48 hours. Liquid was performed.
Water is added to the yeast cells recovered from the culture solution so that the dry yeast weight in the yeast suspension is 180 g / L to obtain a self-digestion solution. For 48 hours to extract the yeast extract. Extraction of hot water before the start of self-digestion (0 hours), 3, 6, 24, and 48 hours after the start of self-digestion, fractionate 5 mL of auto-digestion solution, The succinic acid production amount (% by weight) per unit and the succinic acid content (% by weight) per dry weight of the yeast extract were measured. Further, as a control, a yeast extract was prepared from yeast cells recovered from the culture solution by the hot water extraction method in the same manner as in Reference Example 1, and the amount of succinic acid produced (% by weight) per yeast and yeast The succinic acid content (% by weight) per dry weight of the extract was measured. Table 3 shows the measurement results.

As a result, it was confirmed that the amount of succinic acid produced increased depending on the autolysis time and reached a plateau. That is, the succinic acid content in the yeast extract can be adjusted by adjusting the self-digestion time.
Further, the succinic acid content (0.2% by weight) per dry yeast cell cultured at 300 rpm by hot water extraction method was cultured at 600 rpm in AB933 strain of Reference Example 1 and dried yeast by hot water extraction method. It was equivalent to the succinic acid content per body (0.2% by weight). That is, the succinic acid content in the yeast immediately after the culture was not so different depending on the culture conditions such as KLa. It is a finding for the first time by the present inventors that the conditions at the time of culture such as KLa affect not the amount of succinic acid produced by yeast immediately after cultivation but the amount of succinic acid produced during autolysis.

[Example 3]
In the yeast extract production method of the present invention, autolysis was performed under pH control to produce a yeast extract.
First, in the same manner as in Example 2, a precultured yeast suspension of AB933 strain was prepared. Subsequently, the main culture was performed in the same manner as in Example 2 at a stirring speed of 400 rpm. Furthermore, water was added to the yeast cells recovered from the culture solution so that the weight of dry yeast in the yeast suspension was 180 g / L to obtain a self-digestion solution. The yeast extract was extracted with a mentor (manufactured by Maruhishi Bioengineering) at 40 ° C. and 25% sodium hydroxide solution for 28 hours under pH 5.2 control.
With respect to the obtained yeast extract, the production amount (% by weight) of succinic acid per dry yeast cell was measured in the same manner as in Reference Example 1, and it was 4.3% by weight. This value was almost the same as that shown in Table 2 of Example 1 when autolysis was performed under pH non-control. That is, from this result, it is possible to produce a yeast extract having a high succinic acid content by the method for producing a yeast extract of the present invention, regardless of whether the pH is controlled or not during the self-digestion reaction. it is obvious.

[Example 4]
A yeast extract was prepared from the Saccharomyces cerevisiae AB933 strain as a raw material by the method for producing a yeast extract of the present invention.
First, in the same manner as in Example 2, a precultured yeast suspension of AB933 strain was prepared. Subsequently, main culture was carried out in the same manner as in Example 2 except that the stirring speed was 300 rpm and the pH was not controlled for 48 hours.
Water is added to the yeast cells recovered from the culture solution so that the dry yeast weight in the yeast suspension is 180 g / L to obtain a self-digestion solution. For 48 hours to extract the yeast extract.
About the obtained yeast extract, it carried out similarly to the reference example 1, and measured the succinic-acid production amount (weight%) per dry yeast cell and the succinic-acid content (weight%) per dry weight of yeast extract. Table 4 shows the measurement results. In Table 4, Samples 1A to 1C, Samples 2A to 2C, Samples 3A to 3C, Samples 4A to 4B, and Sample 5A are sample groups having the same implementation date. As a result, in all 12 samples, the amount of succinic acid produced per dry yeast cell was 3.9 to 5.8, and the succinic acid content per dry weight of yeast extract was 15.4 to 26. .4% by weight.

[Example 5]
In the same manner as in Example 2, except that Saccharomyces cerevisiae Delft strain (ATCC 6037), Saccharomyces cerevisiae Winsor strain (ATCC 96473), and Candida utilis strain C90 (IAM0626) were used as raw materials. A cultured yeast suspension was prepared. Subsequently, main culture was performed in the same manner as in Example 4 except that the stirring speed was 300 rpm, and the yeast extract was extracted.
About each yeast extract, it carried out similarly to the reference example 1, and measured succinic-acid content (weight%) in a yeast extract. The succinic acid content in the yeast extract of each strain when the autolysis temperature is 40 ° C. is 10.5% by weight for Delft strain, 12.2% by weight for Winsor strain, and 3.9% by weight for C90 strain. Met.
Furthermore, with respect to Saccharomyces cerevisiae Winsor strain, when the succinic acid production amount (% by weight) per dry yeast cell and the succinic acid content (% by weight) in the yeast extract were measured at an autolysis temperature of 52 ° C., the dried yeast The amount of succinic acid produced per body was 2.2% by weight, and the content of succinic acid in the yeast extract was 5.2% by weight.
As a result, in both strains registered in ATCC and strains of the genus Candida, yeast cultured under conditions where the stirring speed is 300 rpm and KLa is in the range of 0.9 to 195 hr-1 It was confirmed that the succinic acid production amount per dry yeast cell and the succinic acid content in the yeast extract increase.

[Example 6]
The yeast extract produced by the yeast extract production method of the present invention was added to the clam chowder, and the relationship between the succinic acid content in the yeast extract and the yeast extract addition effect was examined.
First, in the same manner as in Reference Example 1, a preculture solution of AB933 strain was prepared. Subsequently, the main culture was performed in the same manner as in Reference Example 1 except that the stirring speed was 300 rpm and the pH was not controlled for 48 hours.
Next, water is added to the yeast cells recovered from the culture solution so that the dry yeast weight in the yeast suspension is 180 g / L to obtain a self-digestion solution. Holding for 28 hours under control, the yeast extract was extracted. Solid-liquid separation was performed on the extracted yeast extract to obtain a yeast extract (A) having a succinic acid content per dry weight of 23%.

As shown in Table 5, the obtained yeast extract (A) and a commercially available yeast extract (trade name: Exl Prime LS, Alltech Serbia) are mixed, and the succinic acid content in the yeast extract is 2-6. % Yeast extracts 1 to 4 were prepared. The succinic acid content per dry weight of Exl Prime LS used in this example was 0.5% by weight. In Table 5, “succinic acid content (% by weight)” is the succinic acid content (% by weight) per dry weight of the yeast extract.

One powder of commercially available powdered crumb chowder (trade name: carefully crushed chow chowder, manufactured by Pokka Corporation) (previously sieved and without the ingredients) dissolved in 266 ml of hot water, yeast extract 1 ~ 4 were blended so that each amount would be 0.15% to obtain clam chowder (samples 1 to 4).
For each clam chowder, 11 sensory panelists conducted a comparative sensory test on saltiness, sweetness, umami, acidity, taste, aftertaste, seafood odor, richness, astringency, and palatability. Specifically, out of 6 stages (1 is the weakest and 6 is the strongest), the evaluation of the sample 1 to which the yeast extract 1 was added was evaluated as 4, and the samples 2 to 4 to which the yeast extract 2 to 4 were added were evaluated. . Table 6 and FIG. 8 show the average values of the evaluations of the specialized panelists. In FIG. 8, “2% succinic acid in extract” shows the result of sample 1, “3% succinic acid in extract” shows the result of sample 2, “4% succinic acid in extract” shows the result of sample 3, “ “6% succinic acid in the extract” indicates the results of Sample 4.

As a result, compared to Sample 1 to which yeast extract having a succinic acid content of 2% by weight was added, in Samples 2 to 4 to which yeast extract having a succinic acid content of 3 to 6% by weight was added, umami, taste, The aftertaste and richness were significantly enhanced, and the palatability was also good. On the other hand, sweetness did not differ much between samples 1 to 4. In samples 3 and 4, the seafood odor and astringent taste were further significantly enhanced.

From these results, the taste enhancing effect of the yeast extract is affected by the succinic acid content in the yeast extract, and is produced by the yeast extract having a low succinic acid content and the method for producing the yeast extract of the present invention. It is apparent that a yeast extract having a desired succinic acid content can be prepared by appropriately mixing with the yeast extract.

[Example 7]
The yeast extract (A) prepared in Example 6 (succinic acid content per dry weight: 23%) was added to the clam chowder, and the relationship between the succinic acid content in the yeast extract and the yeast extract addition effect was examined. It was.
As shown in Table 7, the yeast extract (A) and a commercially available yeast extract (trade name: Exl Prime LS, Alltech Serbia) are mixed, and the succinic acid content in the yeast extract is 2, 6, or 10. % Yeast extracts 1 to 3 were prepared. The succinic acid content per dry weight of Exl Prime LS used in this example was 0.3% by weight. In Table 7, “succinic acid content (% by weight)” is the succinic acid content (% by weight) per dry weight of the yeast extract.

After dissolving 1 powder of commercially available powdered crumb chowder (trade name: carefully crushed chow chow chow, manufactured by Pokka Corporation) in 266 mL of hot water, the yeast extracts 1 to 3 are each 0.15%. Blended to obtain crumb chowder (samples 1 to 3).
For each crumb chowder, 13 sensory panelists conducted a comparative sensory test on salty taste, sweetness, umami, bitterness, sourness, taste, aftertaste, seafood odor, richness, astringency, and palatability. Specifically, out of 6 stages (1 is the weakest and 6 is the strongest), the evaluation of the sample 1 to which the yeast extract 1 was added was 4, and the samples 2 and 3 to which the yeast extract 2 and 3 were added were evaluated. . Table 8 and FIG. 9 show the average values of the evaluations of the specialized panelists. In FIG. 9, “2% succinic acid in extract” shows the result of sample 1, “succinic acid 6% in extract” shows the result of sample 2, and “succinic acid 10% in extract” shows the result of sample 3. Show.

As a result, there is almost no difference in any taste between the sample 2 to which the yeast extract having a succinic acid content of 6% by weight and the sample 3 to which the yeast extract having a succinic acid content of 10% by weight was added. Even when the succinic acid content in the extract was as high as 10% by weight, no negative effect on the taste was observed. Samples 2 and 3 have significantly enhanced umami, taste, aftertaste, richness, seafood odor, and astringency compared to sample 1 to which yeast extract having a succinic acid content of 2% by weight was added. The palatability was also good. On the other hand, sweetness and bitterness were not so different between samples 1 to 3. From these results, it was confirmed that by using a yeast extract having a high succinic acid content, umami, seafood odor, and the like can be enhanced without excessively affecting sweetness and bitterness.

Since the yeast extract having a high succinic acid content can be easily produced by the method for producing a yeast extract of the present invention, it can be used in the field of foods using the yeast extract.

Claims

A method for producing a yeast extract, comprising extracting yeast extract by self-digestion from yeast cultured under a condition of KLa (oxygen transfer capacity coefficient) of 0.9 to 195 hr −1 .
The method for producing a yeast extract according to claim 1, wherein the yeast is a yeast grown under conditions where the respiratory quotient is 1.3 or more.
The method for producing a yeast extract according to claim 1 or 2, wherein the self-digestion is carried out under a condition that the temperature of the autolysis liquid is 30 to 55 ° C.
The method for producing a yeast extract according to any one of claims 1 to 3, wherein the amount of succinic acid produced by self-digestion is 1.7 to 6.5 wt% per dry yeast cell weight before self-digestion. .
The method for producing a yeast extract according to any one of claims 1 to 4, wherein the yeast is a genus Saccharomyces or a genus Candida.
The method for producing a yeast extract according to any one of claims 1 to 5, wherein the succinic acid content of the produced yeast extract is 3.0 to 30.0% by weight per dry yeast extract weight.
A yeast extract produced by the method for producing a yeast extract according to any one of claims 1 to 6 and having a succinic acid content of 3.0 to 30.0% by weight per dry yeast extract weight.
Yeast extract having a succinic acid content produced from yeast of 3.0 to 30.0% by weight per dry yeast extract weight.
A method for producing a seasoning composition, comprising the yeast extract according to claim 7 or 8.
A method for producing a food or drink, comprising the yeast extract according to claim 7 or 8.