Method for culturing yeast and method for producing fermented food and beverages using the cultured yeast cells

Abstract

To provide methods for producing fermented foods and drinks with more characteristic and good flavor, especially bread and the like.SOLUTION: In the production of fermented foods and drinks using sucrose as a sugar source, especially bread, by using a mixed cultured cells obtained by inoculating Hanseniaspora vineae, which is a sucrose non-fermentable yeast, and Saccharomyces cerevisiae, which is a sucrose fermentable yeast at a ratio of a specific range and performing mixed culture using sucrose as a carbon source, fermented foods and drinks with an unprecedented characteristic and favorable flavor can be produced.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for mixed culture of yeasts belonging to different genera, and a method for producing fermented foods and beverages using the cells obtained by such mixed culture, particularly breads, and other methods for producing wine, sake, shochu, beer, and the like.

Background Art

[0002] Recently, food preferences have diversified in the food field. Among them, the flavor characteristics of fermented foods and beverages are important factors leading to product differentiation. Particularly in breads involving the kneading of yeast and wheat flour in the dough fermentation process, bread-making yeast expands the dough with carbon dioxide gas generated when converting the sugar contained in the bread dough into ethanol, and gives the bread a unique flavor due to the by-produced higher alcohols, esters, organic acids, and the like.

[0003] Most of the bread-making yeasts that have been industrially put into practical use are classified as Saccharomyces cerevisiae. Therefore, in these same species, there is a limit to producing unprecedented differentiated scents and tastes.

[0004] In order to impart characteristics not found in Saccharomyces cerevisiae in the production of breads, studies have been conducted using species other than Saccharomyces cerevisiae within the genus Saccharomyces. For example, attempts have been made to produce breads with more characteristic and better flavor and shape by cross-breeding strains belonging to Saccharomyces bayanus or Saccharomyces bayanus var. uvarum used in wine brewing and strains belonging to Saccharomyces cerevisiae (Patent Document 1, Patent Document 2).

[0005] Furthermore, in pursuit of obtaining fermented foods and beverages with more characteristic flavors that are unprecedented, techniques using yeasts other than the genus Saccharomyces have been reported.

[0006] For example, the genus Hanseniaspora is used in winemaking due to its distinctive fermentation properties. Specifically, while Saccharomyces yeasts, which are excellent at ethanol production, are dominant in the mid-to-late stages of fermentation in winemaking where there is no sterilization process for the raw materials, it has been reported that wild yeasts derived from grape fruit, such as those of the Hanseniaspora genus, proliferate in the early stages of fermentation, producing various higher alcohols and higher alcohol esters, which contribute to the diversification of wine quality (Non-Patent Literature 1).

[0007] Furthermore, there are reports that when a strain of Hanseniaspora vineae isolated from grape juice is cultured using grape juice and then separately inoculated into wine fermentation mash from the middle of the fermentation stage, the concentration of aromatic compounds increases, producing a desirable aroma (Non-Patent Literature 2).

[0008] Furthermore, regarding Hanseniaspora binee, there are reports that using isomerized sugar solution (a liquid sugar mainly composed of glucose and fructose) as a sugar source in the production of bread increases the volume of baked bread, as well as increasing the aromatic components such as acetoin, 2-phenethyl acetate, and acetic acid in the bread, and also results in a more desirable taste (Patent Document 3).

[0009] Incidentally, many yeast strains belonging to genera other than Saccharomyces lack the ability to ferment (sometimes referred to as assimilation and decomposition) sucrose (sometimes referred to as sucrose or sugar). Such yeasts are called sucrose-non-fermenting yeasts, but in the production of fermented foods and beverages, especially bread, sucrose is commonly used as a sugar source. Although the yeast cells can be utilized by using isomerized sugar solution as in prior art, their use is limited, which is a drawback in practical terms.

[0010] This drawback also has a significant impact on the preparation of yeast cells for use in the production of fermented foods and beverages, that is, when cultivating sucrose-non-fermenting yeast to obtain large quantities of cells. When culturing yeast, it goes without saying that a carbon source must be supplied to the culture medium, but sucrose-non-fermenting yeast does not have the ability to utilize sucrose, so sucrose cannot be used as a carbon source. When culturing yeast on a large scale industrially, the use of inexpensive molasses as a carbon source is common, but since the main sugar in molasses is sucrose, it cannot be used in the same way for cultivating sucrose-non-fermenting yeast.

[0011] Thus, although there are challenges to practical application, yeasts of the genus Hanseniaspora have been reported to produce excellent flavor in the production of fermented foods and beverages. In other words, to create unprecedented flavors and aromas, it is possible to focus on and utilize yeasts of genera other than Saccharomyces. These genera include sucrose-non-fermenting yeasts, and their distinctive fermentation characteristics offer the potential to differentiate them from conventional products. However, when utilizing sucrose-non-fermenting yeasts, specifically in the cultivation of the bacterial cells and the production of fermented foods and beverages using the resulting cells, it is necessary to apply sucrose, which is commonly used in the food manufacturing industry, as the carbon source and sugar source for the bacterial strain.

[0012] To date, a method has been developed to culture Saccharomyces cerevisiae, a sucrose-non-fermenting baker's yeast, using molasses as a carbon source, by co-culturing it with a sucrose-fermenting baker's yeast of the same genus (Patent Document 4). This technique utilizes the invertase activity of the sucrose-fermenting yeast to enzymatically decompose sucrose, making the resulting monosaccharides usable by the sucrose-non-fermenting yeast. However, its purpose is limited to the production of fructooligosaccharide-containing bread using fructooligosaccharides as a sugar source instead of sucrose. In order to minimize the decomposition of fructooligosaccharides by the baker's yeast, the invertase activity is kept very low. Therefore, the resulting mixed culture does not meet the level of invertase activity required in the present invention, and sucrose fermentation is insufficient, making it unsuitable for the production of fermented foods and beverages that use sucrose as a sugar source. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Japanese Patent Publication No. 2019-088199 [Patent Document 2] Japanese Patent Publication No. 2019-33737 [Patent Document 3] Japanese Patent Publication No. 2020-191800 [Patent Document 4] Japanese Patent Application Publication No. 62-220185 [Non-patent literature]

[0014] [Non-Patent Document 1] Frontiers in Microbiology, 8, 532, 2017 [Non-Patent Document 2] Journal of Agricultural and Food Chemistry, 64, 4574-4583, 2016; Frontiers in Microbiology, 7, 338, 2016 [Overview of the project] [Problems that the invention aims to solve]

[0015] The present invention relates to a mixed cell culture of sucrose non-fermenting yeast and sucrose fermenting yeast, characterized in that when used in the production of various fermented foods and beverages, particularly bread, as well as wine, sake, shochu, beer, etc., products with a uniquely distinctive and superior flavor can be obtained. The invention also relates to a method for culturing the mixed cell culture using sucrose as a carbon source, and a method for producing fermented foods and beverages, particularly bread, using the mixed cultured cell culture with sucrose as a sugar source. [Means for solving the problem]

[0016] To achieve the above objectives, it is possible to apply a mixed culture of sucrose-non-fermenting yeasts from genera other than Saccharomyces, particularly Hanseniaspora yeasts, and sucrose-fermenting Saccharomyces yeasts to the production of various fermented foods and beverages. The inventors have diligently researched these points and have established a method for mixed culture of Hanseniaspora yeasts, particularly Hanseniaspora binee, which do not utilize sucrose, using sucrose, which is commonly used as a carbon source, with sucrose-fermenting Saccharomyces yeasts in a specific inoculation ratio, and a method for producing fermented foods and beverages using sucrose as a sugar source from mixed cultured cells with a specific ratio, thereby completing the present invention.

[0017] (1) A method for culturing yeast, characterized by co-culturing sucrose-non-fermenting yeast with sucrose-fermenting yeast belonging to the genus Saccharomyces when culturing sucrose-non-fermenting yeast using a medium containing sucrose as a carbon source. And, The aforementioned sucrose-non-fermenting yeast is Hanseniaspora binee TW15 strain (NITE P-04484), The inoculation ratio of the sucrose-non-fermenting yeast to the sucrose-fermenting yeast in the seed culture medium at the start of cultivation is 90-99:1-10. The method wherein the proportion of sucrose-non-fermenting yeast cells in the total number of cells of a mixed cell body consisting of sucrose-non-fermenting yeast obtained by the culture and sucrose-fermenting yeast belonging to the genus Saccharomyces is 80-92%. . ( 2) The method for culturing yeast according to (1), characterized in that the sucrose-fermenting yeast is at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces bayanus var. uvarum, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces arboricola, Saccharomyces pastorianus, Saccharomyces kudriavzevii, and Saccharomyces uvarum. ( 3 ) A mixed cell mass comprising a sucrose-non-fermenting yeast and a sucrose-fermenting yeast belonging to the genus Saccharomyces And, The aforementioned sucrose-non-fermenting yeast is Hanseniaspora binee TW15 strain (NITE P-04484), The present invention relates to a bacterial cell for use in the production of fermented food and beverages using sucrose as a sugar source, characterized in that the proportion of the number of sucrose-non-fermenting yeast cells in the total number of cells of the mixed bacterial cell population is 80-92%. 。 ( 4 ) The mixed cell mass according to ( 3 ), characterized in that the sucrose-fermenting yeast is at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces bayanus var. uvarum, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces arboricola, Saccharomyces pastorianus, Saccharomyces kudriavzevii, and Saccharomyces uvarum. ( 5 ) (1) or (2) the culture method described above, or (3) or (4) A method for producing a fermented food or beverage, characterized by using the cell mass according to mixture . ( 6 ) (1) or (2) the culture method described above, or (3) or (4) A method for producing a fermented food or beverage, characterized by using the cell mass according to mixture and using sucrose as a sugar source. ( 7 ) The method for production according to any one of ( 5 ) or ( 6 ), characterized in that the fermented food or beverage is bread.

Advantages of the Invention

[0018] According to the present invention, by mixing and culturing sucrose-nonfermenting yeasts of genera other than Saccharomyces, particularly Hanseniaspora, with sucrose-fermenting Saccharomyces yeasts in a specific range of inoculation ratios in a culture medium using sucrose as the carbon source, and using the resulting mixed culture cells with a specific range of relative abundance in the production of fermented foods and beverages, especially bread, it becomes possible to produce bread and other products with a unique and superior flavor unlike anything before. In other words, by utilizing the characteristic fermentation properties of sucrose-nonfermenting yeast in the production of fermented foods and beverages, it is possible to obtain differentiated products unlike anything before, and furthermore, the present invention provides a method for applying sucrose, which is commonly used as a carbon source in the cultivation of sucrose-nonfermenting yeast and as a sugar source in the production of fermented foods and beverages using the culture cells, to conventionally used sucrose. [Brief explanation of the drawing]

[0019] [Figure 1] This figure compares the sequence of the 26S rDNA-D1 / D2 region (570 base pairs) of strain TW15 with the sequence of Hanseniaspora binere CBS 2171 (Japan DNA Databank accession number KY107860), which was detected by homology search. [Modes for carrying out the invention]

[0020] This invention relates to the production of fermented foods and beverages, particularly bread, with a unique and distinctive flavor using sucrose-non-fermenting yeast, especially yeast of the genus Hanseniaspora. The means to achieve this is to inoculate sucrose-non-fermenting yeast and sucrose-fermenting yeast in a specific ratio using sucrose as a carbon source, and then culture them together. The resulting mixed culture cells, obtained in a specific ratio, are then used to produce fermented foods and beverages, particularly bread, using sucrose as a sugar source. In this invention, the specific role of sucrose-non-fermenting yeast in the culture of the cells and the production of fermented foods and beverages is that fermentation occurs based on the sugar source supplied by the invertase present in the sucrose-fermenting yeast. In order to maximize the characteristics of sucrose-non-fermenting yeast, it is desirable to increase the proportion of sucrose-non-fermenting yeast other than the genus Saccharomyces. However, in order to supply a sufficient amount of sugar source that can be utilized by these strains, the presence of sucrose-fermenting yeast with invertase activity in a specific ratio is necessary.

[0021] In other words, during the culture process, when sucrose-non-fermenting yeast and sucrose-fermenting yeast are appropriately mixed and inoculated, and cultured in a medium with sucrose as the carbon source, the invertase present in the sucrose-fermenting yeast breaks down sucrose into glucose and fructose, and this can be used to propagate not only the sucrose-fermenting yeast but also the sucrose-non-fermenting yeast. Furthermore, in the manufacturing process of fermented food and beverages using this mixed culture of microbial cells with sucrose as the sugar source, fermentation of the sucrose-non-fermenting yeast occurs based on the sugar source supplied by the invertase present in the sucrose-fermenting yeast.

[0022] In other words, in order to create the characteristic flavor of fermented foods and beverages using sucrose-non-fermenting yeast, invertase activity of sucrose-fermenting yeast is essential in the culture process using sucrose as a carbon source (or sugar source) and in the manufacturing process of fermented foods and beverages. Furthermore, in order to maximize fermentation by the sucrose-non-fermenting yeast, it is necessary to increase the proportion of sucrose-non-fermenting yeast while ensuring adequate invertase activity that can supply the sugar source necessary for its fermentation. To achieve this, it is essential to include sucrose-fermenting yeast in the minimum proportion, and the ratio of the two strains must be adjusted to an appropriate range.

[0023] The "invertase activity" that serves as an indicator for achieving the objectives of this invention refers to the value measured using the methods described in Examples 1 and 3 for the cultured bacterial cells. Furthermore, in this invention, "lacking invertase activity" and "non-fermentable by sucrose" include not only cases where the invertase activity is 0 units, but also cases where it is less than 70 units.

[0024] Unlike the Saccharomyces species commonly used in the production of fermented foods and beverages, the sucrose-non-fermenting yeast used in this invention is not particularly limited as long as it is a yeast that does not possess invertase activity, but yeasts of the Hanseniaspora genus, which have a proven track record of use in winemaking and the production of fermented mash, can be suitably used. For example, Hanseniaspora vineae, Hanseniaspora uvarumu, Hanseniaspora occidentalis, and Hanseniaspora occidentalis can be cited, and in particular, strain Hanseniaspora vineae TW15, isolated from Yamasachi grapes collected in October 2016 in Ikeda-cho, Nakagawa-gun, Hokkaido, can be suitably used.

[0025] Furthermore, the Hanseniaspora binee TW15 strain has been deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). 04484 It is being held in deposit as such.

[0026] Hanseniaspora binee TW15 strain possesses the ability to produce a unique and distinctive flavor in fermented foods and beverages, particularly bread, and also has the following myomicrobial properties.

[0027] (A) Morphological properties When cultured in YPD liquid medium (1.0% dried yeast extract, 2.0% high polypeptone, 2.0% glucose) at 30°C for 1 day, the cells are lemon-shaped or oval, measuring 4-6 μm × 8-13 μm, and exhibit bipolar budding. When cultured in YPD agar medium at 30°C for 1 day, the colonies are light brown and glossy. (B) Physiological properties It grows at temperatures of 20-30°C. (C) Fermentability of sugars [Table 1] (D) Assimilation of carbon sources [Table 2] (E)26S rDNA-D1 / D2 region sequence The sequence of the 26S rDNA-D1 / D2 region (570 base pairs) was determined, and when this information was entered into the BLAST program on the internet for homology searching, it was found to be a perfect match with the sequence of Hanseniaspora binee CBS 2171 (Japan DNA Databank accession number KY107860).

[0028] The yeast belonging to the genus Saccharomyces used in this invention is not particularly limited as long as it is a yeast possessing invertase activity, including commercially available baker's yeast, as well as Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces bayanus var. uvarum, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces arboricola, Saccharomyces pastorianus, and Saccharomyces kudriafuzebii. Saccharomyces eubayanus and other strains can be suitably used. Examples include NBRC2044, which possesses invertase activity that utilizes sucrose, and MP yeast (product name: Nitten MP Yeast, manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.). Note that the NBRC strain is a stock strain of the NBRC (National Institute of Technology and Evaluation, Biotechnology Center).

[0029] The culture of the mixed yeast in this invention can be carried out under normal yeast culture conditions, i.e., in a culture medium containing a nitrogen source, inorganic substances, amino acids, vitamins, etc., under aerobic conditions at a temperature of 25-35°C, except that the use of sucrose, which is commonly used as a carbon source, is essential.

[0030] Here, the term "carbon source" in descriptions such as "containing sucrose as a carbon source (e.g., culture medium)" or "using sucrose as a carbon source (e.g., culture)" is not particularly limited as long as it contains sucrose. In addition to sucrose, other carbon sources such as sucrose syrup, sugar refining process juices (hot water leaching juices from the sugar refining process of sugars such as beet sugar, post-carbonation juices, storage molasses, ion exchange chromatography waste liquid, waste molasses, etc.), beet molasses, and sugarcane molasses, as well as combinations thereof, can be used as appropriate.

[0031] Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, urea, yeast extract, Bact yeast extract, Bact peptone, and corn liquor.

[0032] Examples of inorganic substances include magnesium phosphate, potassium phosphate, dipotassium hydrogen phosphate, and magnesium sulfate hydrate; examples of amino acids include glutamic acid; examples of vitamins include pantothenic acid and thiamine; and examples of antifoaming agents include Adekanol LG-294 (a product of ADEKA Corporation).

[0033] Culture methods include shaking culture, swirling shaking culture, reciprocating shaking culture, fed-batch culture, aerated stirring culture, and static culture. Culture time varies depending on the culture scale, but is generally between 3 and 72 hours.

[0034] In mixed culture, each yeast cell is inoculated into the culture medium in a specific range of inoculation ratios (expressed as volume ratios) in the form of a seed culture solution. The seed culture solution is a liquid containing yeast cells that have reached the stationary stage of growth, and is obtained by culturing each yeast cell in a seed medium (1.0% yeast extract, 2.0% polypeptone, 2.0% glucose) at 28-30°C with shaking at a rate of 100-150 rpm for 24-48 hours.

[0035] Next, the relative abundance (expressed as cell count) of each strain at the completion of culture does not fluctuate significantly from the inoculation ratio in the seed culture medium at the start of culture, provided that sucrose-fermenting yeast is inoculated within a specific range at the start of culture. In other words, the relative abundance (expressed as cell count) of each yeast in the mixed culture at the completion of culture can be roughly maintained from the ratio inoculated in the seed culture medium at the start of culture, provided that there is a certain amount of invertase activity that can supply the carbon source necessary for the growth of sucrose-non-fermenting yeast. Specifically, when sucrose-fermenting yeast is inoculated in the seed culture medium at the start of culture in a volume ratio of 0.1-30.0%, there is no significant fluctuation in the relative abundance (expressed as cell count) of both strains at the completion of mixed culture, and sucrose-non-fermenting yeast can always be cultured at a high ratio.

[0036] In the production of fermented foods and beverages, particularly bread, that use sucrose as a sugar source, it is necessary to maximize the proportion of sucrose-non-fermenting yeasts other than those of the genus Saccharomyces, especially yeasts of the genus Hanseniaspora, in order to cultivate the characteristic flavors of these fermented foods and beverages. On the other hand, in order to supply the sugar source necessary for the fermentation of these sucrose-non-fermenting yeasts, the mixed culture cells must possess a certain amount of invertase activity, and the effects of the present invention can be obtained by using a mixed culture cell in which the proportions of the organisms are appropriately balanced in terms of their interactions. Here, in order to obtain a mixed culture cell suitable for realizing the effects of the present invention, the ratio of the organisms inoculated into the culture medium at the start of cultivation is particularly important. Specifically, in order to obtain the effects of the present invention, the inoculation ratio of the sucrose-non-fermenting yeast seed culture solution volume inoculated at the start of cultivation is preferably 70.0 to 99.9%, more preferably 85.0 to 99.5%, and even more preferably 90.0 to 99.0%.

[0037] Furthermore, the proportion of sucrose-non-fermenting yeasts other than the genus Saccharomyces in the mixed culture cells at the time the mixed culture is completed, that is, the proportion expressed as the number of sucrose-non-fermenting yeast cells in the mixed culture cells used in the production of fermented food and beverages, is preferably 50-95%, more preferably 70-94%, and even more preferably 80-92%.

[0038] In this invention, the phrase "using sucrose as a sugar source (for example, in the production of fermented food and beverages)" means not only using sucrose as the entire sugar source, but also using a sugar source that contains at least 50% by weight of sucrose. Other sugar sources that can be used include glucose, fructose, galactose, lactose, raffinose, trehalose, etc.

[0039] In this invention, "bread with better flavor" and "bread with superior flavor" mean that at least one aspect of the aroma and taste of the baked bread is equivalent to or better than that of the same type of bread made with commercially available bread yeast. Furthermore, "bread with a high overall evaluation" means that the overall evaluation of the shape (including internal shape), aroma, taste, color, and hue of the baked bread is significantly superior to that of the same type of bread made with commercially available bread yeast.

[0040] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention. [Examples]

[0041] Hanseniaspora bieneer TW15 strain and Saccharomyces cerevisiae NBRC 2044 were cultured together in a medium with sucrose as the carbon source, and bread was produced using these mixed cultures. The quality of the resulting bread was then compared.

[0042] (seed culture) Each strain was inoculated using a platinum loop into 10 ml of YPD medium (1.0% Bacterium yeast extract, 2.0% polypeptone, 2.0% glucose) in a 50 ml Erlenmeyer flask, and cultured for 24 hours at 30°C with swirling shaking at a rate of 150 rpm to obtain seed cultures that had reached a stationary growth stage.

[0043] (main culture) Ten ml of each seed culture solution was inoculated into 100 ml of the main culture medium {1.0% Bact yeast extract, 2.0% Bactopeptone, 0.2% Potassium dihydrogen phosphate, 0.1% Magnesium sulfate heptahydrate, 0.05% Adekanol LG-294 (ADEKA Corporation product) and 2.0% Sucrose} in four ratios: TW15 strain:NBRC 2044 = 99.9:0.1, 99:1, 90:10, and 0:100 in 500 ml baffled Erlenmeyer flasks. The flasks were then cultured for 24 hours at 30°C with swirling shaking at a shaking speed of 150 rpm to obtain bacterial cell suspensions (mixed culture cells). The relative abundance of TW15 strain in the mixed culture cells was determined by identifying the cells based on their morphology under a microscope and counting the number of cells. Although the solid content of the cultured bacterial cells is approximately 30%, a portion was dried to calculate the exact value, and in Examples 1 and 2, the weight converted to a solid content of 33% was used.

[0044] (Measurement of invertase activity) The invertase activity of a bacterial cell suspension (200 mg / ml) was measured using the following method. A substrate solution containing 187.5 mM sucrose in 100 mM acetate buffer (pH 5.0) was dispensed into 1.6 × 12 cm test tubes, each containing 0.20 ml. The substrate solution and bacterial cell suspension were incubated at 30°C for approximately 10 minutes. 0.05 ml of the bacterial cell suspension was added, and the mixture was reacted at 30°C for 3 minutes. 0.25 ml of DNS reagent (3,5-dinitrosalicylic acid 1.0%, sodium hydroxide 1.7%, sodium potassium tartrate 30%) was added to stop the reaction. The mixture was then heated in boiling water for 5 minutes, cooled, and 5 ml of distilled water was added. A535 was measured. A standard curve was prepared using glucose. Under these conditions, one unit of activity was defined as the production of 1 nmol of invert sugar per minute by 1 mg (dry cell equivalent) of yeast cells.

[0045] The culture results are shown in Table 3 below. As the inoculation ratio of NBRC 2044 seed culture volume increased, invertase activity and cell yield increased, and the abundance ratio of TW15 strain, as indicated by the number of cells, tended to decrease, but a high abundance ratio of over 80% was achieved. Furthermore, the cell yield was equivalent to that when NBRC 2044 alone was cultured with an inoculation ratio of 90% of TW15 strain seed culture volume.

[0046] [Table 3] [Examples]

[0047] (Bread quality verification test) To compare and confirm the quality of the bread produced using each cultured bacterial cell culture obtained in Example 1 at the four inoculation ratios, the following tests were conducted.

[0048] 250g of strong wheat flour, 10.0g of butter, 17.0g of sucrose, 6.0g of skim milk, 5.0g of salt, 7.0g of cultured yeast cells (33% solids), and 170ml of distilled water were placed in a Panasonic SD-BMT1000 bread maker and the entire process, including baking, was automatically performed using the bread-making mode (approximately 4 hours). After the baked bread cooled to room temperature, its weight and volume were measured, and the specific volume (ml / g) was calculated. Furthermore, these were stored in plastic bags at room temperature for one day, and their volume, shape, browning, internal shape, softness, hue, aroma, and taste were evaluated on a three-point scale (Excellent: ◎, Good: ○, Slightly Poor: △).

[0049] The results are shown in Table 4 below. Compared to the bacterial cultures cultured with an inoculation ratio of TW15 strain:NBRC 2044 = 0:100 by the volume of seed culture medium, the bread made with inoculation ratios of TW15 strain:NBRC 2044 = 99:1 and 90:10 received equivalent or better evaluations in all items, and had a high overall evaluation. The bread made with an inoculation ratio of TW15 strain:NBRC 2044 = 99:1 was particularly outstanding in the items of aroma and taste. The aroma and taste were also good in the case of an inoculation ratio of TW15 strain:NBRC2044 = 99.9:0.1.

[0050] [Table 4] [Examples]

[0051] Using a culture medium with molasses, which is primarily composed of sucrose (a more practical sugar), as the carbon source, we performed solitary cultures of the Hanseniaspora bieneer TW15 strain, as well as mixed cultures of the Hanseniaspora bieneer TW15 strain with a commercially available baker's yeast strain (MP Yeast; a product of Nippon Beet Sugar Manufacturing Co., Ltd.). We investigated the cell yield and measured invertase activity using the cultured cells.

[0052] (seed culture) Each strain was inoculated using a platinum loop into 10 ml of YPD medium (1.0% Bacto yeast extract, 2.0% Bacto polypeptone, 2.0% glucose) in a 50 ml Erlenmeyer flask, and cultured for 48 hours at 28°C with reciprocating shaking at a shaking speed of 100 rpm to obtain seed cultures that had reached a stationary growth stage.

[0053] (preculture) Using 10 ml of each seed culture solution, two ratios were determined: TW15 strain:MP yeast = 100:0 and 99:1. These were then inoculated into 200 ml of molasses medium 1 {mixed molasses (imported sugarcane sugar and domestic beet molasses mixed appropriately, adjusted to 34% fermentable sugar content and Bx43), 0.3% ammonium sulfate, 0.2% urea, 0.08% potassium dihydrogen phosphate, 0.08% magnesium sulfate heptahydrate, and 1.0% Bact yeast extract} in 500 ml round-bottom flasks. The cultures were then incubated for 48 hours at 28°C with reciprocating shaking at a shaking speed of 100 rpm. Specifically, for TW15 strain:MP yeast = 100:0, the inoculation amounts were 10 ml:0 ml of seed culture solution, and for 99:1, they were 9.9 ml:0.1 ml. The obtained culture solution was inoculated entirely into 1 L of molasses medium 2 {380 g of mixed molasses (imported sugarcane sugar and domestic beet molasses mixed appropriately, adjusted to 34% fermentable sugar content and Bx43), 0.7% ammonium sulfate, and 0.07% ammonium dihydrogen phosphate} in a 2 L jar fermenter, and cultured with aeration for 13 hours at 26°C with an aeration rate of 1 vvm. Next, this culture solution was transferred entirely to a 10 L jar fermenter, and batch-type fed-batch culture was performed for 18 hours at 30°C with an aeration rate of 1 vvm and a stirring speed of 450 rpm. 2423 g of mixed molasses was supplied as the carbon source in divided portions, 60 g of urea as the nitrogen source, and 37.5 g of ammonium dihydrogen phosphate as the inorganic salt. After the culture was completed, the bacterial cells were collected by centrifugation and washed twice with sterile water to obtain the inoculum for this culture.

[0054] (main culture) Using the single-culture or mixed-culture inoculum obtained in the pre-culture, batch-type fed-batch culture was performed in a 2L jar fermenter at 30°C, pH 3-7, aeration rate of 1 vvm, and stirring speed of 800 rpm for 12 hours. The initial inoculum amount was 10g (dry cell equivalent), and the carbon source was supplied by dividing 224.6g of mixed molasses. The nitrogen source was 4.0g of urea, and the inorganic salts added were 1.0g of ammonium dihydrogen phosphate, 0.6g of magnesium sulfate heptahydrate, and 6.0mg of zinc sulfate. After the culture was completed, the cells were collected by centrifugation and washed to obtain the main culture cells. The proportion of TW15 strain in the culture cells was determined by taking a portion of the culture solution after the culture was completed, identifying TW15 and MP yeast based on cell morphology under a microscope, and counting the number of cells.

[0055] The culture results are shown in Table 5 below. The cell yield in single culture was 9.4 g (dried cells) / L culture medium, and the cell yield in mixed culture was 32.3 g (dried cells) / L culture medium, indicating a 3.4-fold increase in cell yield with mixed culture. Furthermore, in mixed culture, the abundance of TW15 reached 81% at the end of the culture, demonstrating that a high level of TW15 abundance was maintained even in mixed culture using molasses as a carbon source and MP yeast. Moreover, while the cells recovered by centrifugation in single culture exhibited agglutination, which could negatively affect production, this was not observed in mixed culture, indicating that the properties of the bacterial suspension improved with mixed culture.

[0056] [Table 5]

[0057] (Measurement of invertase activity) The invertase activity of the bacterial cells obtained from this culture was measured using the method described in Example 1.

[0058] The results are shown in Table 6 below. The invertase activity of mixed culture cells (cultured with an inoculation ratio of TW15 strain:MP yeast = 99:1) increased by approximately 80 times compared to single culture cells (cultured with an inoculation ratio of TW15 strain:MP yeast = 100:0), reaching a practical level.

[0059] [Table 6] [Examples]

[0060] (Bread dough fermentation test) To compare and confirm the dough fermentation ability of each cultured bacterial cell culture obtained in Example 3 using the two inoculation ratios, the following tests were conducted.

[0061] (Experiment 1) Fermentation power of sugar-free dough 100g of strong wheat flour, 50ml of water containing 2.0g of NaCl, and 15ml of a suspension containing 2.0g of cultured yeast cells (33% solids) were kneaded for 2 minutes. 33.8g of the prepared unsweetened dough was placed in a measuring bottle, and the amount of carbon dioxide generated was measured every 5 minutes at 30°C for 100 minutes using a fermograph. The amount of carbon dioxide generated up to 60 minutes after the start of fermentation was then compared with the amount of carbon dioxide generated from 60 minutes to 100 minutes after the start of fermentation.

[0062] (Experiment 2) Fermentation power of a low-sugar dough with 5% sucrose 100g of strong wheat flour, 50ml of water containing 5.0g of sucrose and 2.0g of NaCl, and 12ml of a suspension containing 2.0g of cultured yeast cells (33% solids) were kneaded for 2 minutes. 34.2g of the prepared unsweetened dough was placed in a measuring bottle, and the amount of carbon dioxide produced was measured every 5 minutes at 30°C for 140 minutes using a fermograph. The amount of carbon dioxide produced up to 60 minutes after the start of fermentation, from 60 to 100 minutes after the start of fermentation, and from 100 to 140 minutes after the start of fermentation were compared.

[0063] (Experiment 3) Fermentation power of dough with 30% sucrose (high sugar content) 100g of strong wheat flour, 40ml of water containing 30g of sucrose and 0.5g of NaCl, and 12ml of a suspension containing 3.0g of yeast culture cells (33% solids) were kneaded for 2 minutes. 37.1g of the prepared unsweetened dough was placed in a measuring bottle, and the amount of carbon dioxide generated was measured every 5 minutes at 30°C for 80 minutes using a pharmograph.

[0064] The results of Experiments 1-3 are shown in Table 7 below. Compared to the case using single-cultured cells (cells cultured with an inoculation ratio of TW15 strain:MP yeast = 100:0), the test group using mixed cultured cells with commercially available baker's yeast strains (cells cultured with an inoculation ratio of TW15 strain:MP yeast = 99:1) showed an increase in carbon dioxide generation in the dough at each sugar concentration of approximately 10 to 15 times, demonstrating excellent fermentability and confirming its practicality.

[0065] [Table 7]

[0066] In summary, the present invention is as follows:

[0067] This invention makes it possible to produce fermented foods and beverages with a unique and superior flavor profile, unlike anything before, by mixing and culturing sucrose-nonfermenting yeasts other than those of the Saccharomyces genus, particularly Hanseniaspora, with sucrose-fermenting Saccharomyces yeasts in a specific ratio using sucrose as a carbon source, and then using the resulting cultured cells in the production of fermented foods and beverages, especially bread. [Accession Number]

[0068] The accession numbers of the microorganisms deposited in this invention are shown below. (1) Hanseniaspora vineae TW15 strain (NITE P- 04484 ).

Claims

1. A method for culturing yeast, characterized in that when culturing sucrose-non-fermenting yeast using a medium containing sucrose as a carbon source, sucrose-fermenting yeast belonging to the genus Saccharomyces is mixed in, The sucrose-non-fermenting yeast is Hanseniaspora binee TW15 strain (NITE P-04484), The inoculation ratio of the sucrose-non-fermenting yeast to the sucrose-fermenting yeast in the seed culture medium at the start of cultivation is 90-99:1-10. The method wherein the proportion of sucrose-non-fermenting yeast cells in a mixed cell body consisting of sucrose-non-fermenting yeast obtained by the culture and sucrose-fermenting yeast belonging to the genus Saccharomyces is 80 to 92% of the total number of cells.

2. The method for culturing yeast according to claim 1, characterized in that the sucrose-fermenting yeast is at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces bayanus bar ubarum, Saccharomyces micatae, Saccharomyces paradoxus, Saccharomyces arboricola, Saccharomyces pastorianus, Saccharomyces cudriahuzebii, or Saccharomyces eubayanus.

3. A mixed bacterial cell consisting of a sucrose-non-fermenting yeast and a sucrose-fermenting yeast belonging to the genus Saccharomyces, The sucrose-non-fermenting yeast is Hanseniaspora binee TW15 strain (NITE P-04484), The microbial cells for use in the production of fermented food and beverages using sucrose as a sugar source, characterized in that the proportion of sucrose-non-fermenting yeast cells in the total number of microbial cells of the mixed cells is 80 to 92%.

4. The mixed bacterial cell according to claim 3, characterized in that the sucrose-fermenting yeast is at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces bayanus bar ubarum, Saccharomyces micatae, Saccharomyces paradoxus, Saccharomyces arboricola, Saccharomyces pastorianus, Saccharomyces cudriahuzebii, or Saccharomyces eubyanus.

5. A method for producing fermented food and beverages, characterized by using a mixed bacterial cell obtained by the culture method described in claim 1 or 2, or a mixed bacterial cell described in claim 3 or 4.

6. A method for producing fermented food and beverages, characterized by using a mixed bacterial cell obtained by the culture method described in claim 1 or 2, or a mixed bacterial cell described in claim 3 or 4, and using sucrose as a sugar source.

7. The manufacturing method according to either 5 or 6, characterized in that the fermented food or beverage is bread.

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