Temperature-sensitive flavor-producing saccharomyces cerevisiae and use thereof in preparing umami seasoning

Abstract

To provide a temperature-sensitive flavor-producing Saccharomyces cerevisiae and its use in the preparation of an umami seasoning.SOLUTION: A temperature-sensitive flavor-producing Saccharomycescerevisiae, which was deposited in the Chinese Type Culture Collection Center on May 31, 2024, with a deposit number of CCTCCNO: M20241116. In the present invention, the temperature-sensitive flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum (Accession No. CCTCCNO: M2024431) are mixed and fermented to obtain a fermentation broth containing high concentrations of glutamate and nucleotides, and the fermentation broth further contains a variety of other amino acids, organic acids, β - glucan and other functional nutrients and has higher functional nutritional value.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biotechnology, and specifically relates to a thermosensitive flavor-producing Saccharomyces cerevisiae and its use in the preparation of umami seasonings. [Background technology]

[0002] As living standards improve, demand for natural umami seasonings is increasing. Currently, there are many methods for producing natural umami seasonings, but they still have several drawbacks. (1) Corynebacterium glutamicum requires pure fermentation, and the resulting product is monosodium glutamate, which has a single component and an unsatisfactory flavor. (2) Two-stage fermentation technology, which mainly uses mold and bacteria, has been adopted by some people, but the process involves complicated steps, is prone to bacterial contamination, and is expensive.

[0003] Chinese Invention Patent Application No. 202180054828.3 provides a flavor enhancing composition derived from plants of the Umbelliferae family and having a content of flavor-effective glutamate, and a method for producing the flavor enhancer by reducing biotin in fermentable carbohydrates prior to bacterial fermentation. The method uses carrot juice, carrot puree, carrot residue, or a combination thereof as a raw material and ferments it with microorganisms such as Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium casei, Brevibacterium lactofermentum, Bacillus subtilis, or a combination thereof to produce the flavor enhancer. This flavor enhancer contains glutamate as well as organic acids such as lactic acid, citric acid, acetic acid, and succinic acid, or a combination thereof. While this method improves fermentation efficiency, it still does not solve the problems of umami seasonings, such as single ingredients and insufficient flavor. Furthermore, this flavor enhancer requires the addition of exogenous auxiliary ingredients, such as sodium chloride (salt), ribonucleotides, inosine monophosphate, guanosine monophosphate, monosodium glutamate, yeast, amino acid blends, peptides, arginine hydrochloride, arginine ammonium chloride, lysine hydrochloride, and lysine-ornithine hydrochloride. The addition of these ingredients destroys the product's pure, natural, and green image. Invention Patent WO2015 / 020292 ferments vegetable proteins (e.g., soybean protein, wheat protein, corn protein, etc.) with mold (e.g., Aspergillus sojae CJCC_080124P (KCCM11026P)) to produce umami amino acids and polypeptides, and then adds a carbon source, phosphorus source, and trace elements, and ferments the mixture with bacteria (e.g., Corynebacterium ammoniagenes CJIP009 (KCCM-10226), Corynebacterium glutamicum Bacillus, etc.) to produce more umami substances.This method can increase the variety of fermentation products and improve flavor and umami, but it has problems such as complex steps and high production costs. Chinese Invention Patent No. 201410790020.3 discloses a Saccharomyces cerevisiae strain with excellent anti-autolysis properties. The cell wall of this strain is twice as thick as that of the original strain. Autolysis simulation data and 60-hour scanning electron microscope observations all show that the autolysis properties of the recombinant strain are significantly superior to those of the original strain, making it valuable for improving beer quality, such as flavor and foam stability. Because this strain is genetically modified, it cannot currently be used in food production. Chinese Invention Patent Application No. 201610538817.3 discloses an autolysis process that produces highly active yeast autolysate by mixing yeast with a specific single-cell protein in a specified ratio, autolyzing it, and then drying and sterilizing the mixture. The process retains the original enzyme activity without sterilization. Autolysis is divided into a low-temperature stage and a high-temperature stage, with the low-temperature stage maintained at 45-60°C for 20-60 minutes, and the high-temperature stage maintained at 75-95°C for 30-120 minutes. This technology has the problem of high processing temperatures and large energy consumption, which can easily damage the flavor of the product.

[0004] Therefore, establishing a technology for preparing natural umami seasonings that are low cost, have simple operation steps, and have a rich flavor is an important issue that needs to be resolved as soon as possible. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the problems existing in the prior art, the present invention provides a thermosensitive flavor-producing Saccharomyces cerevisiae that can be used to prepare umami seasonings, and a method for preparing the same in the preparation of umami seasonings. By using carrot juice as the main raw material and mixing this yeast with Corynebacterium glutamicum to ferment the mixture, products such as glutamic acid and flavor nucleotides can be obtained, thereby reducing production costs, improving production efficiency, and producing natural umami seasonings with a higher umami level and a richer flavor. [Means for solving the problem]

[0006] The technical solution adopted by the present invention to solve the technical problems is as follows: A temperature-sensitive flavor-producing Saccharomyces cerevisiae, which was deposited with the China Center for Typical Cultures Depositary on May 31, 2024, and has the deposit number CCTCC NO:M 20241116.

[0007] The present invention also provides use of the yeast in preparing an umami seasoning, which comprises mixing the yeast with Corynebacterium glutamicum and carrying out fermentation to prepare an umami seasoning using carrot juice as a main ingredient.

[0008] The present invention provides (1) inoculating the activated thermosensitive flavor-producing Saccharomyces cerevisiae into a sterilized fermentation medium, controlling the fermentation temperature at 28±1°C, the pH range at 4.5 to 6.5, and controlling the dissolved oxygen at 10% to 30% by a combination of rotation speed and ventilation, and fermenting for 12 to 24 hours; When the wet weight of the yeast reaches 50 g / L or more and the total sugar content drops to 0.5% (w / v) or less, add pre-sterilized carrot juice and concentrated sugar solution to the fermentation tank until the total sugar content reaches 7% (w / v) or more, heat the tank to 40-75°C, maintain the temperature for 30-120 minutes, then quickly lower the temperature to 30°C, inoculate with activated fresh Corynebacterium glutamicum CCTCC NO:M 2024431, and continuously ferment for 12-24 hours while controlling the temperature of the mixed fermentation at 30-33°C, the dissolved oxygen content at 30-50% by a combination of rotation speed and ventilation, and the pH at 6.5-8.0 during fermentation (2). The present invention further provides a method for preparing the umami seasoning, which comprises step (3) of raising the temperature to 100°C after the fermentation is completed and maintaining the temperature for 10 to 60 minutes.

[0009] Preferably, during mixed fermentation, sugar supplementation begins when the total sugar content drops to 0.2% to 2.0% (w / v), and the sugars supplemented are one or more selected from glucose, sucrose, fructose, corn syrup, and high-fructose corn syrup in any combination ratio, and the sugar solution supplemented by feeding has a sugar:water ratio of 1:1 by weight.

[0010] Preferably, the fermentation medium uses carrot juice as a main ingredient and contains, in mass ratios, 500 to 1000 g / kL of carrot juice, 5.0 to 15 g / kL of dipotassium hydrogen phosphate, 0.05 to 2.0 g / kL of magnesium sulfate heptahydrate, 0.005 to 0.2 g / kL of ferrous sulfate heptahydrate, 0.001 to 0.2 g / kL of manganese sulfate monohydrate, 0 to 3.0 g / kL of aspartic acid, and 0 to 3.0 g / kL of threonine.

[0011] Preferably, the initial total sugar content in the fermentation medium is controlled in the range of 3% to 5% (w / v), the initial pH is 4.5 to 6.5, and sterilization is carried out after uniform mixing under sterilization conditions of 115°C to 121°C for 15 to 30 minutes.

[0012] Preferably, the solids content of the carrot juice is 6.0° in terms of Brix.

[0013] The present invention further provides an umami seasoning prepared by the above method, which includes a fermentation product of a mixture of the flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum CCTCC NO:M 2024431.

[0014] Preferably, the product has a glutamic acid concentration of 80 to 156 g / L and a total nucleotide concentration of 250 mg / L or more.

[0015] Preferably, the product further comprises β-glucan and organic acids. [Effects of the Invention]

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: When produced in carrot juice, the thermosensory flavor-producing Saccharomyces cerevisiae provided by the present invention has high-temperature autolysis properties; that is, it grows and proliferates normally at 25-28°C, and grows best at 28°C. However, growth is significantly inhibited at temperatures above 30°C. When the temperature reaches 35°C or higher, the fungal cells break down their cell walls and initiate autolysis, simultaneously releasing intracellular endolysates such as RNA and proteins.

[0017] The present invention utilizes a mixture of temperature-sensitive flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum (Deposit Number: CCTCC NO: M 2024431) for fermentation, resulting in a fermentation broth containing high concentrations of glutamic acid and nucleotides. This fermentation broth also contains various functional nutrients, such as other amino acids, organic acids, and β-glucans. At the end of the mixed-bacteria fermentation, the glutamic acid concentration in the fermentation broth can reach 80 g / L or more (maximum 156 g / L), and the total nucleotide concentration can reach 250 mg / L or more. These two substances synergistically enhance the umami flavor, resulting in a stronger umami flavor, a richer and more harmonious flavor, a richer ingredient composition, and higher functional nutritional value compared to a simple glutamic acid fermentation broth. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows the colony morphology of the temperature-sensitive flavor-producing Saccharomyces cerevisiae provided in an embodiment of the present invention. [Figure 2] 1 shows a growth curve of a temperature-sensitive flavor-producing Saccharomyces cerevisiae provided in an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] To facilitate understanding of the present invention, the present invention will now be described in more detail with reference to the drawings and specific examples. However, the present invention may be embodied in various forms and is not limited to the examples set forth herein. Rather, these examples are provided to provide a more thorough and comprehensive understanding of the present disclosure. Example 1

[0020] The present invention provides a thermosensible flavor-producing Saccharomyces cerevisiae yeast, which is of marine origin.

[0021] 1. Screening and isolation of strains (1) Strain isolation and purification: Initially, representative seaweed (mainly kelp, Sargassum, and Enteromorpha spp.) was collected from the waters off Qingdao, Shandong Province, and then the collected samples were quickly packed into sterile sampling bottles. Seaweed samples were collected from multiple locations under sterile conditions in the laboratory and then immersed in sterile saline and shaken. After a certain period of time, bacterial suspension samples of various concentrations were prepared by gradient dilution. Each gradient suspension sample was sequentially plated onto nutrient agar (NA), MRS agar, potato dextrose agar (PDA), and yeast extract peptone dextrose agar (YPDA) media and cultured under various conditions to isolate microbial resources such as bacteria, yeast, and mold. Ten single colonies containing the relevant bacterial strains, including three yeast strains, were isolated from the YPDA plates and purified by plate streaking. They were then streaked onto test tube slopes and stored at 4°C. (2) Strain Screening: First, three yeast strains, including Y01, were selected and analyzed for their growth and reproduction ability in a specific medium (sterilized carrot juice), the sensory characteristics of the fermentation liquid, the strain's RNA content, and mixed fermentation performance. The final results showed that, compared with other strains, this strain was temperature-sensitive, grew rapidly in carrot juice medium, and the fermentation liquid had a unique, pleasant aroma. Furthermore, this strain had a high RNA content and could coexist and synergistically ferment with Corynebacterium glutamicum (CCTCC accession number: M 2024431), and was therefore used to prepare an umami seasoning.

[0022] 2. Molecular biological identification and biological deposit The strain obtained by screening was identified by molecular biology techniques, and its 18S rDNA nucleotide sequence is shown in Sequence Listing SEQ ID No. 1. Through sequence homology analysis by the National Center for Biotechnology Information (NCBI), the 18S rDNA nucleotides of the flavor-producing Saccharomyces cerevisiae of the present invention were aligned using the BLAST tool, and the result showed that Y01 belongs to Saccharomyces cerevisiae. The yeast strain Y01 was deposited at the China Center for Typical Cultures Depositary on May 31, 2024, with the deposit number CCTCC NO:M 20241116. The depository address is Wuhan University, Wuhan, Hubei Province.

[0023] 3. Measurement of cultural, biological, and functional properties

[0024] 1. Biological characteristics: This strain is easy to cultivate, grows rapidly, and reproduces by budding under normal culture conditions. When cultivated on YPDA medium, typical colonies form in approximately 30 hours. They are milky white, flat, moist, and easy to pick (see Figure 1). Long-term cultivation develops a distinctive floral and fruity scent. The strain has a wide pH adaptability range, growing at pH 3.5 to 7.5, meaning it can maintain a rapid growth and reproduction rate at the optimal pH for Corynebacterium glutamicum (approximately 7.0).

[0025] 2.Culture characteristics: Experimental studies have shown that this yeast strain can utilize a variety of carbon sources, including glucose, fructose, sucrose, mannitol, and galactose. It utilizes glucose most efficiently and fructose and sucrose rapidly. The strain can grow normally between 25°C and 30°C, with growth optimal at around 28°C. This strain is temperature-sensitive; growth slows and cells begin to deform above 30°C. At temperatures above 35°C, autolysis begins. This strain can grow over a pH range of 3.5 to 7.5, with optimal growth at pH 4.5 to 5.0. During shake flask culture, the maximum bacterial concentration initially increases with increasing rotation speed, then decreases, with the optimal rotation speed being 180 rotations per minute. When cultured in pure carrot juice (6.0°Brix), the lag period for growth is approximately 4 hours. After 6 to 8 hours, the cells enter the logarithmic growth phase and grow rapidly, and after 14 to 16 hours, the cell biomass reaches its maximum, the growth rate slows down, and the cells gradually enter a plateau phase. FIG. 2 shows the growth curve of the strain under the optimum temperature and rotation speed conditions (the medium composition is, by weight, 1% yeast extract, 2% peptone, and 2% glucose).

[0026] 3. Functional property measurements: The yeast strain of the present invention has excellent flavor-producing properties. When cultured alone in carrot juice, the culture broth has a distinct floral and fruity aroma. When mixed with Corynebacterium glutamicum and fermented, new aromas such as milky and sweet aromas are added to the floral and fruity aromas.

[0027] Measurement of RNA content: (1) After yeast cultivation, the mixture is centrifuged and dried to obtain dry yeast powder. (2) Weigh out approximately 2 g of dry yeast powder (m0), suspend it in 12 ml of 0.04 mol / L sodium hydroxide solution, and grind it uniformly in a mortar. (3) Transfer the bacterial suspension to a test tube, heat it in a boiling water bath for 30 minutes, cool it, transfer it to a centrifuge tube, and centrifuge it at 4000 rpm for 10 minutes. (4) Slowly pour the supernatant into 4 ml of acidic ethanol and mix thoroughly. After the addition is complete, leave the mixture to stand until the RNA is completely precipitated, then centrifuge at 3,000 rpm for 5 minutes and discard the supernatant. (5) Wash the precipitate twice with 95% ethanol and then once with ethyl ether. (6) Transfer the precipitate to a Buchner funnel using ethyl ether, filter it under suction, dry the precipitate in air, and weigh the weight of the crude RNA (ml, g). (7) Prepare a 5-50 μg / L solution of the sample, measure the absorbance at 260 nm and 280 nm, and calculate the RNA content (%) using the following formula. RNA content (%) = (RNA content x m1) / (1000 x m0) x 100% RNA content (μg / g) = (OD 260 × dilution factor) / (0.024 × L) where OD 260 where m is the absorbance of the sample, m is the mass of dry yeast powder (g), L is the thickness of the colorimetric cup, and 0.024 is the absorbance of 1 microgram RNA / ml.

[0028] Measurements have shown that the RNA content of the yeast strains of the present invention can reach 10% (w / w) or more, whereas the RNA content of the synchronously cultured control strains (commercially available baker's yeast strains BY1 and BY2) is only 5% to 7% (w / w). Example 2

[0029] In this example, a natural umami seasoning is prepared by mixing flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum and fermenting them in carrot juice. The specific steps are as follows:

[0030] (1) Preparation of raw materials and activation of bacterial strains (i) Preparation of carrot juice Fresh, non-softened and non-rotten carrots were selected as raw materials, washed, peeled, trimmed, cut, parboiled for 5-10 minutes, and then crushed in a juicer. The resulting carrot pulp was separated into juice and residue, and then sterilized. If the test results passed, as shown in Table 1, it was ready for use.

[0031] Table 1: Carrot juice quality detection items and results [Table 1] (ii) Activation of bacterial species Preparation of yeast seed medium: 300 g / L carrot juice (solids 6.0°Brix), 1.65% (w / w) initial total sugars, 115°C, 15 min. After sterilization, the medium was cooled to the appropriate temperature, and flavor-producing Saccharomyces cerevisiae was inoculated at a 1% (v / v) inoculum and cultured at 28°C and 180 rpm for 24 hours. The resulting strain was subcultured once in the above medium at a 5% (v / v) inoculum size for 18 hours for further use. Preparation of Corynebacterium glutamicum seed medium: 200 g / L carrot juice (solids 6.0°Brix), 20 g / L sugar (equivalent to glucose), 0.3 g / L threonine, 3.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, and 0.01 g / L manganese sulfate monohydrate. The initial pH was adjusted to 7.5 with aqueous ammonia, and the initial total sugar concentration was 2.9% (w / v). After homogeneous mixing, the medium was sterilized at 115°C for 30 minutes. After sterilization, the medium was cooled to the appropriate temperature and inoculated with Corynebacterium glutamicum (CCTCC No. M 2024) at a 1.2% inoculum concentration. The medium was then cultured at 32°C for 16 hours at 200 rpm. The obtained bacterial strain was subcultured once in the above medium at an inoculation amount of 5% (v / v) for a culture time of 12 hours, and was prepared for use.

[0032] (2) Fermentation (i) Preparation of fermentation medium: carrot juice (solids content 6.0°Brix) 500 g / kg, dipotassium hydrogen phosphate 5.0 g / kg, magnesium sulfate heptahydrate 0.5 g / kg, ferrous sulfate heptahydrate 0.1 g / kg, manganese sulfate monohydrate 0.01 g / kg, aspartic acid 1.5 g / kg, threonine 1.5 g / kg, 121°C, 20 minutes. A concentrated sugar solution (sugar:water = 1:1 by weight) was prepared separately and stored in a separate sterilized tank for future use. (ii) Inoculation and fermentation The yeast seed solution obtained in step (i) of (1) above was inoculated into the fermentation medium at a 3% (v / v) inoculum level. Fermentation was continued for 12 hours, with the pH controlled at 4.5 and the dissolved oxygen controlled at 10% by a combination of rotation speed and ventilation. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermentation tank via a pipeline to adjust the total sugar content to 7% (w / v). The temperature was then raised to 40°C and maintained for 120 minutes. The temperature was then rapidly lowered to 30°C, and freshly activated Corynebacterium glutamicum was inoculated. The temperature was controlled at 31±1°C, the dissolved oxygen controlled at 30% by a combination of rotation speed and ventilation, and the pH controlled at 6.5. Sugars were then added as a feedstock when the total sugar content dropped to 0.2% (w / v). Fermentation was continued for 24 hours and then held at 100°C for 100 minutes.

[0033] (3) Detection of fermentation liquid After removing the bacterial cells from the fermentation broth obtained in step (2) of Example 2, the relevant indicators were detected. The detection results are shown in Table 2.

[0034] Table 2: Detection of components of the fermentation broth obtained in Example 2 [Table 2] Example 3

[0035] In this example, a natural umami seasoning is prepared by mixing flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum and fermenting them in carrot juice. The specific steps are as follows:

[0036] (1) Preparation of raw materials and activation of bacterial strains were the same as in Example 1.

[0037] (2) Fermentation (i) Preparation of fermentation medium: carrot juice 700 g / kg, dipotassium hydrogen phosphate 3.5 g / kg, magnesium sulfate heptahydrate 0.5 g / kg, ferrous sulfate heptahydrate 0.1 g / kg, manganese sulfate monohydrate 0.01 g / kg, aspartic acid 0.5 g / kg, threonine 0.5 g / kg, 121°C, 20 minutes. A concentrated sugar solution (sugar:water = 1:1 by weight) was prepared separately and stored in a separate sterilized tank for future use. (ii) Inoculation and fermentation The yeast seed solution obtained in step (i) of (1) above was inoculated into the fermentation medium at a 5% (v / v) inoculum level. Fermentation was continued for 12 hours, with the pH controlled at 5.5 and the dissolved oxygen controlled at 25% by a combination of rotation speed and ventilation. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermentation tank via a pipeline to adjust the total sugar content to 12% (w / v). The temperature was then increased to 75°C and maintained for 30 minutes, after which it was rapidly reduced to 30°C and freshly activated Corynebacterium glutamicum was inoculated. The temperature was controlled at 31±1°C, the dissolved oxygen controlled at 50% by a combination of rotation speed and ventilation, and the pH controlled at 7.5. Sugars were then added as a feedstock when the total sugar content dropped to 0.2% (w / v). Fermentation was continued for 24 hours followed by a 10 minute hold at 100°C.

[0038] (3) Detection of fermentation liquid After removing the bacterial cells from the fermentation broth obtained in step (2) of Example 3, the relevant indicators were detected. The detection results are shown in Table 3.

[0039] Table 3: Detection of components of the fermentation broth obtained in Example 3 [Table 3] Example 4

[0040] In this example, a natural umami seasoning is prepared by mixing flavor-producing Saccharomyces cerevisiae and Corynebacterium glutamicum and fermenting them in carrot juice. The specific steps are as follows:

[0041] (1) Preparation of raw materials and activation of bacterial strains were the same as in Example 1.

[0042] (2) Fermentation (i) Fermentation medium (800 g / kg carrot juice, 3.5 g / kg dipotassium hydrogen phosphate, 0.5 g / kg magnesium sulfate heptahydrate, 0.1 g / kg ferrous sulfate heptahydrate, 0.01 g / kg manganese sulfate monohydrate, 0.5 g / kg aspartic acid, and 0.5 g / kg threonine) was prepared and fermented at 121°C for 20 minutes. A concentrated sugar solution (sugar:water = 1:1 by weight) was prepared separately and stored in a separate sterilized tank for future use. (ii) Inoculation and fermentation: The yeast seed solution obtained in step (i) of (1) above was inoculated into the fermentation medium at a 5% (v / v) inoculum level. Fermentation was continued for 16 hours, with the pH controlled at 5.5 and the dissolved oxygen controlled at 30% by a combination of rotation speed and ventilation. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermentation tank via a pipeline to achieve a total sugar content of 10% (w / v). The temperature was raised to 55°C and held for 30 minutes, after which it was rapidly lowered to 30°C, and freshly activated Corynebacterium glutamicum was inoculated. The temperature was controlled at 31±1°C, the dissolved oxygen controlled at 30% by a combination of rotation speed and ventilation, and the pH controlled at 7.0. Sugars were added as a feedstock when the total sugar content dropped to 1%. Fermentation was continued for 24 hours, after which the temperature was increased to 100°C and held for 20 minutes.

[0043] (3) Detection of fermentation liquid After removing the bacterial cells from the fermentation broth obtained in step (2) of Example 4, the relevant indicators were detected. The detection results are shown in Table 4.

[0044] Table 4: Detection of components of the fermentation broth obtained in Example 4 [Table 4] Example 5

[0045] In this example, a natural umami seasoning was prepared by pure fermentation of carrot juice using Corynebacterium glutamicum Gu2#, and the specific steps are as follows:

[0046] (1) Preparation of raw materials and activation of bacterial strains (i) As raw materials for preparing carrot juice, fresh carrots that were not soft or spoiled were selected, washed, peeled, trimmed, cut into pieces, parboiled for 5-10 minutes, and then crushed in a juicer. The resulting carrot pulp was separated into juice and residue, and then sterilized. If the test results passed, it was ready for use. (ii) Activation of bacterial species Activation of primary fungal species: Prepare primary seed medium (g / L) according to the following formulation, and culture conditions are as follows: The mixture was prepared using 300 g / L carrot juice, 20 g / L sugar (equivalent to glucose, a monosaccharide), 0.3 g / L threonine, 3.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, and 0.01 g / L manganese sulfate monohydrate. The initial pH was adjusted to 7.5 with aqueous ammonia, and the initial total sugar concentration was 3.35% (w / v). The mixture was then homogenized and sterilized at 115°C for 15 minutes. After sterilization, the mixture was cooled to an appropriate temperature and used for inoculation. The inoculum volume was 2% (v / v), the liquid volume was 15% (v / v), and the incubation conditions were 32°C, 200 rpm, and 14 hours. Activation of secondary bacterial seeds: Prepare secondary seed medium (g / L) according to the following formulation, and the culture conditions are as follows: Carrot juice (400 g / L), sugar (equivalent to glucose, a monosaccharide), threonine (0.5 g / L), dipotassium hydrogen phosphate (5.0 g / L), magnesium sulfate heptahydrate (0.5 g / L), ferrous sulfate heptahydrate (0.01 g / L), and manganese sulfate monohydrate (0.01 g / L) were used. The initial pH was adjusted to 7.5 with aqueous ammonia, and the initial total sugar content was 5.06% (w / v). The mixture was then homogenized and sterilized at 115°C for 15 minutes. After sterilization, the mixture was cooled to an appropriate temperature and used for inoculation. Inoculum size: 10% (v / v), liquid volume: 15%, and incubation conditions were 32°C, 200 rpm, and 10 hours.

[0047] (2) Fermentation The initial fermentation medium consisted of 800 g / kg carrot juice, 55 g / kg sugar (equivalent to glucose, a monosaccharide), 0.5 g / kg threonine, 3.5 g / kg dipotassium hydrogen phosphate, 0.5 g / kg magnesium sulfate heptahydrate, 0.01 g / kg ferrous sulfate heptahydrate, and 0.001 g / kg manganese sulfate monohydrate. Antifoam was added as needed. The initial total sugar content in the medium was 9.8% (w / v). After homogeneous mixing, the mixture was sterilized at 115°C for 30 minutes. After sterilization, the mixture was cooled to the appropriate temperature, the initial pH was adjusted to 7.0 with aqueous ammonia, and fermentation was carried out using a 10% inoculum. The fermentation temperature was controlled at 30°C, and the dissolved oxygen level was controlled at 50% by a combination of rotation speed and ventilation. The fermentation time was 36 hours. The key to pH control during the fermentation process is to maintain pH at 6.5-7.0 during the logarithmic growth phase, pH at 7.0-7.2 during the acid production phase, and around 7.5 6-8 hours before the end of fermentation. During this period, when the total sugar content dropped to 1.5% (w / v), sugar was added as a feedstock to maintain the total sugar content above 1% (w / v). During this process, the sugar-acid conversion rate was controlled at approximately 50%.

[0048] (3) Detection of fermentation liquid After removing the bacterial cells from the fermentation broth obtained in step (2) of Example 5, the relevant indicators were detected. The detection results are shown in Table 5.

[0049] Table 5: Detection of components of the fermentation broth obtained in Example 5 [Table 5]

[0050] The substance composition and content in the final fermentation broth obtained in the two processes of glutamic acid pure fermentation and mixed fermentation in Example 5 were compared, and the results are shown in Table 6.

[0051] Table 6: Comparison of components of single-cell fermentation broth (Corynebacterium glutamicum) and mixed fermentation broth [Table 6]

[0052] As can be seen from Table 6, multi-bacteria mixed fermentation produces a fermentation broth containing high concentrations of glutamic acid and nucleotides. This fermentation broth also contains various functional nutrients, such as other amino acids, organic acids, and beta-glucans. At the end of the mixed-bacteria fermentation, the glutamic acid concentration in the fermentation broth can reach 80 g / L or more (maximum 156 g / L), and the total nucleotide concentration can reach 250 mg / L or more. These two substances synergistically enhance the umami flavor, resulting in a stronger umami flavor compared to simple glutamic acid fermentation broth.

Claims

1. A temperature-sensitive flavor-producing Saccharomyces cerevisiae Y01 Saccharomyces cerevisiae, which was deposited at the China Typical Culture Depository Center on May 31, 2024, and has the deposit number CCTCC NO: M 20241116.

2. 2. Use of the thermosensory flavor-producing Saccharomyces cerevisiae according to claim 1 in preparing an umami seasoning, characterized in that the yeast is mixed with Corynebacterium glutamicum CCTCC NO: M 2024431 and fermented to prepare an umami seasoning using carrot juice as a main raw material.

3. Step (1): inoculating the activated thermosensitive flavor-producing Saccharomyces cerevisiae into a sterilized fermentation medium, controlling the fermentation temperature at 28±1°C, the pH range at 4.5 to 6.5, and controlling the dissolved oxygen at 10% to 30% by a combination of rotation speed and ventilation, and fermenting for 12 to 24 hours; When the wet weight of yeast reaches 50g / L or more and the total sugar content falls to 0.5% (w / v) or less, add pre-sterilized carrot juice and concentrated sugar solution to the fermentation tank until the total sugar content reaches 7% (w / v) or more, heat the tank to 40-75°C, maintain the temperature for 30-120 minutes, then quickly lower the temperature to 30°C, inoculate with activated fresh Corynebacterium glutamicum CCTCC NO:M 2024431, control the mixed fermentation temperature to 30-33°C, control the dissolved oxygen content to 30-50% by combining rotation speed and ventilation, and control the pH to 6.5-8.0 during fermentation, and continue fermentation for 12-24 hours (step (2)); The method for preparing an umami seasoning using the temperature-sensitive flavor-producing Saccharomyces cerevisiae according to claim 1, further comprising: (3) a step of maintaining the umami seasoning at 100°C for 10 to 60 minutes after completion of fermentation.

4. The method according to claim 3, wherein, during mixed fermentation, when the total sugar content falls to 0.2% to 2.0% (w / v), sugar supplementation is started, the sugars to be supplemented are one or a combination of two or more sugars selected from glucose, sucrose, fructose, corn syrup, and high-fructose corn syrup in any blending ratio, and the sugar solution of the sugars to be supplemented has a sugar:water ratio of 1:1 by weight.

5. The method according to claim 3, characterized in that the fermentation medium is made primarily from carrot juice and contains, in mass ratios, 500 to 1000 g / kL of carrot juice, 5.0 to 15 g / kL of dipotassium hydrogen phosphate, 0.05 to 2.0 g / kL of magnesium sulfate heptahydrate, 0.005 to 0.2 g / kL of ferrous sulfate heptahydrate, 0.001 to 0.2 g / kL of manganese sulfate monohydrate, 0 to 3.0 g / kL of aspartic acid, and 0 to 3.0 g / kL of threonine.

6. 4. The method according to claim 3, wherein the initial total sugar content in the fermentation medium is controlled in the range of 3% to 5% (w / v), the initial pH is 4.5 to 6.5, and sterilization is carried out after uniform mixing, under sterilization conditions of 115°C to 121°C and 15 to 30 minutes.

7. 4. The method of claim 3, wherein the solids content of the carrot juice is 6.0° Brix.

8. 4. The umami seasoning prepared by the method according to claim 3, wherein the umami seasoning contains a fermentation product obtained by mixing the thermosensitive flavor-producing Saccharomyces cerevisiae according to claim 1 with Corynebacterium glutamicum CCTCC NO: M 2024431.

9. 9. The umami seasoning according to claim 8, wherein the product has a glutamic acid concentration of 80 to 156 g / L and a total nucleotide concentration of 250 mg / L or more.

10. The umami seasoning according to claim 8, wherein the product further contains β-glucan and organic acids.

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