Temperature-sensitive aroma-producing saccharomyces cerevisiae and use thereof in preparation of flavor enhancer

Abstract

A temperature-sensitive aroma-producing Saccharomyces cerevisiae and use thereof in the preparation of a flavor enhancer. The temperature-sensitive aroma-producing Saccharomyces cerevisiae was deposited in the China Center for Type Culture Collection on May 31, 2024, under the accession number of CCTCC NO: M 20241116. By means of using the temperature-sensitive aroma-producing Saccharomyces cerevisiae and Corynebacterium glutamicum (accession number: CCTCC NO: M 2024431) for mixed fermentation, a fermentation broth containing high concentrations of glutamic acid and nucleotides can be obtained. Meanwhile, the fermentation broth also contains various other functional nutrients, such as amino acids, organic acids, and β-glucan, and has a higher functional nutritional value.

Description

A temperature-sensitive aroma-producing brewing yeast and its application in the preparation of umami agents Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a temperature-sensitive aroma-producing brewing yeast and its application in the preparation of umami agents. Background Technology

[0002] With the improvement of living standards, people's demand for natural flavor enhancers is increasing. At present, a variety of production methods for natural flavor enhancers have emerged, but there are still some shortcomings: (1) Corynebacterium glutamicum requires pure culture fermentation, and the product is monosodium glutamate, which has a single composition and insufficient flavor; (2) Two-step fermentation technology has been adopted by some people, which mainly uses mold and bacteria fermentation. The steps are complicated, easy to be contaminated by bacteria and have high cost.

[0003] Chinese invention patent application 202180054828.3 discloses a flavor-enhancing composition containing glutamate from plants of the Apiaceae family and having a flavor-effective glutamate content, and a method for producing a flavor enhancer by reducing biotin in fermentable carbohydrates prior to bacterial fermentation. The method uses carrot juice, carrot puree, carrot pomace, or combinations thereof as raw materials, and utilizes microorganisms such as *Corynebacterium glutamicum*, *Corynebacterium ammoniagenes*, *Corynebacterium casei*, *Brevibacterium lactofermentum*, *Bacillus subtilis*, or combinations thereof for fermentation to produce the flavor enhancer. In addition to glutamate, the flavor enhancer also contains organic acids including lactic acid, citric acid, acetic acid, succinic acid, and combinations thereof. While this method improves fermentation efficiency, it still fails to address the issues of simple umami components and insufficient flavor. Furthermore, this flavor enhancer still requires the addition of exogenous auxiliary components 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 components undermines the product's pure, natural, and green image. Invention patent WO 2015 / 020292 utilizes mold (such as Aspergillus sojae CJCC_080124P(KCCM11026P)) to ferment plant proteins (such as soybean protein, wheat protein, corn protein, etc.) to produce flavor-enhancing amino acids and peptides. After adding carbon sources, phosphorus sources, and trace elements, further fermentation with bacteria (such as Corynebacterium ammonium chloride CJIP009(KCCM-10226), Corynebacterium glutamicum, Bacillus, etc.) produces even more umami substances. While this method can increase the variety of fermentation products and improve freshness and flavor, it suffers from problems such as cumbersome procedures and high production costs. Chinese invention patent 201410790020.3 discloses a brewing yeast strain with excellent anti-autolysis properties. This strain has a cell wall twice as thick as the original strain. Simulated autolysis experiment data and 60-hour scanning electron microscopy results both show that the autolysis performance of the recombinant strain is significantly better than that of the original strain, indicating good application value in improving beer flavor, foam stability, and other quality aspects. However, because this strain has been genetically modified, it cannot currently be used in food production.Chinese invention patent application 201610538817.3 discloses a self-dissolving process for producing highly active yeast autolysates. The process involves mixing a specific single-cell protein (which retains its original endogenous enzyme activity without sterilization) with yeast in a specific ratio, followed by autolysis, drying, and sterilization. The autolysis process is divided into a low-temperature stage (45–60°C for 20–60 minutes) and a high-temperature stage (75–95°C for 30–120 minutes). This technology suffers from high processing temperatures, high energy consumption, and a tendency to damage the flavor of the product.

[0004] Therefore, establishing a low-cost, simple-to-operate, and flavorful natural umami agent preparation technology is a key issue that urgently needs to be addressed. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a temperature-sensitive aroma-producing brewing yeast and its application in the preparation of umami agents. This yeast can be used to prepare umami agents. Using carrot juice as the main raw material, the yeast is mixed with Corynebacterium glutamicum for fermentation to obtain products such as glutamic acid and flavor nucleotides. This reduces production costs, improves production efficiency, and yields natural umami agents with higher umami content and richer flavor.

[0006] The technical solution adopted by the present invention to solve its technical problem is: a temperature-sensitive aroma-producing brewing yeast Saccharomyces cerevisiae, which was deposited at the China Center for Type Culture Collection on May 31, 2024, with accession number CCTCC NO: M 20241116.

[0007] The present invention also provides the application of the yeast in the preparation of umami agents, wherein the yeast is mixed with Corynebacterium glutamicum for fermentation, and carrot juice is used as the main raw material to prepare umami agents.

[0008] The present invention further provides a method for preparing the umami agent, comprising:

[0009] (1) Inoculate the activated temperature-sensitive aroma-producing brewing yeast into the sterilized fermentation medium; control the fermentation temperature at 28±1℃, the pH range at 4.5~6.5, control the dissolved oxygen at 10%~30% by combining the rotation speed and ventilation, and the fermentation time at 12 hours~24 hours;

[0010] (2) When the yeast wet weight reaches 50 g / L or more and the total sugar drops to below 0.5% (w / v), add pre-sterilized carrot juice and concentrated sugar solution to the fermenter to make the total sugar reach 7% (w / v) or more. Raise the temperature to 40℃~75℃ and maintain it for 30 minutes to 120 minutes. Then quickly cool it down to 30℃ and inoculate it with fresh activated Corynebacterium glutamicum CCTCC NO: M 2024431. Control the mixed fermentation temperature at 30℃-33℃, control the dissolved oxygen at 30%~50% by combining rotation speed and ventilation, control the pH at 6.5~8.0 during fermentation, and continue fermentation for 12 hours to 24 hours.

[0011] (3) After fermentation, raise the temperature to 100℃ and maintain it for 10 to 60 minutes.

[0012] Preferably, during the mixed fermentation process, when the total sugar content drops to 0.2% to 2.0% (w / v), sugar replenishment begins. The added sugar is selected from one or more of glucose, sucrose, fructose, corn syrup, and high-fructose corn syrup in any ratio. The sugar solution added is in a sugar:water ratio of 1:1 by weight.

[0013] Preferably, the fermentation medium uses carrot juice as the main raw material and includes the following components in the following mass ratios: 500-1000 g / kg carrot juice, 5.0-15 g / kg potassium dihydrogen phosphate, 0.05-2.0 g / kg magnesium sulfate heptahydrate, 0.005-0.2 g / kg ferrous sulfate heptahydrate, 0.001-0.2 g / kg manganese sulfate monohydrate, 0-3.0 g / kg aspartic acid, and 0-3.0 g / kg threonine.

[0014] Preferably, the initial total sugar content in the fermentation medium is controlled within the range of 3% to 5% (w / v), the initial pH is 4.5 to 6.5, and the mixture is sterilized after thorough mixing. The sterilization conditions are 115℃ to 121℃ and the time is 15 minutes to 30 minutes.

[0015] Preferably, the solids content of the carrot juice is calculated as 6.0°Brix.

[0016] The present invention further provides an umami agent prepared using the method described above, wherein the umami agent comprises the product of mixed fermentation of the aroma-producing brewing yeast and Corynebacterium glutamicum CCTCC NO: M 2024431.

[0017] Preferably, the concentration of glutamic acid in the product is 80-156 g / L and the concentration of total nucleotides is 250 mg / L or more.

[0018] Preferably, the product further comprises β-glucan and organic acids.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The temperature-sensitive aroma-producing brewing yeast provided by the present invention has the characteristic of high-temperature autolysis when generated in carrot juice, that is, it can grow and proliferate normally at 25℃~28℃, and grows best at 28℃. When the temperature exceeds 30℃, growth is significantly inhibited. When the temperature reaches above 35℃, the cell walls begin to break and autolyze, releasing intracellular RNA and protein and other endosomal substances at the same time.

[0020] This invention employs a mixed fermentation method using a temperature-sensitive aroma-producing brewing yeast and Corynebacterium glutamicum (preservation number CCTCC NO: M 2024431) to obtain a fermentation broth containing high concentrations of glutamic acid and nucleotides. This broth also contains various other amino acids, organic acids, β-glucan, and other functional nutrients. At the end of the mixed fermentation, the glutamic acid concentration in the resulting broth can reach over 80 g / L (up to 156 g / L), and the total nucleotide concentration can reach 250 mg / L or higher. These two substances synergistically enhance the umami flavor, resulting in a stronger, richer, and more harmonious taste compared to fermentation broth made solely with glutamic acid, and possessing a more diverse composition and higher functional nutritional value. Attached Figure Description

[0021] Figure 1 shows the colony morphology of the temperature-sensitive aroma-producing brewing yeast provided in the embodiment of the present invention;

[0022] Figure 2 shows the growth curve of the temperature-sensitive aroma-producing brewing yeast provided in the embodiment of the present invention. Detailed Implementation

[0023] To facilitate understanding of the present invention, it will be described in more detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0024] Example 1: This invention provides a temperature-sensitive aroma-producing brewing yeast, which is derived from the ocean.

[0025] I. Screening and Isolation of Strains

[0026] (1) Isolation and purification of bacterial strains: Representative seaweeds (mainly kelp, Sargassum, and Ulva) were collected from the waters off Qingdao, Shandong Province. The collected samples were then quickly placed into sterile sampling bottles. Under sterile laboratory conditions, the seaweed samples were sampled at multiple points and then immersed in sterile physiological saline with shaking. After a certain period of time, the samples were serially diluted to prepare bacterial suspensions of different concentrations. The bacterial suspensions of each gradient were sequentially spread on nutrient agar (NA), MRS agar, potato dextrose agar (PDA), and yeast extract peptone dextrose agar (YPDA) media and cultured under different conditions to isolate microbial resources such as bacteria, yeast, and mold. Ten single colonies, including this strain, were isolated from the YPDA plates. Three yeast strains were also isolated. After purification by streak plating, the colonies were streaked onto test tube slants and stored at 4°C.

[0027] (2) Screening of yeast strains: Three yeast strains, including Y01, were initially selected for analysis of their growth and reproduction capabilities in a specific culture medium (sterilized carrot juice), sensory characteristics of the fermentation broth, strain RNA content, and mixed fermentation performance. The final results showed that, compared with other strains, this strain exhibited temperature sensitivity, rapid reproduction in carrot juice culture medium, and a distinct, pleasant aroma in the fermentation broth. Furthermore, the strain had a high RNA content and could coexist and co-ferment with *Corynebacterium glutamicum* (preservation number CCTCC NO: M 2024431). Therefore, this strain was selected for the preparation of umami enhancers.

[0028] II. Molecular biological identification and biological preservation

[0029] The selected strains were identified using molecular biology techniques, and their 18S rDNA nucleotide sequences are shown in SEQ ID No. 1 of the sequence listing. Sequence homology analysis by the National Center for Biotechnology Information (NCBI) was performed, and the 18S rDNA nucleotides of the aroma-producing Saccharomyces cerevisiae of this invention were compared using a BLAST tool. The comparison results showed that Y01 belongs to Saccharomyces cerevisiae.

[0030] The yeast strain Y01 was deposited at the China Center for Type Culture Collection (CCTCC) on May 31, 2024, with accession number CCTCC NO: M 20241116. The address of the depository is Wuhan University, Wuhan, Hubei Province.

[0031] III. Determination of Culture Characteristics, Biological Features and Functional Properties

[0032] 1. Biological characteristics:

[0033] This strain is easy to culture and grows rapidly, proliferating via budding under normal culture conditions. When cultured on YPDA medium, typical colonies form in approximately 30 hours; these colonies are milky white, flat, moist, and easily picked up, as shown in Figure 1. A distinct floral and fruity aroma can be detected with prolonged culture. The strain exhibits a wide pH adaptability, growing within a range of pH 3.5–7.5, meaning it can maintain a high growth and reproduction rate even at the optimal pH for Corynebacterium glutamicum (~7.0).

[0034] 2. Cultivation characteristics:

[0035] Experimental studies have shown that this yeast strain can utilize various carbon sources, including glucose, fructose, sucrose, mannitol, and galactose, with glucose showing the highest utilization efficiency, followed by fructose and sucrose. The strain can grow normally at 25℃–30℃, with optimal proliferation around 28℃. When the temperature exceeds 30℃, the reproduction rate begins to decrease, and the cell structure begins to deform. When the temperature continues to rise to 35℃ or higher, cell autolysis begins, indicating that the strain is temperature-sensitive. The strain can grow at pH 3.5–7.5, with optimal growth at pH 4.5–5.0. In shake-flask culture, the maximum cell concentration initially increases and then decreases with increasing rotation speed, with the optimal rotation speed being 180 rpm. When cultured in pure carrot juice (6.0°Brix), the lag phase is approximately 4 hours. From 6–8 hours, the strain enters the logarithmic phase, proliferating rapidly. The biomass reaches its maximum at 14–16 hours, after which the growth rate slows down, gradually entering the stationary phase.

[0036] Figure 2 shows the growth curve of this strain under optimal temperature and rotation speed conditions (the culture medium formula by weight is: 1% yeast extract, 2% peptone, and 2% glucose).

[0037] 3. Functional characteristic testing:

[0038] The yeast strain of the present invention has good aroma-producing characteristics. When it is cultured alone in carrot juice, the culture medium has obvious floral and fruity aroma. When it is mixed with Corynebacterium glutamicum for fermentation, in addition to floral and fruity aroma, it also has new aromas such as milky and sweet aroma.

[0039] RNA content determination:

[0040] (1) After yeast culture, centrifugation and drying are used to obtain dry yeast powder;

[0041] (2) Weigh about 2 grams of dry yeast powder (m0) and suspend it in 12 ml of 0.04 mol / L sodium hydroxide solution, and grind it evenly in a mortar;

[0042] (3) Transfer the bacterial suspension to a test tube, heat in a boiling water bath for 30 minutes, cool and then transfer to a centrifuge tube, centrifuge at 4000 rpm for 10 minutes;

[0043] (4) Slowly add the supernatant to 4 ml of acidic ethanol while mixing. After adding, let stand until the RNA is completely precipitated, then centrifuge at 3000 rpm for 5 minutes and discard the supernatant;

[0044] (5) Wash the precipitate twice with 95% ethanol, and then wash the precipitate once with ether;

[0045] (6) Transfer the precipitate to a Buchner funnel with ether and filter it. Dry the precipitate in air and weigh the crude RNA (m1, g).

[0046] (7) Prepare a solution of 5–50 μg / L for the sample, measure the absorbance at 260 nm and 280 nm, and calculate the RNA content (%). The calculation formula is as follows: RNA content (%) = (RNA content × m1) / (1000 × m0) × 100%; RNA content (μg / g) = (OD) / (m1 / m0) × 100%; 260 (×dilution factor) / (0.024×L);

[0047] Where: OD 260 λ represents the absorbance of the sample; m0 represents the mass of the dry yeast powder (g); L represents the thickness of the cuvette; 0.024 represents the absorbance at 1 μg RNA / mL.

[0048] Measurements showed that the RNA content of the yeast strains of this invention can reach more than 10% (w / w), while the RNA content of the control strains (commercial baker's yeast strains BY1 and BY2) cultured simultaneously was only 5%-7% (w / w).

[0049] Example 2 This example describes a method for preparing a natural umami enhancer by fermenting carrot juice with a mixture of aroma-producing brewer's yeast and Corynebacterium glutamicum. The specific steps are as follows:

[0050] (1) Raw material preparation and strain activation

[0051] (i) Preparation of carrot juice

[0052] Select fresh, non-softened and non-rotten carrots as raw materials. After cleaning, peel, trim and cut into chunks. Pre-cook for 5-10 minutes, then transfer to a juicer to grind into a pulp. After the resulting carrot pulp is separated from the pulp, it is sterilized and tested to ensure it is qualified for use. See Table 1.

[0053] Table 1. Quality Inspection Items and Results of Carrot Juice

[0054] (ii) Activation of microbial strains

[0055] Prepare the yeast seed culture medium: 300 g / L carrot juice (6.0°Brix solids), initial total sugar 1.65% (w / w), 115°C, 15 minutes. After sterilization, cool to a suitable temperature, inoculate with aroma-producing Saccharomyces cerevisiae at a 1% (v / v) inoculation rate, 28°C, 180 rpm, 24 hours. Subculture the resulting culture once in the above medium, with an inoculation rate of 5% (v / v), and incubate for 18 hours before use.

[0056] Prepare the seed culture medium for Corynebacterium glutamicum: 200 g / L carrot juice (solids as 6.0°Brix), 20 g / L sugar (monosaccharide - 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; adjust the initial pH to 7.5 with ammonia, resulting in an initial total sugar content of 2.9% (w / v). Mix thoroughly and sterilize at 115°C for 30 minutes. After sterilization, cool to a suitable temperature and inoculate with Corynebacterium glutamicum (CCTCC No. M 2024) at a 1.2% inoculum, incubating at 32°C and 200 rpm for 16 hours. Subculture the resulting strain once in the above medium at a 5% (v / v) inoculum for 12 hours before use.

[0057] (2) Fermentation

[0058] (i) Preparation of fermentation medium: 500 g / kg carrot juice (solids at 6.0°Brix), 5.0 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, 1.5 g / kg aspartic acid, 1.5 g / kg threonine, 121°C, 20 minutes.

[0059] Prepare a separate concentrated sugar solution (sugar:water = 1:1 by weight) and store it in a sterilized tank for later use.

[0060] (ii) Inoculation and fermentation

[0061] The yeast seed culture obtained in step (1) (i) above was inoculated into the fermentation medium at an inoculation rate of 3% (v / v). During fermentation, the pH was controlled at 4.5, and the dissolved oxygen was controlled at 10% by a combination of agitation and ventilation. The fermentation time was 12 hours. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermenter through pipes to bring the total sugar content to 7% (w / v). The temperature was raised to 40°C and maintained for 120 minutes, then rapidly cooled to 30°C. Freshly activated Corynebacterium glutamicum was inoculated. During fermentation, the temperature was controlled at 31±1°C, and the dissolved oxygen was controlled at 30% by a combination of agitation and ventilation. The pH was controlled at 6.5 during fermentation. Fermentation continued, and when the total sugar content dropped to 0.2% (w / v), sugar was added continuously. Fermentation continued for 24 hours and then stopped at 100°C for 100 minutes.

[0062] (3) Detection of fermentation broth

[0063] After removing bacteria from the fermentation broth obtained in step (2) of Example 2, relevant indicators were tested, and the test results are shown in Table 2.

[0064] Table 2. Detection of Fermentation Broth Components Obtained in Example 2

[0065] Example 3 This example describes a method for preparing a natural umami enhancer by fermenting carrot juice with a mixture of aroma-producing brewer's yeast and Corynebacterium glutamicum. The specific steps are as follows:

[0066] (1) Raw material preparation and strain activation are the same as in Example 1.

[0067] (2) Fermentation

[0068] (i) Preparation of fermentation medium: 700 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, 0.5 g / kg threonine, 121℃, 20 minutes.

[0069] Prepare a separate concentrated sugar solution (sugar:water = 1:1 by weight) and store it in a sterilized tank for later use.

[0070] (ii) Inoculation and fermentation

[0071] The yeast seed culture obtained in step (1) (i) above was inoculated into the fermentation medium at an inoculation rate of 5% (v / v). During fermentation, the pH was controlled at 5.5, and the dissolved oxygen was controlled at 25% by a combination of agitation and ventilation. The fermentation time was 12 hours. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermenter through a pipeline to bring the total sugar content to 12% (w / v). The temperature was raised to 75°C and maintained for 30 minutes, then rapidly cooled to 30°C. Freshly activated Corynebacterium glutamicum was inoculated. During fermentation, the temperature was controlled at 31±1°C, and the dissolved oxygen was controlled at 50% by a combination of agitation and ventilation. The pH was controlled at 7.5 during fermentation. Fermentation continued, and when the total sugar content dropped to 0.2% (w / v), sugar was added continuously. Fermentation continued for 24 hours and then stopped at 100°C for 10 minutes.

[0072] (3) Detection of fermentation broth

[0073] After removing bacteria from the fermentation broth obtained in step (2) of Example 3, relevant indicators were tested, and the test results are shown in Table 3.

[0074] Table 3. Detection of Fermentation Broth Components Obtained in Example 3

[0075] Example 4 This example describes a method for preparing a natural umami enhancer by fermenting carrot juice with a mixture of aroma-producing brewer's yeast and Corynebacterium glutamicum. The specific steps are as follows:

[0076] (1) Raw material preparation and strain activation are the same as in Example 1.

[0077] (2) Fermentation

[0078] (i) Prepare the 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), and incubate at 121°C for 20 minutes. Separately prepare a concentrated sugar solution (sugar:water = 1:1 by weight), and store it separately in a sterilized tank for later use.

[0079] (ii) Inoculation and Fermentation: The yeast seed culture obtained in step (1) (i) above was inoculated into the fermentation medium at an inoculation rate of 5% (v / v). During fermentation, the pH was controlled at 5.5, and the dissolved oxygen was controlled at 30% by a combination of agitation and ventilation. The fermentation time was 16 hours. Pre-sterilized carrot juice (≥30°Brix) and concentrated sugar solution were added to the fermenter through pipes to bring the total sugar content to 10% (w / v). The temperature was raised to 55°C and maintained for 30 minutes, then rapidly cooled to 30°C. Freshly activated Corynebacterium glutamicum was inoculated. During fermentation, the temperature was controlled at 31±1°C, and the dissolved oxygen was controlled at 30% by a combination of agitation and ventilation. The pH was controlled at 7.0 during fermentation. Fermentation continued, and when the total sugar content dropped to 1%, sugar was added continuously. After 24 hours of continued fermentation, the temperature was raised to 100°C and maintained for 20 minutes.

[0080] (3) Detection of fermentation broth

[0081] After removing bacteria from the fermentation broth obtained in step (2) of Example 4, relevant indicators were tested, and the test results are shown in Table 4.

[0082] Table 4. Detection of fermentation broth components obtained in Example 4

[0083] Example 5: This example uses Corynebacterium glutamicum Gu2# pure culture to ferment carrot juice to prepare a natural flavor enhancer. The specific steps are as follows:

[0084] (1) Raw material preparation and strain activation

[0085] (i) Carrot juice preparation: Select fresh carrots that are not soft or rotten as raw materials. After cleaning, peel, trim and cut into pieces, pre-cook for 5-10 minutes, transfer to a juicer to grind into pulp, and sterilize the resulting carrot pulp after separating the juice and pulp. After passing the test, it can be used for future use.

[0086] (ii) Activation of microbial strains

[0087] Activation of primary strain: Prepare primary seed culture medium (g / L) according to the following formula, and culture conditions are as follows:

[0088] Carrot juice 300 g / L, sugar (calculated as monosaccharide - glucose) 20 g / L, threonine 0.3 g / L, dipotassium hydrogen phosphate 3.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, manganese sulfate monohydrate 0.01 g / L. Adjust the initial pH to 7.5 with ammonia. Initial total sugar: 3.35% (w / v). Mix thoroughly and sterilize at 115°C for 15 minutes. After sterilization, cool to a suitable temperature before inoculation. Inoculation volume: 2% (v / v), liquid volume: 15% (v / v), incubation conditions: 32°C, 200 rpm, 14 hours.

[0089] Activation of secondary strains: Prepare secondary seed culture medium (g / L) according to the following formula, and culture conditions are as follows:

[0090] Carrot juice 400 g / L, sugar (calculated as monosaccharide - glucose) 30 g / L, 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, manganese sulfate monohydrate 0.01 g / L. Adjust the initial pH to 7.5 with ammonia. Initial total sugar: 5.06% (w / v). Mix thoroughly and sterilize at 115°C for 15 minutes. After sterilization, cool to a suitable temperature before inoculation. Inoculation volume: 10% (v / v), liquid volume: 15%, incubation conditions: 32°C, 200 rpm, 10 hours.

[0091] (2) Fermentation

[0092] The initial fermentation medium consisted of: 800 g / kg carrot juice, 55 g / kg sugar (calculated as monosaccharide - glucose), 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. Antifoaming agent was added as needed. The initial total sugar content in the medium was 9.8% (w / v). After thorough mixing, the medium was sterilized at 115℃ for 30 minutes. After sterilization, the medium was cooled to a suitable temperature, and the initial pH was adjusted to 7.0 using ammonia. Fermentation was initiated at a 10% inoculum size. The fermentation temperature was controlled at 30℃, and dissolved oxygen was controlled at 50% using a combination of agitation and ventilation. The fermentation time was 36 hours. Key pH control during fermentation was as follows: pH 6.5–7.0 during the logarithmic phase, pH 7.0–7.2 during the acid-producing phase, and approximately 7.5 in the last 6–8 hours. During this period, when the total sugar content drops to 1.5% (w / v), sugar supplementation is started to maintain the total sugar content at no less than 1% (w / v), and the sugar-acid conversion rate is controlled at around 50% during the process.

[0093] (3) Detection of fermentation broth

[0094] After removing bacteria from the fermentation broth obtained in step (2) of Example 5, relevant indicators were tested, and the test results are shown in Table 5.

[0095] Table 5. Detection of components in the fermentation broth obtained in Example 5

[0096] The composition and content of substances in the final fermentation broth obtained from the two production processes of pure culture fermentation and mixed culture fermentation of glutamic acid in Example 5 were compared, and the results are shown in Table 6.

[0097] Table 6. Comparison of components in single-strain fermentation broth (Corynebacterium glutamicum) and mixed-strain fermentation broth.

[0098] As shown in Table 6, a fermentation broth containing high concentrations of glutamic acid and nucleotides can be obtained through mixed fermentation with two microorganisms. This broth also contains various other functional nutrients such as amino acids, organic acids, and β-glucan. At the end of the mixed fermentation, the glutamic acid concentration in the broth can reach over 80 g / L (up to 156 g / L), and the total nucleotide concentration can reach 250 mg / L or higher. These two substances synergistically enhance the flavor, resulting in a stronger, richer, and more harmonious taste compared to fermentation broth made solely with glutamic acid, and thus a more diverse and higher functional nutritional value.

Claims

1. A temperature-sensitive aroma-producing Saccharomyces cerevisiae Y01, characterized in that: The yeast is preserved in China Center for Type Culture Collection on May 31, 2024, and the preservation number is CCTCC NO: M 20241116.

2. The use of the temperature-sensitive aroma-producing Saccharomyces cerevisiae according to claim 1 in the preparation of a fresh flavoring agent, characterized in that: The yeast is mixed with Corynebacterium glutamicum CCTCC NO: M 2024431 to ferment, and carrot juice is used as the main raw material to prepare a flavoring agent.

3. The method for preparing a flavor enhancer using the temperature-sensitive aroma-producing Saccharomyces cerevisiae according to claim 1, characterized in that, It comprises: (1) inoculating the activated temperature-sensitive aroma-producing Saccharomyces cerevisiae into sterilized fermentation medium; The fermentation temperature is controlled at 28±1℃, the pH range is 4.5-6.5, the rotation speed is combined with ventilation to control the dissolved oxygen at 10%-30%, and the fermentation time is 12-24 hours; (2) when the wet weight of the yeast reaches 50 g / L or more and the total sugar decreases to 0.5% (w / v) or less, sterilized carrot juice and concentrated sugar solution are supplemented into the fermentation tank to make the total sugar reach 7% (w / v) or more, the temperature is raised to 40-75℃ and maintained for 30-120 minutes, then the temperature is quickly lowered to 30℃, and fresh activated Corynebacterium glutamicum CCTCC NO: M 2024431 is inoculated; the mixed fermentation temperature is controlled at 30-33℃, the rotation speed is combined with ventilation to control the dissolved oxygen at 30%-50%, the pH is controlled at 6.5-8.0 during the fermentation process, and the fermentation is continued for 12-24 hours; (3) after the fermentation is completed, the temperature is maintained at 100℃ for 10-60 minutes.

4. The method of claim 3, wherein, During the mixed fermentation process, the sugar is added when the total sugar decreases to 0.2%-2.0% (w / v), and the added sugar is selected from one or more than two combinations of glucose, sucrose, fructose, corn syrup, and fructose-glucose syrup in any ratio.

5. The method of claim 3, wherein: The fermentation medium uses carrot juice as the main raw material and comprises the following components in the following mass ratio: carrot juice 500-1000 g / kg, potassium dihydrogen phosphate 5.0-15 g / kg, magnesium sulfate heptahydrate 0.05-2.0 g / kg, ferrous sulfate heptahydrate 0.005-0.2 g / kg, manganese sulfate monohydrate 0.001-0.2 g / kg, aspartic acid 0-3.0 g / kg, and threonine 0-3.0 g / kg.

6. The method of claim 3, wherein: The initial total sugar content in the fermentation medium is controlled in the range of 3%-5% (w / v), the initial pH is 4.5-6.5, and the mixture is sterilized after being uniformly mixed, with the sterilization conditions being 115-121℃ for 15-30 minutes.

7. The method of claim 3, wherein, The solids in the carrot juice are 6.0°Brix.

8. A flavoring agent prepared by the method of claim 3, characterized by: The flavoring agent comprises the product of the mixed fermentation of the temperature-sensitive aroma-producing Saccharomyces cerevisiae and Corynebacterium glutamicum CCTCC NO: M 2024431 as claimed in claim 1.

9. The flavor enhancer according to claim 8, characterized in that: The concentration of glutamic acid in the product is 80-156 g / L, and the total nucleotide concentration is 250 mg / L or more.

10. The flavor enhancer according to claim 8, characterized in that: The product further comprises β-glucan and organic acid.

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