Schizosaccharomyces japonicus capable of efficiently metabolizing malic acid and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to schizosaccharomyces japonicus capable of efficiently metabolizing malic acid and an application of the schizosaccharomyces japonicus. The schizosaccharomyces japonicus strain FBKL2.9SZJ-37 capable of efficiently metabolizing malic acid is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 20251245, the preservation time is June 4, 2025, the strain shows the efficient malic acid metabolizing ability during fermentation, 2.959 g / L of malic acid is consumed after the strain is cultured in an MDL screening culture medium for 7 days, and the highest degradation rate in all strains is 98.64%; 2.965 g / L of malic acid is consumed after fermentation of a simulated malic acid fermentation medium (SMF) for 16 days, and the degradation rate of malic acid is 98.83%; the malic acid consumption after fermentation of the cabernet sauvignon, the rosa sterilis and the rosa roxburghii tratt is 2.93 g / L, 28.73 g / L and 32.40 g / L respectively, and the malic acid degradation rates are 98.32%, 60.50% and 64.98% respectively. The strain is excellent in fermentation performance, stable in ethanol yield, high in tolerance, relatively high in glycerol yield and relatively low in volatile acid level.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms and relates to a high-efficiency malate-metabolizing Schizosaccharomyces japonicus and application thereof. BACKGROUND

[0002] In the process of fruit wine brewing, malic acid is one of the main organic acids in fruit wine, and its content and metabolic state have an important influence on the flavor and taste of fruit wine. High content of malic acid will cause the fruit wine to be astringent and sour, affecting its quality and palatability, especially for fruit wine brewed from high-acid fruits such as kiwi and hawthorn. Malic acid reduction treatment is an important step to improve the quality. At present, the commonly used methods for malic acid reduction in fruit wine brewing mainly include physical malic acid reduction (such as freezing precipitation), chemical malic acid reduction (calcium carbonate treatment, etc.) and biological malic acid reduction. Although the physical and chemical methods have significant malic acid reduction effect, the physical malic acid reduction method has low efficiency and is easy to lose flavor substances, and the chemical malic acid reduction method may introduce other chemical substances, affecting the natural properties and flavor of fruit wine. In contrast, the biological malic acid reduction method reduces the content of malic acid through microbial metabolism, and has the advantages of green, environmental protection, safety, etc.

[0003] However, the current mainstream biological malic acid reduction technology still has many limitations. For example, malic acid-lactic acid fermentation (MLF) is a commonly used biological malic acid reduction method in the grape wine industry, which relies on lactic acid bacteria to convert malic acid into lactic acid. Although this method can effectively reduce the acidity, it has problems such as long fermentation period, sensitivity to environmental conditions (such as pH, SO2 concentration), and easy production of undesirable flavors (such as diacetyl, acetic acid, etc.). In addition, although lactic acid bacteria show high efficiency in biological malic acid reduction, their metabolic process may introduce bacterial contamination, affecting the microbial stability and flavor consistency of fruit wine. Therefore, researchers have begun to explore the use of yeast strains for biological malic acid reduction. Yeast bacteria not only can perform alcohol fermentation in fruit wine brewing, but also have a certain ability to metabolize malic acid. However, most common Saccharomyces cerevisiae strains have low efficiency in metabolizing malic acid, which cannot effectively reduce the content of malic acid in fruit wine, thereby being difficult to significantly improve the taste and flavor of fruit wine.

[0004] Schizosaccharomyces japonicus is a kind of yeast with unique metabolic characteristics, which shows good acid tolerance and high metabolic activity in the fermentation process. The yeast can efficiently metabolize malic acid through the malic acid-ethanol fermentation (MAF) pathway. Studies have shown that Schizosaccharomyces japonicus can produce a variety of flavor substances during malic acid metabolism, such as ester compounds like ethyl acetate and isoamyl acetate. These substances can impart fruit wine with richer fruity and floral aromas, thereby enhancing the overall flavor of fruit wine. However, wild-type Schizosaccharomyces japonicus often has defects such as slow fermentation speed, weak ethanol production ability, and easy production of hydrogen sulfide, which limits its industrial application. In addition, the acid reduction efficiency of different strains varies significantly, and there is currently a lack of strains that combine high acid reduction rate and excellent fermentation characteristics. The flavor quality of fruit wine is closely related to the balance of acidity. Effective degradation of malic acid not only reduces the astringent sensation, but also promotes the accumulation of other flavor substances such as glycerol and esters, making the wine body more mellow and soft.

[0005] Therefore, developing a Schizosaccharomyces japonicus strain that can efficiently metabolize malic acid while maintaining stable fermentation performance is of great significance for improving the quality of high-acid fruit brewing. SUMMARY

[0006] To overcome the shortcomings of the prior art, the present application provides a Schizosaccharomyces japonicus that efficiently metabolizes malic acid and its application, which is as follows: A Schizosaccharomyces japonicus that efficiently metabolizes malic acid, which is classified as Schizosaccharomyces japonicus, has a laboratory strain preservation library number record of FBKL2.9SZJ-37, is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC M 20251245, and was preserved on June 4, 2025.

[0007] The Schizosaccharomyces japonicus that efficiently metabolizes malic acid is 98.64% after being cultured in MDL screening medium for 7 days, and consumes 2.959 g / L of malic acid; is 98.83% after being fermented in simulated malic acid fermentation medium (SMF) for 16 days, and consumes 2.965 g / L of malic acid; is 98.32%, 60.50%, and 64.98% after being fermented in Cabernet Sauvignon, golden rose and rose, respectively, and consumes 2.93 g / L, 28.73 g / L, and 32.40 g / L of malic acid, respectively.

[0008] The identified characteristics of the high-metabolizing-malic-acid Schizosaccharomyces japonicus strain are: growing on WL medium, the colony is light green round ball-shaped convex with a tray-shaped bottom, and the cell is oval or round rod-shaped; the 5.8S-ITS-RFLP analysis PCR product is 700 bp, and the Hae III enzyme digestion product is 550+150 bp; the 26S D1 / D2 region sequence analysis obtains an effective sequence of 600 bp, and the genotype of seven microsatellite loci is Glb-T2a-G3b-Saa-Syc-11a-12a, specifically, the microsatellite locus GA1 amplification product is 190 bp, the microsatellite locus TG2 amplification product is 280 bp, the microsatellite locus CG3 amplification product is 280 bp, the microsatellite locus SaGAA1 amplification product is 110 bp, the microsatellite locus SyGAA2 amplification product is 110+80 bp, the microsatellite locus C11 amplification product is 170 bp, and the microsatellite locus C12 amplification product is 190 bp.

[0009] Further, the yeast has high tolerance and competitiveness in alcohol fermentation, can coexist with Saccharomyces cerevisiae, and has fast fermentation speed, low residual sugar content (which can be reduced to 2.99 g / L or less), and stable ethanol yield (which can reach 10.9 %vol or more).

[0010] Further, the yeast has high-efficiency degradation of malic acid, specifically through the malic acid-alcohol fermentation (MAF) pathway.

[0011] The above-mentioned high-efficiency-metabolizing-malic-acid Schizosaccharomyces japonicus is applied in wine brewing.

[0012] Further, the wine brewing is fruit wine brewing.

[0013] The above-mentioned high-efficiency-metabolizing-malic-acid Schizosaccharomyces japonicus is applied in degradation of malic acid.

[0014] A wine brewing method, which uses the above-mentioned high-efficiency-metabolizing-malic-acid Schizosaccharomyces japonicus as a fermentation strain.

[0015] Further, the above-mentioned high-efficiency-metabolizing-malic-acid Schizosaccharomyces japonicus is used as a single strain for inoculation and fermentation.

[0016] The beneficial effects of the present application are: 1. The Schizosaccharomyces japonicus strain FBKL2.9SZJ-37 provided by the present application has excellent malic acid metabolism ability, and the acid reduction rate is as high as 98% or more, which is significantly better than the common Saccharomyces cerevisiae and wild-type Schizosaccharomyces japonicus strain in the prior art. The strain can efficiently degrade malic acid through the malic acid-alcohol fermentation (MAF) pathway, quickly reduce the astringent feeling of fruit wine, and improve the taste balance.

[0017] 2、The strain shows high tolerance and competitiveness in alcohol fermentation, can coexist with Saccharomyces cerevisiae, and has fast fermentation speed, low residual sugar content (which can be reduced to 2.99 g / L or less), stable ethanol yield (which reaches 10.9 %vol or more), and overcomes the defects of low fermentation efficiency and weak alcohol production capacity of wild-type Schizochytrium sp.

[0018] 3、The Schizochytrium sp. strain FBKL2.9SZJ-37 provided by the present application is suitable for brewing various high-acid fruits such as kiwi, roxburgh rose, and gold roxburgh rose. Single-strain inoculation can complete the simultaneous acid reduction during the alcohol fermentation process, simplifying the traditional step-by-step fermentation process settings, such as the complex process of first brewing Saccharomyces cerevisiae and then lactic acid bacteria, thereby shortening the production cycle and reducing costs.

[0019] 4、The Schizochytrium sp. strain inoculation method provided by the present application replaces the physical and chemical acid reduction method with biological acid reduction, avoiding the loss of flavor substances or the introduction of chemical additives, and meeting the natural and environmentally friendly food processing trend, thereby improving the market competitiveness of the product.

[0020] 5、The present application provides a Schizochytrium sp. strain FBKL2.9SZJ-37 isolated and screened from natural fermentation liquid of water crystal grapes, which has a preservation number of CCTCC M 20251245. The strain has the advantages of high-efficiency acid reduction, excellent fermentation performance, and wide application, and the strain of the present application provides a new microbial resource and technical solution for fruit wine biological acid reduction. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : Colony morphological characteristics of Schizochytrium sp. FBKL2.9SZJ-37; Figure 2 : Cell morphological characteristics of Schizochytrium sp. FBKL2.9SZJ-37. DETAILED DESCRIPTION

[0022] In order to make the technical solutions and beneficial effects of the present application more clear, the present application will be described in detail below in combination with typical embodiments. It should be particularly noted that the following embodiments are only preferred embodiments of the present application, and are not limiting of the present application. Any reasonable adjustment or improvement made by a person skilled in the art based on the core idea of the present application should be included within the protection scope of the present application.

[0023] Example 1 Isolation and identification of Schizochytrium sp. FBKL2.9SZJ-37 1、Strain isolation and purification Samples were collected from crystal grapes in Sandu County and Xifeng County, Guizhou Province, during the natural fermentation process. The specific collection period was from July to September 2020, and 63 wild *Schizosaccharomyces nigra* strains were isolated from crystal grapes in Sandu and Xifeng counties. These samples covered grapes at different ripening stages to ensure strain diversity and representativeness. The collected grape samples underwent natural fermentation. After crushing, the grapes were placed in sterile fermentation bottles for sealed fermentation. During fermentation, the progress was monitored by monitoring carbon dioxide release. In the mid-to-late stages of fermentation (approximately 10 days), samples were taken for microbial isolation. Yeast cells were isolated from the fermentation samples using culturable isolation techniques. The fermentation broth was diluted and spread onto WL medium, then incubated at 28°C. After 3-4 days of incubation, colony morphology was observed, and single colonies with typical yeast colony characteristics were selected for purification. Multiple streak plating was performed to ensure the acquisition of pure cultured yeast strains. From the isolated yeast strains, strains with significant acid-reducing abilities were selected and numbered. Among them, strain FBKL2.9SZJ-37 showed excellent malic acid metabolism and fermentation performance and was selected as the focus of subsequent research.

[0024] 2. Strain identification 2.1 Morphological identification The FBKL2.9SZJ-37 strain was inoculated onto WL medium, and its colony morphology was observed. The results showed that this strain formed green protrusions on WL medium with a flat, tray-like base, and the cells were oval or rod-shaped. These morphological characteristics are consistent with the typical characteristics of *Schizosaccharomyces nigra*.

[0025] 2.2 Molecular identification 5.8S-ITS-RFLP analysis: PCR amplification was performed targeting the 5.8S-ITS region, and the amplification product size was 700 bp. The PCR product was digested with Hae III restriction endonuclease, and the digestion product size was 550 bp + 150 bp.

[0026] 26S rRNA gene D1 / D2 region sequence analysis: Representative strains were selected for 26S rRNA gene D1 / D2 region sequence analysis, and the amplified product size was 600 bp. The sequencing results were compared with the sequence of type strains in the GenBank database. The sequence similarity of strain FBKL2.9SZJ-37 was higher than 99%, further confirming that it is a fission yeast of Japan.

[0027] 2.3 Microsatellite locus analysis Design of microsatellite loci. The whole genome sequence of S. pombe was analyzed by Krait software, and 7 pairs of microsatellite primers with high polymorphism and good specificity were designed, including GA1, TG2, CG3, SaGAA1, SyGAA2, C11 and C12. Through microsatellite locus analysis, the genotype of FBKL2.9SZJ-37 strain was confirmed as Glb-T2a-G3b-Saa-Syc-11a-12a.

[0028] Example 2 Verification of malate degradation ability of S. pombe FBKL2.9SZJ-37 1. MDS and MDL medium screening experiment The strain FBKL2.9SZJ-37 was inoculated in MDS medium containing 3 g / L malic acid (composition: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, L-malic acid 3 g / L, (NH4)2SO4 5 g / L, bromophenol blue 0.4 g / L, agar 20 g / L). After 7 days of culture at 28°C, a clear blue halo appeared around the colonies, indicating that the pH increased and caused the bromophenol blue to develop, indicating that the malic acid was efficiently degraded. Other S. pombe strains also showed acid-reducing color development, while the control strain S. cerevisiae CECA did not show a blue halo, indicating its weak malic acid degradation ability. FBKL2.9SZJ-37 was inoculated in MDL liquid medium (composition same as MDS, without agar) with an initial inoculum of 10 6 cells / mL, and after 7 days of culture at 28°C, the malic acid degradation rate was detected to be 98.64% (Table 1).

[0029] 2. Study of fermentation characteristics in simulated fermentation To further verify the malic acid degradation ability of strain FBKL2.9SZJ-37 in actual fermentation and its fermentation characteristics, and to compare it with commercial S. cerevisiae CECA, this study designed malic acid simulation fermentation (SMF) and mixed acid simulation fermentation (SMTF) experiments. The fermentation conditions were set as follows: malic acid simulation fermentation (SMF): the fermentation medium composition included 100 g / L glucose, 100 g / L fructose, 10 g / L yeast extract, 1 g / L (NH4)2SO4, 1 g / L MgSO4·7H2O, 3 g / L L-malic acid, and the pH was adjusted to 3.3. FBKL2.9SZJ-37 was inoculated at 10 6The FBKL2.9SZJ-37 strain was inoculated into 150 mL of fermentation medium at an inoculum of 1.5 x 107cells / mL in a 250 mL flask and subjected to fermentation at 28°C. Simulated mixed-acid fermentation (SMTF): The fermentation medium was similar to SMF, but the concentrations of L-malic acid and tartaric acid were both 3 g / L, and the pH was adjusted to 3.3. The fermentation conditions and inoculum were consistent with those of SMF.

[0030] 3.1 Fermentation rate detection The weight loss of carbon dioxide during fermentation was monitored by weighing method to evaluate the fermentation rate. During fermentation, the weight of the fermentation flask was weighed every 24 hours, and the cumulative weight loss of carbon dioxide was recorded. The results showed that the fermentation rate of FBKL2.9SZJ-37 was faster in malic acid simulated fermentation, the fermentation time was shorter, and the total carbon dioxide weight loss was higher, showing good fermentation performance.

[0031] 3.2 Alcohol content detection The alcohol content in the fermentation broth was determined by alcohol meter method. The results showed that the ethanol content produced by FBKL2.9SZJ-37 in malic acid simulated fermentation was stable and high, reaching 10.9%vol, which was comparable to that of Saccharomyces cerevisiae CECA and higher than that of wild-type Schizochytrium sp. FBKL2.9SZJ-43. This indicated that FBKL2.9SZJ-37 had comparable performance to commercial yeast CECA in alcoholic fermentation.

[0032] 3.3 Residual sugar content detection The residual sugar content in the fermentation broth was determined by direct titration method. The results showed that after fermentation of FBKL2.9SZJ-37, the residual sugar content in malic acid simulated fermentation was less than 2.99 g / L, indicating complete fermentation and high sugar metabolism capacity of the strain. In contrast, the residual sugar content of CECA was higher, indicating relatively weak sugar metabolism capacity.

[0033] 3.4 Malic acid degradation rate analysis The malic acid degradation rates of Schizochytrium sp. FBKL2.9SZJ-37 and commercial yeast CECA in simulated malic acid fermentation (SMF) and simulated mixed-acid fermentation (SMTF) are shown in Table 1. It can be seen that Schizochytrium sp. FBKL2.9SZJ-37 showed excellent malic acid degradation ability under simulated fermentation conditions, with a malic acid degradation rate much higher than that of commercial yeast CECA.

[0034] Table 1 Analysis of strain characteristics and fermentation performance of Schizochytrium sp. FBKL2.9SZJ-37

[0035] Note: The lower-case superscript letters represent the significant difference analysis between the two strains The study further compared and analyzed the change rule of malic acid content in the fermentation process of Japanese fission yeast and commercial Saccharomyces cerevisiae (Table 2). The results showed that the acid reduction was mainly concentrated in the first 10 days of fermentation, and the acid reduction rate of Japanese fission yeast was much higher than that of Saccharomyces cerevisiae. The acid reduction level of Japanese fission yeast at the second day of fermentation (SMTF) or the fourth day of fermentation (SMF) was similar to that of Saccharomyces cerevisiae at the end of fermentation.

[0036] Table 2 Change of malic acid content (g / L) of Japanese fission yeast FBKL2.9SZJ-37 and commercial yeast CECA at different fermentation stages

[0037] In summary, FBKL2.9SZJ-37 not only performs well in malic acid degradation, but also has significant advantages in alcohol fermentation and sugar metabolism, making it a very potential microbial resource in fruit wine brewing. Its high-efficiency malic acid degradation ability and good fermentation performance can effectively improve the quality of fruit wine, reduce the astringent feeling, enhance the mellow and softness of the wine body, and thus meet the needs of consumers for high-quality fruit wine.

[0038] Example 3 A Japanese fission yeast strain with high-efficiency malic acid degradation for application in fruit wine development; the Japanese fission yeast with high-efficiency malic acid degradation is classified as Schizosaccharomyces japonicus, and the laboratory strain preservation center number is recorded as FBKL2.9SZJ-37, which is preserved in the China Center for Type Culture Collection, with the preservation number being CCTCC M 20251245 and the preservation time being June 4, 2025.

[0039] A fruit wine brewing method, comprising the following steps: Fruit crushing; 2) Enzymatic hydrolysis; 3) Inoculating the Japanese fission yeast strain with high-metabolic malic acid into crushed and enzymatically hydrolyzed grape mash and Rosa roxburghii Tratt mash and Rosa roxburghii Tratt juice, and carrying out alcohol fermentation, with the fermentation process temperature being controlled at 28℃, and the CO2 generation being monitored by weighing method, and the fermentation endpoint being considered when the weight loss is <0.2 g / d; 4) After the fermentation is completed, filtering and centrifuging with sterile gauze, and measuring the malic acid content after standing.

[0040] The inoculation amount of the Japanese fission yeast strain with high-metabolic malic acid is 106 cells / mL.

[0041] Schizosaccharomyces japonicus FBKL2.9 SZJ-37 showed high acid reduction (acid reduction rates were 98.32%, 60.50%, 64.98% respectively) and good fermentation performance in Cabernet Sauvignon wine grape, golden pear, pear wine fermentation through malate-alcoholic fermentation (MAF) pathway. The results showed that the strain could effectively reduce the content of malic acid in fruit wine, significantly improve the taste and flavor of fruit wine, and improve the overall quality of fruit wine under the condition of single strain inoculation. Especially in high acid fruits (such as golden pear, pear), its acid reduction effect was significantly better than that of Saccharomyces cerevisiae, providing efficient strain resources for biological acid reduction of high acid fruit wine.

[0042] Table 3 Changes in the content of malic acid and tartaric acid during the fermentation of three fruits inoculated with Schizosaccharomyces japonicus FBKL2.9 SZJ-37

[0043] Table 4 Fermentation characteristics of Schizosaccharomyces japonicus FBKL2.9 SZJ-37 strain in the fermentation of three fruits

[0044] Note: The superscript lowercase letters represent the significance difference analysis between different fermentations of the same index Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred from the content disclosed in the present application by those of ordinary skill in the art should be considered as falling within the scope of protection of the present application.

Claims

1. A high-metabolic-malic-acid-producing Schizosaccharomyces japonicus, characterized by, It is preserved in China Center for Type Culture Collection, the preservation number is CCTCC M 20251245, and the preservation time is June 4, 2025.

2. The high-metabolizing-malic-acid-producing Ogataea polymorpha according to claim 1, characterized by, The yeast has high tolerance and competitiveness in alcohol fermentation, can coexist with Saccharomyces cerevisiae, and has fast fermentation speed, low residual sugar content, low volatile acid content, high glycerol yield and stable ethanol yield.

3. The high-metabolic-malate-producing S. japonicus according to claim 1, characterized by, The yeast has the property of efficiently degrading malate, specifically through the malate-alcohol fermentation pathway.

4. The *Schizosaccharomyces niger* strain with high malic acid metabolism as described in claim 1, characterized in that, The identified characteristics of the high-metabolic-malate Schizosaccharomyces japonicus strain are: growing on WL medium, the colony is a light green spherical convex with a tray-shaped bottom, and the cell is oval or round rod-shaped. 5.8S ITS RFLP analysis PCR product is 700bp, HaeIII enzyme digestion product is 550+150bp; 26S D1 / D2 region sequence analysis obtains 600bp effective sequence, seven microsatellite site analysis its genotype is Glb T2a G3b Saa Syc 11a 12a, specifically, microsatellite site GA1 amplification product is 190bp, microsatellite site TG2 amplification product is 280bp, microsatellite site CG3 amplification product is 280bp, microsatellite site SaGAA1 amplification product is 110bp, microsatellite site SyGAA2 amplification product is 110+80bp, microsatellite site C11 amplification product is 170bp, and microsatellite site C12 amplification product is 190bp.

5. The high-malate-metabolizing Schizosaccharomyces japonicus of claim 1 is applied in wine brewing.

6. Use according to claim 5, characterized in that, The wine brewing is fruit wine brewing.

7. The high-malate-metabolizing Schizosaccharomyces japonicus of claim 1 is applied in degrading malate.

8. A method of brewing an alcoholic beverage, characterized by, The high-malate-metabolizing Schizosaccharomyces japonicus of claim 1 is used as a fermentation strain.

9. The brewing method according to claim 8, characterized by, The high-malate-metabolizing Schizosaccharomyces japonicus of claim 1 is used as a single-strain inoculation fermentation.

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