Acid-tolerant high-ester-aroma-producing saccharomyces cerevisiae and application thereof
By screening out the acid-resistant Cyberlindnera jadinii strain GJLJ20, the problems of unstable ester formation and fermentation obstacles under high acid conditions were solved, achieving stable and efficient fermentation and flavor enhancement under high acid conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI GUJING DISTILLERY CO LTD
- Filing Date
- 2026-03-25
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, the formation of esters in alcohol production is unstable, resulting in poor flavor output. Furthermore, yeast growth and fermentation efficiency are low in high-acid environments, leading to delayed or stopped fermentation and disordered flavor metabolism.
A resistant acid-tolerant strain of *Cyberlindnera jadinii* GJLJ20 was screened, which can efficiently synthesize ester compounds such as phenylethyl acetate and isoamyl acetate under high acid conditions, and can be used as a food starter to stabilize the fermentation process.
It maintains the growth activity and fermentation efficiency of yeast in a high-acid environment, significantly enhancing the flavor richness and complexity of fermented foods. It is particularly suitable for brewing and fermentation processes in high-acid environments, such as the production of baijiu and fruit wine.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of industrial microbial technology, specifically relating to an acid-resistant yeast that produces high levels of ester aromas, and its applications. Background Technology
[0002] The production of traditional fermented foods (such as baijiu, soy sauce, and vinegar) relies on the synergistic effect of complex microbial systems. During fermentation, various microorganisms secrete a variety of enzyme systems, including oxidoreductases, hydrolases, and transferases, to catalyze the degradation of carbohydrates, proteins, and lipids in the raw materials, forming primary metabolites such as free amino acids, monosaccharides, and fatty acids, which are further converted into flavor compounds such as alcohols, aldehydes, acids, and esters. Yeast plays a crucial role in flavor metabolism and can be divided into two main categories: brewing yeast and non-brewing yeast. Brewing yeast primarily functions in ethanol production, while non-brewing yeast is widely involved in the synthesis and regulation of flavor components such as esters, higher alcohols, and organic acids, making significant contributions to the diversity and complexity of the flavor profile of the beverage.
[0003] Phenylacetyl acetate and isoamyl acetate are key flavor esters in alcoholic beverages, exhibiting aromas of rose and honey, and banana and pear, respectively. Their content directly affects the richness and harmony of the aroma. Currently, the formation of esters in alcoholic beverage production relies mainly on natural microbial communities or the addition of exogenous esterifying enzymes, which suffers from unstable flavor output and low efficiency. Although some studies have involved the screening of ester-producing yeasts, most have focused on conventional genera such as Saccharomyces cerevisiae or Saccharomyces esculenta. The exploration of non-Saccharomyces cerevisiae resources, which are highly acid-resistant and especially capable of simultaneously and efficiently synthesizing phenylacetyl acetate and isoamyl acetate, remains insufficient.
[0004] During alcoholic fermentation, an appropriate amount of lactic acid is an important organic acid that contributes to the acidity and flavor balance of the wine, and is also a precursor for the derivation of ethyl lactate. However, excessive accumulation of lactic acid leads to a sharp drop in the acidity (pH value) of the mash, creating a high-acid stress environment. This environment severely inhibits the growth of most brewing yeasts (…). Saccharomyces cerevisiae The growth, metabolic activity, and reproductive efficiency of yeasts compared to non-saccharifying yeasts lead to: 1) delayed or stopped fermentation: Low pH conditions result in poor cell membrane stability and inhibited enzyme activity, leading to insufficient fermentation kinetics and reduced alcohol conversion efficiency; 2) dysregulation of flavor metabolism: Disruptions in the synthesis pathways of core esters (such as ethyl acetate and ethyl hexanoate) result in insufficient ester production and a bland aroma in the wine. Therefore, screening for yeast strains with excellent acid resistance, especially tolerance to high concentrations of lactic acid, is one of the core strategies for coping with acidity fluctuations during natural fermentation and achieving stable and efficient fermentation. Summary of the Invention
[0005] This invention addresses the shortcomings of existing technologies by providing a *J. 'C' 'S. 'Lindnerella'* yeast strain that is acid-resistant and produces high levels of ester compounds such as phenethyl acetate and isoamyl acetate, obtained by screening *J. 'C' 'Lindnerella'* yeast from strong-aroma baijiu mash. This strain can be used as a fermentation starter to cope with acidity fluctuations during natural fermentation and maintain food flavor.
[0006] The *J. selaginella* provided by this invention is classified and named as follows: Cyberlindnera jadinii GJLJ20 has been deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCC NO: M 20252589.
[0007] The ncbi comparison results of the 18S sequence of *G. selaginella leucis* provided by this invention are shown in the appendix. Figure 2 .
[0008] This invention relates to the application of Jeddings Brønsted's yeast in food fermentation.
[0009] Furthermore, this invention relates to the application of *Gerdinia sacchariformis* in the fermentation of alcoholic beverages.
[0010] This yeast, as an ester-producing yeast, can be used in various wine brewing environments with a pH value of 2.5-7.0.
[0011] The application process includes activation, expansion, and fermentation culture, specifically including the following steps:
[0012] Single colonies of *Saccharomyces cerevisiae* were streaked and inoculated into YPD medium and cultured at 30°C for 48 h to activate the second generation. The activated and expanded *Saccharomyces cerevisiae* seed culture was then inoculated into liquid fermentation medium and cultured at 30°C for 120 h under static anaerobic conditions at pH 4.0. After fermentation, the cultures were centrifuged and the supernatant was collected.
[0013] The YPD medium is composed of: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, pH 7.0, and autoclaved at 115°C for 20 minutes. If YPD solid medium is used, the formulation also includes 15 g / L agar.
[0014] The liquid fermentation medium consists of: sorghum juice saccharification liquid with a sugar content of 10-13°Bx, and 4% lactic acid added to adjust the pH of the system to 5.0.
[0015] The present invention also verified the acid tolerance of the described Jeddings cerebral nauplius in a conventional fermentation system.
[0016] The lactic acid-tolerant *J. jeddingensis* strain of this invention, even in a culture medium containing 4% lactic acid at pH 3.0, still maintains an OD600 of 2.27 ± 0.4, effectively overcoming fermentation obstacles caused by a high-acid environment. The results indicate that the addition of lactic acid had no effect on the growth of this yeast.
[0017] The ester compounds produced by the YPD fermentation broth of *G. lingenensis* of this invention include ethyl acetate, isoamyl acetate, phenylethyl acetate, isoamyl acetate, ethyl hexanoate, and furfuryl acetate.
[0018] The flavor compounds produced by *G. jellyfish* in sorghum juice fermentation broth with 4% volumetric lactic acid were as follows: ethyl acetate 369.97 mg / L, isoamyl acetate 8.31 mg / L, phenethyl acetate 111.15 mg / L, isoamyl alcohol 14.86 mg / L, phenethyl alcohol 4.52 mg / L, ethyl hexanoate 0.51 mg / L, and hexyl hexanoate 0.35 mg / L.
[0019] The biological characteristics of the *G. geraniol* yeast of this invention are as follows: when cultured on Bengal Red solid medium at 30°C for 2-3 days, the colonies are round and reddish with neat edges, smooth and moist surface, and are sticky and easily picked up.
[0020] The beneficial effects of this invention are reflected in:
[0021] This invention screened a non-Saccharomyces cerevisiae strain from brewing mash that is tolerant to lactic acid and produces high levels of esters such as phenylethyl acetate, named *Saccharomyces cerevisiae*. Cyberlindnera jadinii The strain, designated GJLJ20, exhibits outstanding flavor synthesis capabilities, efficiently synthesizing two key flavor esters: phenethyl acetate (yield 111.15 mg / L), contributing rose and honey aromas, and isoamyl acetate (yield 8.31 mg / L), contributing banana and pear-like aromas. It also produces high yields of ethyl acetate (369.97 mg / L) and other flavor compounds, significantly enhancing the richness and complexity of fermented foods. Furthermore, this strain demonstrates excellent acid tolerance, maintaining normal growth and flavor production even in environments with lactic acid concentrations as high as 4% and pH values as low as 3.0, effectively overcoming fermentation obstacles caused by high-acid environments. It is particularly suitable for brewing and fermentation processes in high-acid environments, such as solid-state brewing of baijiu (Chinese liquor), fruit wine brewing, and the production of fermented foods like kimchi, offering unique application advantages.
[0022] The non-brewing yeast strains screened by this invention can also be applied to other traditional fermented foods, broadening the selection of yeast strains in food fermentation, improving the flavor and quality of traditional fermented foods, and promoting the standardized industrial production of traditional fermented foods. Attached Figure Description
[0023] Figure 1 This is a colony morphology diagram of *G. seldomochia* GJLJ20.
[0024] Figure 2 The results of the ncbi comparison of the 18S sequence of *J. seldomochia* in *L. seldomochia* are presented in this invention. Detailed Implementation
[0025] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.
[0026] The culture medium formulations involved in the examples are as follows:
[0027] Screening medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar 15g / L, add 2% lactic acid to control the pH to be slightly acidic between 5.5 and 6, autoclave at 115℃ for 20 minutes.
[0028] Acid tolerance verification medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, lactic acid in different volume ratios added, autoclaved at 115°C for 20 minutes.
[0029] Fermentation medium: sorghum juice saccharification liquid, sugar content 10-13°Bx, pH=4.5 (pH becomes 2.7 after adding lactic acid), filtered and then autoclaved at 115℃ for 20 minutes.
[0030] Example 1: Screening of yeast strains resistant to lactic acid and ester-producing compounds
[0031] Take 5g of fermented mash sample and place it into a 250mL Erlenmeyer flask containing glass beads and 50mL of sterile water. Shake overnight on a shaker at 150r / min at 30℃. Take 5mL of the culture medium and continuously dilute it to 10 with sterile water. -5 For each dilution gradient, 0.2 mL was spread onto screening medium plates and incubated at 30°C for 2-3 days. Then, vigorous single colonies were picked, isolated and purified by multiple streak strips, and inoculated into acid-resistant verification medium. The culture temperature was 30°C, and after static culture for 5 days, the types of volatile products were qualitatively determined using headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS).
[0032] The specific methods for HS-SPME / GC-MS are as follows:
[0033] (1) Inoculate the seed culture into the acid-resistant verification medium with a 5% inoculation amount, add 100 mL of the 250 mL sample bottle solution, incubate at 30 °C, and let it stand for 120 hours.
[0034] (2) HS-SPME extraction conditions: Insert the extraction head into the headspace of the sample vial and adsorb at 60℃ for 60 min. After adsorption, remove the extraction head and insert it into the gas chromatograph injection port for desorption at 230℃ for 5 min.
[0035] (3) GC analysis conditions: Gas chromatography conditions: DB-WAX column (60m×0.25mm×0.25μm); Temperature program: Initial temperature 40℃, hold for 5min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min, hold for 10min, carrier gas is high-purity helium (1.0mL / min); Injector temperature 250℃, splitless.
[0036] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40-450 m / z.
[0037] HS-SPME / GC-MS analysis revealed that this strain possesses the ability to synthesize flavor compounds such as isoamyl alcohol, phenethyl alcohol, ethyl hexanoate, ethyl acetate, as well as isoamyl acetate, phenethyl acetate, and benzyl acetate. The flavor compounds present in higher concentrations were mainly isoamyl acetate, phenethyl acetate, isoamyl alcohol, and phenethyl alcohol. The strain was preserved at -80°C in 30% glycerol for further analysis.
[0038]
[0039] Example 2: Molecular identification of the target yeast
[0040] Molecular biological identification method (26S sequence amplification): DNA extraction was performed using the Shanghai Sangon Biotech Rapid Fungal Genome Extraction Kit, and the extraction process was carried out in accordance with the kit instructions.
[0041] The primers were 26S universal sequences, and the PCR amplification method used the recommended temperature program of Novizan rapid amplification enzyme.
[0042] Sequencing was performed by Shanghai Sangon Biotech Co., Ltd. The 18S fragment gene sequence obtained from the sequencing was compared with NCBI's BLAST algorithm to determine the strain species information, identifying it as *Giardinardia yeast*. Cyberlindnera jadinii ), and named it Jedinger Seberlindnerella vaginalis GJLJ20.
[0043] Example 3: Verification of acid resistance of yeast
[0044] (1) Sample preparation: The sample was prepared using the *G. jetting* sacchariformis GJLJ20 (…). Cyberlindnera jadiniiAfter dissolving the glycerol in the preservation tubes, streaking was used to isolate single colonies, which were then inoculated into 100 mL of LYPD medium and cultured on a shaker at 30°C for 24 h. After activation, the colonies were inoculated into 250 mL of acid-resistant verification medium at a 5% (v / v) inoculation rate and cultured at 30°C for 72 h. After fermentation, the fermentation broth was obtained, and the supernatant was collected after centrifugation at 10,000 rpm for 5 min.
[0045] (2) Verification of fermentation capacity and growth: The alcohol content of different lactic acid fermentation broths was tested by liquid phase.
[0046]
[0047] As shown in Table 1, adding different proportions of lactic acid does not affect the normal growth and fermentation of this yeast. It can still grow normally and produce alcohol at low pH levels.
[0048] (3) Detection of volatile metabolites
[0049] Volatile metabolites in the samples were detected using the HS-SPME / GC-MS method described in Example 1, with 0.326 mg / L 2-6-dimethylphenol as an internal standard. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of over 80% were confirmed as target compounds. A culture broth without added bacteria was used as a blank control group, and the relative contents of each volatile substance were calculated (converted to the internal standard multiple).
[0050] The test results are shown in Table 3. The ester compounds produced by fermentation of *G. jettinger* 'Sacchariformis' GJLJ20 with 4% lactic acid added included ethyl acetate, isoamyl acetate, phenylethyl acetate, propyl acetate, and furfuryl acetate, with contents 13.64 times, 16.26 times, 12.29 times, 0.12 times, and 0.48 times that of the internal standard, respectively. Adding different volume ratios of lactic acid did not affect the fermentation effect of the fermentation broth.
[0051]
[0052] Example 4: Validation of yeast fermentation performance
[0053] As described in Example 3, this yeast exhibits high tolerance to lactic acid and possesses a stable ability to produce esters such as phenethyl acetate, ethyl acetate, and isoamyl acetate. A sorghum juice fermentation broth with 4% (v / v) lactic acid and a seed culture with 5% (v / v) lactic acid were prepared. Gas chromatography-FID (Gas Chromatography-Induced Ionization) was used to perform absolute quantification of flavor compounds such as phenethyl acetate, ethyl acetate, and isoamyl acetate. The absolute quantification method is as follows: An appropriate amount of fermentation broth was diluted 1:1 with anhydrous ethanol. After alcohol precipitation, centrifugation, and membrane filtration, 5 ml of the supernatant was collected. 50 μL of a mixed internal standard (tert-amyl alcohol, n-amyl acetate, and 2-ethylbutyric acid) was added, and the mixture was thoroughly mixed before injection for analysis. The chromatographic column was a DB-wax 57CB capillary column (50 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The gas injection port temperature was 230℃, the detector temperature was 230℃, the carrier gas was high-purity nitrogen, the flow rate was 1.0 mL / min, the injection volume was 1 μL, and the split ratio was 30:1. The temperature program conditions were as follows: initial temperature 35℃, hold for 0 min, increase to 60℃ at 4℃ / min, hold for 4 min, then increase to 195℃ at 6℃ / min, hold for 20 min.
[0054] The absolute quantitative test results are shown in Table 4. The content of phenethyl acetate synthesized by this strain is 111.15 mg / L, which is much higher than the yield reported in the current literature.
[0055] Absolute quantitative results further confirmed that this strain has a strong ability to synthesize ethyl acetate, isoamyl acetate, and phenethyl acetate, indicating its good application potential in improving and enhancing the flavor of traditional fermented foods. This strain is particularly suitable for brewing and fermentation processes under high-acid conditions, such as in solid-state brewing of baijiu, winemaking, and the production of fermented foods like kimchi, showing unique application advantages.
[0056]
Claims
1. A type of acid-resistant, high-ester-producing *Jerdinia sacchariformis*, characterized by: The classification of the yeast J.C. Seberlindnerella vaginalis is named Cyberlindnera jadinii GJLJ20 has been deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCC NO: M20252589.
2. The application of the *J. seldom-C. Lindnerella vaginalis* as described in claim 1 in food fermentation.
3. The application according to claim 2, characterized in that: The pH range of the fermentation system is 2.5-7.
0.
4. The application according to claim 2, characterized in that: The food products mentioned include alcoholic beverages.
5. The application according to claim 2, characterized in that: The aforementioned *Jedingsberlindnerella vaginalis* produces high levels of ester compounds during fermentation.
6. The application according to claim 5, characterized in that: The ester compounds include one or more of ethyl acetate, isoamyl acetate, phenethyl acetate, isoamyl acetate, ethyl hexanoate, and furfuryl acetate.