Corynespora aspera YJHT233 and application thereof

Abstract

The invention relates to the technical field of industrial microorganisms, and particularly discloses a yeast strain and application thereof. The invention provides clavipora lusitaniae YJHT233, which is obtained through space mutation breeding, can tolerate 9% of alcohol, can tolerate an acid environment with the lowest pH value of 2.5, can tolerate a 12% high NaCl system, a 55% high glucose system and a 44 DEG C high temperature environment, and has excellent ester production capacity. The strain is used for brewing rice-flavor liquor or yellow wine, the total content of ester substances in the obtained product is greatly increased, the content of main volatile ester substances such as ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl caprylate and ethyl lactate is remarkably increased, the whole flavor is coordinated and pleasant, and the fruit fragrance is rich and rich in layers.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Portuguese corynespora yeast YJHT233 and application thereof. Background Art

[0002] As a traditional drink in my country, Huangjiu has a production history of more than 3,000 years. Huangjiu is made from glutinous rice and water as the main raw materials, with the addition of koji and saccharifying agents, and is brewed under the action of hundreds of microorganisms. Because it is rich in active peptides, amino acids, organic acids, vitamins, polyphenols, esters and other ingredients that are beneficial to health, it has always been known for its rich nutrition and health preservation, and is highly praised by experts.

[0003] The flavor characteristics of rice wine come from aroma substances. When the content of a certain aroma substance exceeds the threshold, the corresponding smell can be easily perceived. The complex flavor of rice wine mainly comes from three aspects: ① aroma substances inherent in fermentation raw materials such as rice and malt; ② aroma substances produced by microbial metabolism during fermentation; ③ aroma components formed by oxidation, esterification and Maillard reaction during the aging of rice wine. Studies have found that esters are one of the main skeleton components of rice wine aroma. Esters are an important source of the main aroma of rice wine. There are more than 40 common and confirmed esters, such as ethyl acetate, ethyl butyrate, diethyl succinate, isopentyl acetate, ethyl caprylate, ethyl lactate and other esters, which give rice wine a rich fruity and floral aroma.

[0004] The fermentation process is the key to the flavor quality of rice wine, which mainly involves the joint action of koji microorganisms and environmental microorganisms. Yeast is one of the most active microorganisms in the fermentation process of wine. According to its role, it can be divided into brewing yeast (producing alcohol) and non-brewing yeast (ester-producing yeast). There are rich species of non-brewing yeast, and its metabolites and secreted enzymes have an important influence on the aroma and flavor of brewed wine. In recent years, studies have found that some non-brewing yeasts use fermentation raw materials to synthesize aroma compounds such as esters and higher alcohols to increase the complexity of flavor and improve the flavor of the product. However, most strains are not good enough in alcohol tolerance, acid tolerance, and osmotic pressure tolerance, which seriously restricts large-scale fermentation applications. Summary of the invention

[0005] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a strain of Portuguese Clavispora lusitaniae YJHT233, which can produce high ester substances and has strong alcohol resistance, high osmotic pressure resistance, high temperature resistance and acid resistance. It can be used in winemaking to significantly increase the ester content in wine and enhance the product flavor, and has great application value.

[0006] To achieve the above objectives, the inventors conducted in-depth research and completed the solution of the present invention after repeated research and demonstration, which is as follows:

[0007] In the first aspect, an embodiment of the present application provides a strain of Clavispora lusitania e YJHT233, characterized in that the Clavispora lusitania e YJHT233 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 21, 2021, with a deposit number of GDMCC No: 62152.

[0008] The colonies of this strain are milky white and creamy, sticky, with smooth edges, a flat surface, and reflective under light.

[0009] In a second aspect, the present invention provides a fermentation agent, characterized in that the fermentation agent comprises the above-mentioned Portuguese Corynespora yeast YJHT233.

[0010] In a third aspect, the present invention provides the use of the above-mentioned Portuguese Corynespora yeast YJHT233 or fermentation agent in winemaking.

[0011] Furthermore, the application is to increase the content of ester substances in wine.

[0012] Furthermore, the winemaking is the brewing of rice-flavored liquor or yellow wine.

[0013] In a fourth aspect, the present invention provides a method for preparing rice-flavored liquor or yellow wine, the method comprising the step of adding the above-mentioned Portuguese Corynespora yeast YJHT233 or fermentation agent to the mash for fermentation.

[0014] Furthermore, the addition amount of the Portuguese corynespora yeast YJHT233 is 10 5 CFU / mL—10 8 CFU / mL.

[0015] Furthermore, the fermentation temperature is 28-32°C.

[0016] Furthermore, the fermentation time is 10-20 days.

[0017] In a fifth aspect, the present invention provides a rice-flavored white wine or yellow wine prepared using the above-mentioned Portuguese Corynespora yeast YJHT233 or fermentation agent.

[0018] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0019] 1. The Portuguese yeast YJHT233 provided by the present invention is obtained by spaceflight mutagenesis and breeding, can tolerate 9% alcohol, a minimum tolerance acidic environment of pH 2.5, a 12% high NaCl system, a 55% high glucose system and a high temperature environment of 44°C, and has excellent ester production ability. Compared with the original strain, the contents of ethyl octanoate, ethyl butyrate, ethyl lactate and ethyl benzoate are respectively increased by 1-3.25 times, and the contents of ethyl acetate and ethyl heptanoate are also increased by more than 20%.

[0020] 2. The Portuguese Corynosporum YJHT233 provided by the present invention can be used for rice-flavor liquor fermentation to significantly increase the content of major volatile esters and β-phenylethanol in the product, among which the increase rate of major flavor substances such as ethyl acetate, ethyl hexanoate, isopentyl acetate, ethyl nonanoate, ethyl octanoate, β-phenylethanol, etc. is more than 10%, and the fermented rice-flavor liquor has a harmonious and pleasant flavor and a strong fruity aroma.

[0021] 3. The Portuguese corynosporum yeast YJHT233 provided by the present invention has a significant effect on improving the ester flavor substances when used for fermenting yellow rice wine. Among them, phenylethyl acetate, ethyl benzoate, diethyl succinate, ethyl butyrate, ethyl caprate, ethyl laurate, ethyl palmitate, ethyl nonanoate, ethyl caprylate, ethyl lactate, etc. are greatly improved, and the total ester content is more than doubled and reaches 45.401 mg / L, and the fermented yellow rice wine has a strong aroma and rich layers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Portuguese coryneformis YJHT233;

[0023] Figure 2 This is the flavor chromatogram of rice wine fermented by active dry yeast;

[0024] Figure 3 This is the flavor chromatogram of the fermented rice wine of YJHT233;

[0025] The Portuguese Clavispora lusitaniae YJHT233 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 21, 2021, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the collection number is GDMCC No: 62152; the strain was received and registered by the collection center on December 21, 2021, and was detected as a viable strain by the collection center on December 21, 2021. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0029] The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0030] The culture medium used in the specification of the present invention is specifically as follows:

[0031] Wort medium (malt extract powder 13%)

[0032] GPYM medium (glucose 4%, peptone 0.5%, yeast extract powder 0.5%, malt extract 0.5%, MgSO4·7H2O 0.1%)

[0033] High glucose medium (glucose 10%, peptone 2%, yeast extract powder 1%)

[0034] Molasses medium (molasses 20BX°, (NH4)2SO4 0.1%, KH2PO4 0.1%, MgSO4 0.04%)

[0035] Starch matrix medium ((NH4)2SO4 0.25%, KH2PO4 0.25%, soluble starch 2%)

[0036] High protein matrix culture medium (soybean powder 5%)

[0037] Example 1 Screening of strains

[0038] (1) Screening of ester-producing functional strains: Weigh 25g of rice-flavor liquor koji sample, add it to a triangular flask containing 225ml of sterile water, shake and incubate at 150rpm / min for 15min, and dilute it in a gradient at a ratio of 1:10. Take 100μL of bacterial suspension with different dilution multiples and apply it to the sample dilution and then apply it to the Bengal red medium plate, and culture it at 28℃ for 48h. Observe the colony morphology, pick typical yeast single colonies with moist and smooth surfaces, streak them on PDA medium plates added with bromocresol green, and invert and culture them at 28℃ for 48h. Pick the colonies with larger yellow circles for plate streak purification. Use an inoculation loop to take multiple yeast colonies obtained by purification, and expand them in malt extract medium, GPYM medium, high glucose matrix medium, molasses matrix medium, starch matrix medium, and high protein matrix medium, respectively. The culture conditions are 28℃ and 48h. The aroma of multiple yeast culture broths was evaluated separately, and the strain with the best ester aroma production ability was comprehensively evaluated. This strain was selected as the ester-producing functional strain and named YJ26.

[0039] (2) Mutagenesis: A single colony of the ester-producing strain YJ26 was placed in a cryopreservation tube and carried into a spacecraft for 67 hours. The orbit was a large elliptical orbit with an altitude of 324 to 7970 km. During the orbit, it crossed the Van Allen radiation belt with an inclination of 42.5°. The cabin temperature was (20±5)°C. After returning to the ground, the bacterial solution was diluted to 10 with 50% glycerol. -1 The solution was dispensed into new centrifuge tubes and stored at -80°C for later use.

[0040] (3) Target strain screening: Take 200 μL of the preserved bacterial solution and add it to 5 mL of YPD liquid medium for recovery, and culture it at 28°C, 150 r / min for 48 h. Take 500 μL of the recovered bacterial solution and add it to YPD liquid medium containing 9% ethanol concentration, and culture it at 28°C, 150 r / min for 48 h. Take 200 μL of the bacterial solution and spread it on YPD solid medium, and culture it at 28°C for 48 h. Add 0.4% TTC dye and 9% ethanol to the sterilized and cooled YPD liquid medium, mix well and dispense it into 48-well plates, 2 mL per well. Inoculate all single colonies on the above YPD solid plate into 48-well plates, culture them at 28°C, 150 r / min for 48 h, and select the strains with darker red in the fermentation liquid and pleasant smell as the target strains.

[0041] (4) Identification and preservation of target strain: The strain was identified as Clavispora lusitaniae by 26S rDNA sequencing and named YJHT233. The colony morphology of the strain is as follows: Figure 1As shown, the colony is milky white and creamy, viscous, with smooth edges, a flat surface, and reflective under light. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 21, 2021, with the deposit number GDMCC No: 62152.

[0042] Example 2 Performance test of Portuguese corynespora yeast YJHT233

[0043] (1) Test on the ester production ability of Portuguese coryneformis YJHT233:

[0044] After the YJHT233 strain was activated, it was inoculated into the wort medium with 10% of the seed liquid inoculum. After fermentation for 72 hours, GC-MS was used to determine the proportion of major ester substances in the fermentation broth of the culture medium. The YJ26 strain was used as a control. The results are recorded in Table 1.

[0045] Gas chromatography conditions: DB-WAX UI chromatographic column (30m×0.25mm×0.25μm), Agilent, USA, injection port temperature 220℃, injection volume 1μL, split ratio 100:1, flow rate 1mL / min, FID detector temperature 220℃, hydrogen (H2): air (O2): tail gas (N2) 40:400:25. Heating program: starting temperature 30℃, hold for 2min, increase to 100℃ at 5℃ / min, hold for 10min, increase to 200℃ at 10℃ / min, hold for 10min.

[0046] Table 1 Content of main ester substances in fermentation broth (mg / L)

[0047] Esters YJHT233 YJ26 Increase the ratio Ethyl acetate 53 44 20％ Ethyl butyrate 151 44 243％ Ethyl heptanoate 66 53 25％ Ethyl lactate 27 8 238％ Ethyl Caprylate 119 28 325％ Ethyl benzoate 14 7 100％

[0048] The results in Table 1 show that YJHT233 has excellent ester production ability. Compared with the YJ26 fermentation broth, the main ester components of the malt juice fermentation broth inoculated with YJHT233 have been greatly improved, especially ethyl octanoate, ethyl butyrate, ethyl lactate and ethyl benzoate, the contents of which have increased by 1-3.25 times, respectively. The contents of ethyl acetate and ethyl heptanoate have also increased by more than 20%.

[0049] (2) Tolerance test of Portuguese coryneformis YJHT233:

[0050] A single colony of grape yeast YJHT233 was selected and activated, and then inoculated with 10% of the seed solution into YPD medium containing different concentrations of alcohol, glucose, NaCl and different pH values. The medium was cultured at 150 r / min for 72 h under different temperature gradients of 28°C-50°C. The turbidity of the culture medium under each condition was observed, and the OD of the turbid culture medium was measured. 600 , testing the tolerance of YJHT233.

[0051] The test results showed that strain YJHT233 could tolerate a maximum alcohol content of 9% vol, a minimum acidic environment with a pH of 2.5, a 12% high NaCl system, a 55% high glucose system and a high temperature environment of 44°C.

[0052] Example 3 Application of Portuguese corynosporum YJHT233 in rice-flavor liquor

[0053] Take 250g of Northeast selected rice, wash it, add 190mL of mineral water, and cook it in a pressure cooker for 20min. After the steamed rice is cooled, weigh 1% of Guangdong rice-flavored liquor koji, evenly sprinkle it on the cooled rice, mix the koji and rice evenly, and mix them into granules, pour them into a 500mL cleaned wide-mouth bottle, place it in a constant temperature incubator at 34℃ and let it stand for 40h, weigh 0.3% of baking powder (wine cake powder), add 10% of Portuguese coryneformis YJHT233 bacterial solution, and add 300mL of water at the same time, ferment it at 28℃ in a constant temperature incubator for 10d, and set a control with YJ26 bacteria at the same time. The final concentration of Portuguese coryneformis YJHT233 or YJ26 is controlled at 10 6 -10 7 CFU / mL. After the fermentation of the liquor was completed, the fermented liquor mash was poured into a distiller, and 300 mL of water was added to distill 300 mL of liquor. The content of various ester substances in the liquor was determined using the method described in Example 2, and the results are recorded in Table 2.

[0054] Table 2 Content of flavor substances in fermented rice-flavor liquor (mg / L)

[0055] Flavor substances YJ26 Fermentation YJHT233 fermentation Increase the ratio Ethyl acetate 17.7 22.2 25.4％ Ethyl lactate 5.7 6.2 8.8％ Ethyl butyrate 142.6 156.7 9.9％ Ethyl hexanoate 367.6 440.3 19.8％ Isoamyl acetate 318.4 364.1 14.4％ Ethyl nonanoate 26.4 33.8 28.0％ Ethyl Caprylate 51.1 56.7 11.0％ β-Phenylethanol 57.6 71.4 24.0％

[0056] As shown in the results of Table 2, when Portuguese coryneformis YJHT233 is used in rice-flavor liquor, the contents of ethyl acetate, ethyl nonanoate and β-phenylethanol are increased by more than 20%, which are 25.4%, 28.0% and 24.0% respectively; compared with liquors of other flavors, the types of flavor components of rice-flavor liquor are relatively few, and ethyl acetate and β-phenylethanol are its main aroma components. In addition, the contents of ethyl lactate, ethyl butyrate, ethyl caproate, isoamyl acetate and ethyl octanoate in rice-flavor liquor brewed by Portuguese coryneformis YJHT233 are also increased to varying degrees, with an increase ratio of 8.8%-19.8%. The increase in the content of these ester substances and β-phenylethanol main flavor substances makes the aroma of rice-flavor liquor pleasant, which is of great significance to improving the aroma of rice-flavor liquor.

[0057] Example 4 Application of Portuguese Corynosporum YJHT233 in Yellow Rice Wine

[0058] 1. Preparation of rice wine

[0059] (1) Rice liquefaction: according to the ratio of rice to water of 1:1 to 3.5 (w / w), a certain amount of rice and water are weighed, and 0.5‰ to 2‰ (w / w) of liquefaction enzyme is added. The temperature is raised to 90 to 98°C and kept warm for 60 to 90 min. The whole process is stirred fully to avoid sticking in the pot, and the rice liquefaction juice is prepared.

[0060] (2) Activation of Portuguese Corynosporum YJHT233: Take one spoonful of Portuguese Corynosporum YJHT233 bacterial slurry and inoculate it into 100 mL YPD liquid medium. Incubate it at 30°C and 200 r / min for 48 h. Take 1% and inoculate it into rice liquefied juice cooled to room temperature. Incubate it in a 30°C constant temperature incubator for 1 day. Adjust the bacterial liquid concentration to 10 6 -10 8 CFU / mL is reserved.

[0061] (3) Saccharification: Prepare rice liquefied juice according to step (1), cool it to 60-70°C with cold water, add 0.5‰-2‰ (w / w) saccharifying enzyme for rice, keep warm for 1h-2h, and stir well during the whole process to avoid sticking.

[0062] (4) Cooling and feeding: Cool the saccharification mash to 28-35°C, weigh and replenish the water evaporated by liquefaction and saccharification. Add the following materials according to the mass ratio to 1 kg of fermentation mash: one group of samples is inoculated with 3%-8% of rice wine malt koji for production, 1‰-6‰ of active dry yeast; the other group of samples is inoculated with 3%-8% of rice wine malt koji for production, 1‰-5‰ of active dry yeast and 1%-10% of Portuguese coryneformis YJHT233 culture medium, and the final concentration of Portuguese coryneformis YJHT233 is 10 5 CFU / mL—10 8 CFU / mL.

[0063] (5) Fermentation: Ferment at 28-32°C for 15-20 days.

[0064] (6) Pressing: Filter using perlite as a filter aid to collect the sake.

[0065] (7) Decoction: Keep the mixture at 70-90°C for 10-40 minutes for sterilization to obtain yellow wine. The yellow wine obtained by fermentation with active dry yeast and YJHT233 was used as the test group, and the yellow wine obtained by fermentation with active dry yeast was used as the control group.

[0066] 2. Detection and analysis

[0067] The physical and chemical indexes of the experimental group and the control group rice wine were measured, the content of volatile flavor substances was detected, and sensory evaluation was performed. The results are recorded in Tables 3-5.

[0068] Table 3 Physical and chemical indicators of fermented rice wine

[0069] Alcohol content %vol Total acid g / L Amino acid nitrogen g / L pH Control group 16.0 5.5 0.56 4.83 Experimental Group 16.6 5.61 0.62 4.81

[0070] The results are shown in Table 3. The total acid, alcohol content, and amino acid nitrogen content of the rice wine obtained by adding YJHT233 yeast fermentation were higher than those of the rice wine fermented with active dry yeast, and the pH value was lower. All physical and chemical indicators met the national standard requirements for traditional rice wine.

[0071] Table 4 Content of key flavor substances in fermented rice wine (mg / L)

[0072] Flavor substances Control group Experimental Group Increase the ratio Phenethyl acetate 0.039 0.087 123％ Ethyl benzoate 0.076 0.17 124％ Diethyl succinate 0.086 0.197 129％ Ethyl butyrate 0.087 0.195 124％ Ethyl decanoate 1.385 3.127 126％ Ethyl laurate 0.117 0.24 105％ Ethyl palmitate 0.223 0.448 101％ Ethyl hexanoate 1.869 3.032 62％ Ethyl linoleate 0.665 0.951 43％ Ethyl nonanoate 0.166 0.345 108％ Ethyl Caprylate 0.104 0.224 115％ Ethyl valerate 0.340 0.404 19％ Ethyl lactate 0.055 0.122 122％ Ethyl propionate 0.0000 0.039 - Total esters 21.753 45.401 109％

[0073] The results are shown in Table 4. Compared with the rice wine fermented with active dry yeast, the ester flavor substances of the rice wine fermented with Portuguese coryneformis YJHT233 were greatly improved. Esters are an important source of the main aroma of rice wine, which give the rice wine a strong fruity and floral aroma. In the rice wine fermented with YJHT233, the contents of phenylethyl acetate and ethyl nonanoate with rose aroma increased by 123% and 108% respectively; ethyl benzoate and diethyl succinate with mild fruity aroma increased by 1.24% and 1.29% respectively. Ethyl butyrate, ethyl hexanoate, ethyl octanoate, and ethyl valerate with pineapple aroma increased by 124%, 62%, 115%, and 19% respectively; and ethyl propionate with pineapple aroma was introduced into rice wine for the first time through YJHT233. The content of palmitic acid ethyl ester and lactic acid ethyl ester with cream aroma increased by 101% and 122% respectively, and the content of lauric acid ethyl ester, linoleic acid ethyl ester and capric acid ethyl ester with light fruit aroma increased by 105%, 43% and 126% respectively, and the total ester content increased by 109%. It can be seen that YJHT233 yeast can greatly increase the content of flowery and fruity aroma substances in rice wine fermentation, and has a significant promoting effect on the increase of the content of key ester flavor substances in rice wine.

[0074] Table 5 Sensory evaluation of fermented rice wine

[0075]

[0076]

[0077] The results are shown in Table 5. The rice wine fermented with Portuguese Corynosporum YJHT233 has better sensory aroma and taste than the rice wine fermented with active dry yeast, and has richer floral and fruity aroma.

[0078] In summary, the physical and chemical indicators of the rice wine fermented with Portuguese Corysosporium YJHT233 all meet the national standard requirements for traditional rice wine. Compared with traditional rice wine, it has better aroma and the amount of ester flavor substances is significantly increased, among which phenylethyl acetate, ethyl benzoate, diethyl succinate, ethyl butyrate, ethyl caprate, ethyl laurate, ethyl palmitate, ethyl caproate / ethyl linoleate, ethyl nonanoate, ethyl caprylate, ethyl valerate, ethyl lactate, etc. are greatly increased, and the total ester content is more than doubled, reaching 45.401 mg / L, so that a rice wine with rich aroma and rich layers can be obtained.

[0079] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Clavispora lusitaniae strain YJHT233, characterized in that: The deposit number of the Portuguese corynespora yeast YJHT233 is GDMCC No: 62152.

2. A fermentation agent, characterized in that: The fermentation agent comprises the Portuguese Corynespora yeast YJHT233 as claimed in claim 1.

3. Use of the Portuguese Corynosporum YJHT233 according to claim 1 or the fermentation agent according to claim 2 in winemaking.

4. The use according to claim 3, characterized in that The application is to increase the content of ester substances in wine.

5. The use according to claim 3, characterized in that The wine making is to make rice-flavored liquor or yellow wine.

6. A method for preparing rice-flavored liquor or yellow wine, characterized in that: The method comprises the step of adding the Portuguese Corynespora yeast YJHT233 according to claim 1 or the fermentation agent according to claim 2 to the mash for fermentation.

7. The preparation method according to claim 6, characterized in that: The addition amount of the Portuguese corynespora yeast YJHT233 is 10 5 CFU / mL—10 8 CFU / mL.

8. The preparation method according to claim 6, characterized in that: The fermentation temperature is 28-32°C.

9. The preparation method according to claim 6, characterized in that: The fermentation time is 10-20 days.

10. A rice-flavored liquor or yellow wine, characterized in that: It is prepared using the Portuguese corynespora yeast YJHT233 described in claim 1 or the fermentation agent described in claim 2.

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