An abnormal Wickham yeast Wa K-5 and its application in winemaking

Abstract

The invention discloses an abnormal Wickham yeast Wa K-5 and its application in brewing, belonging to the field of microbial fermentation and food technology. The invention separates and purifies 4 strains of abnormal Wickham yeast from a variety of yellow wine koji, screens the tolerance, aroma production characteristics, etc. of the 4 yeasts, and finally obtains strain Wa K-5 that can tolerate at least 100ppm iso-α acid and 5%vol ethanol, and has excellent aroma production characteristics. The fermentation of 12.0°P wort can produce low-alcohol beer with an ethanol concentration of 0.95%vol, and the fermentation of 6.0°P wort can obtain non-alcoholic beer with an ethanol concentration of 0.48%vol. When mixed with brewer's yeast, the alcohol content can be similar to that of beer fermented by pure strain brewer's yeast, the flower aroma and wort flavor are enhanced, and the foam properties and killing taste are significantly improved. The strain WaK-5 of the present invention has a very broad application prospect in the field of beer brewing.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and food technology, and in particular relates to an abnormal Wickham yeast WaK-5 and application thereof in wine making. Background Art

[0002] In recent years, non-brewery yeast has become one of the research hotspots in the field of beer brewing, and many studies have proved the feasibility of non-brewery yeasts including Torulaspora, Lachancea, Zygosaccharomyces, Mrakia, Saccharomycodes, Schizosaccharomyces, Williopsis, Hyphopichia, etc. for beer production. The rich and diverse non-brewery yeast species provide more possibilities for the innovation of beer varieties and styles, and promote the diversified development of the beer industry. At present, among the non-brewery yeast species involved in beer brewing, the most relevant research is on Brettanomyces bruxellensis, Saccharomycodes ludwigii, Torulaspora delbrueckii and Lachancea thermotolerans. There are few reports of beer brewed with abnormal Wickham yeast.

[0003] Beer is an alcoholic beverage with malt, hops, water and yeast as main ingredients. With the improvement of living standards, people expect more personalized new flavors of beer, and pay more and more attention to the nutritional and health benefits of food, such as non-alcoholic beer and low-alcohol beer that conform to the concept of health.

[0004] Therefore, the present invention is proposed to study how to utilize abnormal Wickham yeast to make low-alcohol or alcohol-free beer and improve the beer flavor. Summary of the invention

[0005] In order to solve the above technical problems, the present invention collects abnormal Wickham yeast from rice wine koji, and obtains a strain WaK-5 with the best performance through iso-α acid tolerance screening, ethanol tolerance screening, aroma preference screening, etc., which is used for the development of low-alcohol and alcohol-free beer. At the same time, the taste, flavor and other aspects are improved through mixed fermentation.

[0006] The invention provides an abnormal Wickerham yeast, which is named as abnormal Wickerham yeast (Wickerhamomyces anomalus) WaK-5 and has a preservation number of CGMCC No.31026.

[0007] The present invention provides a culture containing the above-mentioned abnormal Wickham yeast or a processed product thereof.

[0008] The present invention provides a food composition containing the abnormal Wickham yeast, culture or processed product thereof.

[0009] The present invention provides a microbial agent comprising the abnormal Wickham yeast.

[0010] Furthermore, the microbial agent also includes Saccharomyces cerevisiae. Preferably, the ratio of abnormal Wickham yeast to Saccharomyces cerevisiae in the microbial agent is (1-100):1.

[0011] Furthermore, the microbial agent is a liquid agent.

[0012] Furthermore, the microbial agent is a solid agent.

[0013] Another object of the present invention is to provide the use of the above-mentioned abnormal Wickham yeast, culture or processed product thereof, food composition, microbial agent, etc. in the preparation of alcoholic products.

[0014] Another object of the present invention is to provide a wine product, wherein the wine product contains the above-mentioned culture or its processed product or food composition.

[0015] Furthermore, the above wine product is obtained by fermentation with the above abnormal Wickham yeast or microbial agent.

[0016] Furthermore, the preparation of the wine product includes the step of fermenting wort using the abnormal Wickham yeast or microbial inoculant.

[0017] Furthermore, the wine includes but is not limited to beer, such as low-alcohol beer, non-alcohol beer, mixed fermentation beer, etc.

[0018] Furthermore, the wort may be of any concentration, preferably 6.0-18.0°P, such as 6.0°P, 7.0°P, 8.0°P, 9.0°P, 10.0°P, 11.0°P, 12.0°P, 13.0°P, 14.0°P, 15.0°P, 16.0°P, 17.0°P, 18.0°P, etc., including but not limited to the values ​​listed above.

[0019] Most preferably, when preparing low-alcohol beer, the wort can be selected from a variety of concentrations, including but not limited to 12.0°P, 13.0°P, 14.0°P, 15.0°P, 16.0°P, 17.0°P, 18.0°P, etc.; when preparing non-alcoholic beer, the concentration of the wort does not exceed 6.0°P.

[0020] The above technical solution of the present invention has the following advantages compared with the prior art:

[0021] (1) The abnormal Wickerhamomyces anomalus strain Wa K-5 of the present invention is a non-brewery yeast derived from yellow wine koji. The strain can tolerate 100 ppm of iso-α acid and 5% vol of ethanol, can metabolize glucose, fructose and sucrose in wort, but cannot metabolize maltose and maltotriose, and can obtain 0.95% vol of low-alcohol beer by fermenting wort at 12.0°P. In addition, the strain has excellent aroma production characteristics, can increase the content of acetic acid, 4-vinylguaiacol (4VG) and ethyl ester compounds such as ethyl acetate, ethyl nonanoate, ethyl laurate, etc. in beer, and the beer brewed by the strain has obvious floral and fruity aromas.

[0022] (2) The strain Wa K-5 of the present invention can also be used to brew mixed fermentation beer and non-alcoholic beer. The mixed fermentation beer brewed by sequentially inoculating brewer's yeast ScUS-05 and WaK-5 at a yeast concentration ratio of 1:100 combines the fermentation advantages of the two yeasts and has both flavor and alcohol content. Compared with the beer fermented by pure strains of WaK-5 and Sc US-05, the mixed fermentation beer brewed by this process has significantly increased ester and organic acid content, similar 4VG content to the beer fermented by pure strain Wa K-5, similar alcohol content to the beer fermented by pure strain ScUS-05, enhanced floral aroma and wort flavor, and significantly improved foam properties and mouthfeel. In addition to mixed fermentation beer, Wa K-5 fermented wort at 6.0°P can also obtain non-alcoholic beer with an ethanol concentration of 0.48%vol, and its overall aroma performance is far better than the non-alcoholic beer produced by the dealcoholization method. Compared with the Luddite yeast commercially used for brewing non-alcoholic beer, the products fermented by this strain have significantly higher contents of esters, acetic acid, lauric acid, and 4VG, and a stronger fruity aroma.

[0023] Biomaterial Deposit

[0024] Abnormal Wickham yeast (Wickerhamomyces anomalus) Wa K-5 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 20, 2024, with the deposit number CGMCC No.31026, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 The colonies of WaK-5 grown on WL nutrient medium.

[0027] Figure 2 Aroma sensory evaluation results of WaK-5 brewed beer and control.

[0028] Figure 3 Sensory evaluation results of mixed fermentation beer and control.

[0029] Figure 4 These are the sensory evaluation results of non-alcoholic beer. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0031] The experimental method involved in the present invention is as follows:

[0032] Yeast activation and wort preparation:

[0033] Pick a single yeast colony, inoculate it into YPD liquid medium, and culture it in a shaking incubator at 28°C for 48 hours for expansion. Use an automatic yeast counter to measure the yeast concentration of each bacterial liquid sample. Dilute the light ale concentrated wort appropriately, boil it for 20 minutes for sterilization, and make the original wort concentration 12.0°P.

[0034] Preparation of 12.0°P pale ale wort:

[0035] Dilute the pale ale concentrated wort appropriately, heat to boiling, then add 0.5g / L of Citra hops and boil for 60 minutes. After boiling, the original wort concentration is 12.0°P, pH value is 5.30, and bitterness value is about 20IBU.

[0036] Iso-alpha acid tolerance screening:

[0037] 10 mL of sterile wort with iso-α acid concentrations of 0 ppm, 50 ppm, and 100 ppm (equivalent to International Bitterness Units, IBU) was added to 15 mL centrifuge tubes with lids, and yeast solution was inoculated to make the initial yeast concentration of the wort 1×10 7After the wort was cultured at 20°C for 3 days, the yeast concentration of each sample was measured and recorded. Each experiment was repeated 3 times.

[0038] Ethanol tolerance screening:

[0039] Add 10 mL of sterile wort with 0%, 5%, and 10% ethanol concentrations to 15 mL centrifuge tubes with lids, and inoculate with yeast solution to make the initial yeast concentration of the wort 1×10 7 After the wort was cultured at 20°C for 3 days, the yeast concentration of each sample was measured and recorded. Each experiment was repeated 3 times.

[0040] Aroma preference screening:

[0041] Add 30 mL of sterile wort to a 50 mL centrifuge tube with a cap and inoculate with yeast solution to make the initial yeast concentration of the wort 1×10 7 After the wort was incubated at 20°C for 10 days, all samples were numbered with a random three-digit code and poured into a transparent tasting cup. Ten sensory evaluators (5 men and 5 women) who had received beer sensory evaluation training smelled the wort fermentation liquid and rated the aroma of the fermentation liquid on a 10-point intensity level according to their preferences. The average score was taken as the final score.

[0042] Data processing and statistical analysis:

[0043] Data processing was performed using Excel 2019, SPSS 21.0 and other software. Data significance analysis was performed using one-way analysis of variance and Duncan method, with p < 0.05 indicating a significant difference.

[0044] Sugar composition determination:

[0045] After fermentation, the sugar composition in beer samples and wort was determined by liquid chromatography.

[0046] Preparation and pretreatment of standard solutions: prepare 20 g / L fructose, 20 g / L glucose, 20 g / L sucrose, 40 g / L maltose, and 20 g / L maltotriose solutions, and dilute them 2 times, 4 times, 8 times, and 16 times. All standard solutions are sterilized by filtration through a 0.22 μm water filter membrane.

[0047] Sample pretreatment: Degas the sample by ultrasonic for 15 min, dilute appropriately, and filter through a 0.22 μm water filter to sterilize.

[0048] Mobile phase pretreatment: prepare the mobile phase in the ratio of acetonitrile: water = 85:15, filter through a 0.45 μm organic filter membrane for sterilization, and mix thoroughly by ultrasonication for 15 min.

[0049] Chromatographic conditions: The detector is a differential refractive index detector, the column temperature is set to 40°C, the detector temperature is 40°C, the flow rate is 1.5 mL / min, the elution time is 16 min, and the injection volume is 10 μL. A standard curve is drawn based on the results of the standard solution for compound quantification.

[0050] Ethanol production characteristics determination:

[0051] Sample pretreatment: Ultrasonic degassing of beer sample for 15 min, 4 mL was taken into an extraction bottle, 50 μL of n-propanol was added, and a magnetic stirrer was placed.

[0052] Preparation and pretreatment of standard solution: pipette 20 μL, 60 μL, 100 μL, 140 μL, and 180 μL of ethanol into the extraction bottle respectively, add 50 μL of n-propanol as the internal standard, make up to 4 mL with deionized water and add a magnetic stirrer.

[0053] Extraction method: The extraction bottle was magnetically stirred (100 rpm) in a 70°C water bath and balanced for 10 minutes. The solid phase extraction head was inserted into the extraction bottle to extract the volatile components for 20 minutes. After the extraction, the extraction head was inserted into the gas phase injection port for injection and analysis for 5 minutes.

[0054] Gas chromatography conditions: DB-INOWAX column (30m x 0.25mm, 0.5μm), carrier gas is high-purity nitrogen, nitrogen flow rate: 1mL / min, sample port temperature 250℃, injection mode is splitless, desorption time is 5min. The heating program is the initial temperature of 50℃, maintained for 1min, and then increased to 200℃ at 20℃ / min.

[0055] The ethanol standard curve was drawn based on the determination results of the standard solution for compound quantification.

[0056] Volatile flavor compounds detection:

[0057] Determination of volatile flavor compounds in wort and beer samples without yeast inoculation by headspace solid phase microextraction coupled with gas chromatography-mass spectrometry.

[0058] Sample pretreatment: Take 4 mL of sample into an extraction bottle, add 5 μL of internal standard solution (prepared by adding 2 μL of 2-octanol to 1.2 mL of anhydrous ethanol), put in a magnetic stirrer (100 rpm), and place in a water bath at 40°C for 20 min. Then insert the extraction needle and adsorb for 40 min in a 40°C water bath.

[0059] Chromatographic conditions: desorption for 5 min after injection, injection port temperature of 250 °C, helium column flow rate of 0.8 mL / min.

[0060] The chromatographic program was set to hold at 50 °C for 1 min, then gradually increase to 170 °C at 3 °C / min, hold for 3 min, and finally increase to 215 °C at 7 °C / min, hold for 5 min. The mass spectrometry conditions were set to ionization energy of 70 eV, ion source temperature of 200 °C, and transfer line temperature of 280 °C.

[0061] Example 1 Screening and preservation of strains

[0062] (1) Obtaining abnormal Wickham yeast from rice wine koji

[0063] 16 kinds of yellow wine koji with different characteristics were selected from the five major yellow wine producing areas of Hubei, Zhejiang, Fujian, Jiangxi and Jiangsu, from which abnormal Wickham yeast was screened. Specifically, chloramphenicol was dissolved in ethanol to prepare a 50 mg / mL solution. YPD liquid culture medium was prepared, 0.2% sodium propionate was added to inhibit mold growth, and chloramphenicol solution was added at a final concentration of 100 μg / mL to inhibit bacterial growth, sterilized and cooled. Different wine koji were crushed in a mortar and added to the culture medium at 0.05 g / mL, and cultured in a shaker at 28°C and 200 r / min for 24 hours. The WL nutrient agar culture medium powder was heated and dissolved, and then cooled and poured after sterilization. The yellow wine koji culture solution was moderately diluted, spread on the WL nutrient agar culture medium plate, and cultured at 28°C for three days. WL nutrient medium is a yeast selective medium, and different types of yeast will grow colonies of different colors and morphologies on this medium. Non-brewer's yeast colonies were selected according to color and morphology, and streaked and purified three times to obtain non-brewer's yeast single strains, and the colony color and morphology were recorded. The strains were identified by 26S rDNA sequence analysis, and 4 strains of abnormal Wickerhamomyces anomalus were isolated from 16 traditional Chinese rice wine kojis for subsequent screening.

[0064] (2) Iso-α acid tolerance screening

[0065] Four strains of abnormal Wickham yeast were incubated in worts with iso-α acid concentrations of 0 ppm, 50 ppm, and 100 ppm (equivalently converted to International Bitterness Units, IBU, corresponding to bitterness values ​​of 0 IBU, 50 IBU, and 100 IBU). The initial yeast concentration was 1.00 × 10 7 CFU / mL, the original wort concentration was 12.0°P. The yeast concentration was measured after culturing at 20°C for 3 days, and the results are shown in Table 1.

[0066] Table 1 Growth of strains in worts with different iso-α acid concentrations

[0067]

[0068] Note: Different letters in the same row of the same strain represent significant differences (P<0.05).

[0069] The results showed that the growth of WaK-6 was inhibited to a certain extent in wort with a concentration of 100ppm, and the yeast concentration was significantly lower than that in wort without iso-α acid. The growth of WaK-3 was inhibited in wort with a concentration of 50ppm and 100ppm. Wa H-7 and WaK-5 could tolerate at least 100ppm of iso-α acid, and the tolerance to iso-α acid was strong, especially WaK-5. The yeast concentration increased with the increase of iso-α acid concentration.

[0070] (3) Ethanol tolerance screening

[0071] The yeast concentrations of the four strains of abnormal Wickham yeast were measured after being cultured in worts with ethanol concentrations of 0% vol, 5% vol, and 10% vol for 3 days. The results are shown in Table 2.

[0072] Table 2 Growth of strains in worts with different ethanol concentrations

[0073]

[0074] Note: Different letters in the same row of the same strain represent significant differences (P<0.05).

[0075] When the ethanol concentration was 5% vol, all strains could grow normally. The yeast concentrations of Wa H-7, Wa K-5, and Wa K-6 were even significantly higher than those of wort without ethanol. This may be because this ethanol concentration can reduce the contamination of wort by other microorganisms and provide a more suitable growth environment for Wa yeast. When the ethanol concentration was 10% vol, the growth of all strains was significantly inhibited, but the yeast concentrations of WaK-5 and WaK-6 exceeded 2×10 7 CFU / mL, which was still more than 1 times higher than the initial inoculation amount, indicating that both strains had relatively strong tolerance to ethanol.

[0076] (4) Aroma preference screening

[0077] The aroma preference scores of the wort fermentation broths of the four abnormal Wickham yeast strains are shown in Table 3. The aroma preference score of Wa K-5 is the highest among all strains, indicating that its fermentation produces the best aroma.

[0078] Table 3 Aroma preference scores of wort fermentation broth of strains

[0079]

[0080] In summary, Wa K-5 has stronger tolerance to iso-α acid and ethanol among the four abnormal Wickham yeast strains, and has the best aroma performance. Therefore, Wa K-5 is considered to be the most suitable strain for beer brewing and has been sent for preservation.

[0081] Example 2 Fermentation characteristics of Wa K-5 strain in beer

[0082] The ability of non-brewery yeast to synthesize volatile flavor compounds such as esters and alcohols can improve the aroma of beer, and its metabolism of sugars and ethanol in wort is also crucial. In order to explore the fermentation characteristics of abnormal Wickham yeast Wa K-5 in wort, it was inoculated into 12.0°P pale ale wort. The initial yeast inoculation amount in wort was 1×10 7 CFU / mL, cultured at 20°C until fermentation was complete (sugar content did not change for 3 days), and then stored at 4°C for one week. The sugar composition in the beer was measured to evaluate the sugar metabolism characteristics of the strain, the ethanol concentration was measured to evaluate the ethanol production characteristics, and the volatile flavor substance content was measured and the aroma sensory evaluation was performed to evaluate the aroma production characteristics. Saccharomyces cerevisiae Sc US-05 and Lutheran yeast Sl31238 were used as controls. Each treatment was repeated 3 times. The results are as follows:

[0083] (1) Metabolic sugar characteristics

[0084] Table 4 Sugar composition in wort before and after fermentation (g / L)

[0085]

[0086] Note: Different letters in the same row represent significant differences (P<0.05), and “ / ” means no difference was detected.

[0087] The sugar composition in the wort before fermentation and after fermentation of Wa K-5 was determined, and the results are shown in Table 3. Wa K-5 can utilize almost all the fructose, glucose and sucrose in the wort, but cannot utilize maltose and maltotriose.

[0088] (2) Ethanol production characteristics

[0089] In the national standard "Beer" GB 4927-2008, low-alcohol beer (LAB) is defined as beer with an alcohol content of 0.6% vol to 2.5% vol. After the strain fermentation, the ethanol concentration in the wort was measured to be 0.95% vol, which meets the concept of low-alcohol beer.

[0090] (3)Fragrance-producing characteristics

[0091] The volatile flavor substances in the wort were determined, and it was found that Wa K-5 could increase the production of acetic acid, 4-vinylguaiacol, and ethyl ester compounds such as ethyl acetate, ethyl nonanoate, and ethyl laurate compared with the control Saccharomyces cerevisiae Sc US-05 and Lutheran yeast Sl 31238. Its ethyl acetate production was 14.5 times that of Saccharomyces cerevisiae Sc US-05, and its 4VG production was 34.6 times that of Saccharomyces cerevisiae Sc US-05 (as shown in Table 5).

[0092] Table 5 Content of some volatile flavor substances in Wa K-5 fermented beer and control (μg / L)

[0093]

[0094] Organize evaluators to conduct sensory evaluation on the aroma of fermented beer products (full score 10 points for each item), the results are as follows Figure 2 As shown in the figure, the aroma preference and floral aroma scores of Wa K-5 beer were the highest, and the fruit aroma score was comparable to that of the control brewer's yeast Sc Us-05, indicating that the beer fermented by this strain has obvious floral and fruity aromas, and is more popular with sensory evaluators overall.

[0095] Example 3 Wa K-5 strain mixed fermentation beer

[0096] Dilute the pale ale concentrate wort appropriately, heat it to boiling, then add 0.5g / L of Citra hops and boil for 60 minutes. After boiling, the original wort concentration is 12.0°P, pH value is 5.30, and bitterness value is about 20IBU. After the wort is cooled, inoculate Wa K-5 (yeast inoculation concentration is 1×10 7 CFU / mL), and then inoculated with Sc US-05 (yeast inoculation concentration of 1×10 5 CFU / mL), continue to culture at 20℃ until fermentation is complete (sugar content does not change for 3 days), and store at 4℃ for one week. The beer obtained by this process was evaluated, and the beer fermented by pure strains of brewer's yeast Sc US-05 and Wa K-5 was used as a control. The results are as follows:

[0097] (1) Ethanol metabolism

[0098] The ethanol concentration of the mixed fermentation beer brewed by this process is 4.45% vol, which is similar to the beer fermented by pure strain Saccharomyces cerevisiae Sc US-05 (4.69% vol).

[0099] (2) Metabolism of volatile flavor substances

[0100] The content of volatile flavor substances produced by mixed fermentation beer and control group was determined. It was found that mixed fermentation beer combined the aroma-producing characteristics of Sc US-05 and Wa K-5, and could produce a large amount of esters, such as ethyl caprate, ethyl caproate, isobutyl acetate, isoamyl acetate, phenylethyl acetate, ethyl caprylate, ethyl laurate, ethyl propionate, ethyl acetate, etc. The total ester content was 297093.95μg / L, which was higher than Wa K-5 pure fermentation beer (286497.64μg / L) and Sc US-05 pure fermentation beer (25993.66μg / L). In addition, a large amount of organic acids, such as acetic acid, capric acid, caprylic acid, caproic acid, etc., could be produced, and 4-vinylguaiacol, which was equivalent to that of WaK-5 pure fermentation control group (as shown in Table 6).

[0101] Table 6 Content of some volatile flavor substances in mixed fermentation beer and control (μg / L)

[0102]

[0103]

[0104] Note: “ / ” indicates not detected

[0105] (3) Sensory evaluation

[0106] After fermentation, the aroma, appearance, taste and flavor of the mixed fermentation beer and the control beer were evaluated by sensory evaluation (maximum 10 points for each item). Figure 3 As shown. In terms of aroma, mixed fermentation enhances the floral and wort flavors of beer, and retains the fruity aroma of Sc US-05; in terms of appearance, mixed fermentation improves the foam properties of beer (foaming, foam retention, and foam fineness), and the clarity is also improved to a certain extent. In terms of taste, the malt flavor of beer is enhanced and the sourness is weakened. In terms of taste, the mouthfeel is significantly improved.

[0107] Example 4 Preparation of non-alcoholic beer by Wa K-5 strain

[0108] In the national standard "Beer" GB 4927-2008, non-alcoholic beer (NAB) is defined as beer with an alcohol content of less than or equal to 0.5% vol and an original wort concentration of greater than or equal to 3.0°P. At present, most of the non-alcoholic beer on the market is made by physical dealcoholization of ordinary beer, but during the dealcoholization process, many volatile flavor substances in the beer also evaporate along with the ethanol, affecting the overall flavor of the beer.

[0109] In this example, abnormal Wickham yeast Wa K-5 was used to ferment 6.0°P wort to produce non-alcoholic beer. The specific process is to appropriately dilute the pale ale concentrated wort, heat it to boiling, then add 0.5g / L of Citra hops, boil it for 60 minutes, and after the boiling, the original wort concentration is 6.0°P, the pH value is 5.00, and the bitterness value is about 20IBU. After the wort is cooled, Wa K-5 (yeast inoculation concentration is 1×10 7 CFU / mL), cultured at 20°C until fermentation was complete (sugar content did not change for 3 days), and stored at 4°C for one week. The beer obtained by this process was evaluated, and the beer fermented by pure strain Lutheran yeast S1 31238 was used as a control. The experimental results are as follows:

[0110] (1) Ethanol metabolism

[0111] The ethanol concentration of the non-alcoholic beer brewed by this process is 0.48% vol, which meets the requirements of non-alcoholic beer.

[0112] (2) Metabolism of volatile flavor substances

[0113] The content of volatile flavor substances in non-alcoholic beer was determined, and it was found that compared with the control group, the ester content of ethyl acetate, isoamyl acetate, ethyl myristic acid, ethyl palmitate, ethyl laurate, ethyl caprate, etc. in non-alcoholic beer fermented by WaK-5 was significantly increased, and the total ester content was 9612.81μg / L, which was higher than the control group (2285.58μg / L). In addition, the content of acetic acid, lauric acid, and 4VG was also significantly increased (as shown in Table 7).

[0114] Table 7 Content of some volatile flavor substances in non-alcoholic beer and control (μg / L)

[0115]

[0116] (3) Sensory evaluation

[0117] After fermentation, the aroma, appearance, taste and flavor of the non-alcoholic beer were evaluated (maximum 10 points for each item). In addition to Sl 31238, a commercially available non-alcoholic beer produced by physical dealcoholization was selected as a control. The results are as follows: Figure 4 Although the non-alcoholic beer fermented by WaK-5 scored slightly worse in appearance, in terms of aroma, the non-alcoholic beer fermented by WaK-5 enhanced the fruity and alcoholic flavors, and the overall aroma performance was much better than the commercially available non-alcoholic beer; in terms of taste and mouthfeel, the non-alcoholic beer fermented by WaK-5 also had no obvious shortcomings.

[0118] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A strain of abnormal Wickham yeast, characterized in that: Wickham's yeast ( Wickerhamomyces anomalus )Wa K-5, the deposit number is CGMCC No. 31026.

2. A culture comprising the abnormal Wickham yeast of claim 1.

3. A microbial agent, characterized in that: The method comprises the abnormal Wickham yeast according to claim 1.

4. The microbial agent according to claim 3, characterized in that: The microbial agent also includes brewer's yeast.

5. The microbial agent according to claim 4, characterized in that: In the microbial agent, the ratio of abnormal Wickham yeast to brewer's yeast is (1-100):

1.

6. Use of the abnormal Wickham yeast according to claim 1, the culture according to claim 2, and the microbial agent according to any one of claims 3 to 5 in the preparation of wine products, characterized in that: The alcoholic product is beer.

7. A wine product, characterized in that: The alcoholic product is beer, containing the culture according to claim 2.

8. A method for preparing a wine product, characterized in that: The wine product is beer, comprising the step of fermenting wort using the abnormal Wickham yeast according to claim 1 or the microbial agent according to any one of claims 3-5.

9. The preparation method according to claim 8, characterized in that: The beer is low-alcohol beer, non-alcohol beer or mixed-fermentation beer.

10. The preparation method according to claim 9, characterized in that: When preparing non-alcoholic beer, the concentration of the wort does not exceed 6.0 ºP.

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