Thailand hansenula polymorpha YD 32 and application thereof

Abstract

The invention discloses Thailand hansenula polymorpha YD 32 and application thereof. The preservation number of the Thailand hansenula polymorpha YD 32 is CGMCC (China General Microbiological Culture Collection Center) No: 33320. The strain can efficiently utilize sugarcane juice to ferment and produce key flavor substances of rum, including various alcohol, ester, aldehyde and ketone compounds, so that the body character, taste experience, aroma expression and flavor characteristics of the rum are improved, and the palatability is remarkably improved; when the strain is used for brewing rum, the fermentation period can be shortened, the alcohol conversion rate is increased, the production efficiency is effectively improved, and good operability and economic benefits are achieved.

Description

Technical Field

[0001] The invention relates to non-brewer's yeast, in particular to Thai Hansenula sporangiophora YD 32 and application thereof. Background Art

[0002] Rum, as one of the six major distilled spirits in the world, is an alcoholic beverage made from sugarcane juice or sugarcane molasses through fermentation, distillation, and aging.

[0003] The flavor quality of rum is affected by many factors, including the brewing raw materials, fermentation process, distillation method and aging process. The fermentation process is the most critical stage in rum production. Yeast converts sugars into alcohol through fermentation and produces secondary metabolites, such as flavor compounds such as alcohols and esters. Fermentation temperature, humidity and yeast type have an important influence on the final flavor quality. Although traditional brewing yeast dominates high-sugar fermentation, it produces more fusel alcohols such as higher alcohols during the brewing process, which often increases the spiciness of the wine and reduces the balance of the taste.

[0004] In recent years, the application of non-brewery yeast has gradually attracted attention in the brewing of rum. Some strains of non-brewery yeast, such as ester-producing yeast, can secrete esterase, which catalyzes the reaction of alcohols and acids to produce aromatic ester compounds, thereby enhancing the aroma complexity of the wine. Some, such as Candida, Pichia pastoris, and Hansenula, can produce flavor compounds such as alcohols, aldehydes, and esters, which contribute positively to the flavor of the wine. The industry has begun to pay attention to the application of non-brewery yeast in rum production, but its limitations still exist, such as low tolerance to temperature and pH during fermentation, slow fermentation speed, and the variety and layering of aromas are still not enough to meet the requirements of high-quality rum. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a novel Thai spore Hansen yeast YD 32 that can be used for the production of alcoholic beverages; the second purpose is to provide the application of the strain.

[0006] Technical solution: The Hanseniaspora thailandica YD32 described in the present invention has a preservation number of CGMCC No: 33320.

[0007] Deposit description: The strain Hansenula thailandica YD 32 in the present invention is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No: 33320 and a deposit date of January 7, 2025. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0008] The bacterial agent of the present invention contains Hansenula sporeans YD 32 from Thailand.

[0009] The invention discloses an application of the Thai Hansenula sporogenes YD 32 or bacterial agent in producing substances contained in alcoholic beverages.

[0010] Preferably, the substances contained in the alcoholic beverage are one or more of alcohols, esters, ketones, terpenes and aldehyde compounds.

[0011] Preferably, the substance contained in the alcoholic beverage is one or more of isobutanol, isopentanol, phenylethyl alcohol, 2-nonanol, ethyl hexanoate, isopentanol, ethyl caprylate, ethyl caprate, ethyl butyrate, phenylethyl acetate, ethyl palmitate, ethyl laurate, damascenone, 2-nonanone, geraniol, nerolidol, and octanal.

[0012] The invention discloses an application of the Thai Hansenula sporogenes YD 32 or a bacterial agent in brewing alcoholic beverages.

[0013] Preferably, the alcoholic beverage is rum, and the application steps include:

[0014] (1) inoculating 1.0-2.0% volume fraction of Thai spore Hansenula YD 32 bacterial solution into sugarcane juice or sugarcane molasses for fermentation, and aerobically fermenting at 24-34° C. for 24-48 hours,

[0015] Wherein, the sugarcane juice or sugarcane molasses for fermentation has a sugar content of 24-28° Brix and a pH of 4.5-5.5;

[0016] (2) The fermentation product obtained in step 1 is inoculated with 0.2-0.5% by volume of a saccharomyces cerevisiae liquid of sugarcane juice or sugarcane molasses for initial fermentation, and subjected to facultative anaerobic fermentation at 24-34° C. for 4-5 days, and then sealed and subjected to anaerobic fermentation for 2-4 days.

[0017] Preferably, the bacterial solution concentrations of the Thai Hansenula YD 32 and the Saccharomyces cerevisiae are both 0.5-1.5×10 8 CFU / mL, and the brewer's yeast is brewer's yeast CICC 1415.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Thai spore Hansenula YD32 can efficiently utilize sugarcane juice to ferment and produce alcoholic beverages, especially the key flavor substances of rum; 2. Brewing rum with this strain can effectively improve the palatability, including body properties, taste experience, aroma performance and flavor characteristics; 3. Brewing rum with this strain can shorten the fermentation cycle, increase the alcohol conversion rate, and effectively improve the fermentation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the colony morphology of Hansenula sporeans YD 32 from Thailand on YPD plate;

[0020] Figure 2 is the phylogenetic tree of Hansenula sporangiophora YD 32 from Thailand;

[0021] Figure 3 The growth rate graphs of yeast YD 11, YD 16, YD 121 and YD 32;

[0022] Figure 4 Graph showing the ethanol tolerance of yeast strains YD 11, YD 16, YD 121, and YD 32;

[0023] Figure 5 The pH tolerance graph of yeasts YD 11, YD 16, YD 121 and YD 32;

[0024] Figure 6 The temperature tolerance graph of yeasts YD 11, YD 16, YD 121 and YD 32;

[0025] Figure 7 β-glucosidase activity of yeast YD 11, YD 16, YD 121 and YD 32;

[0026] Figure 8 This is a graph showing the ester production capacity of yeasts YD 11, YD 16, YD 121 and YD 32. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below.

[0028] Example 1: Isolation and screening of yeast strains on the surface and endogenous parts of fresh sugarcane

[0029] Yeast was isolated from the surface and endophytic parts of fresh sugarcane. The enrichment medium used was formulated as follows: 20 g / L sucrose, 10 g / L yeast extract, 10 g / L peptone, 100 mg / L ampicillin, prepared with distilled water and adjusted to pH 5.0; the YPD medium for strain isolation was formulated as follows: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract; the seed liquid medium was prepared as follows: the sugarcane juice was squeezed and filtered, glucose was added to the sugarcane juice to adjust the sugar content to 28°Brix, citric acid was added to adjust the pH to 5.0, and sterilized at 115°C for 20 min.

[0030] The specific steps for isolating yeast strains are as follows:

[0031] 1. Isolation of endogenous yeast from fresh sugarcane:

[0032] (1) Pretreatment of fresh sugarcane: Use sterile water to wash away the soil and debris on the surface of the sugarcane, then rinse the plants thoroughly with sterile water, dry the surface moisture and then squeeze the juice;

[0033] (2) Separation and purification: Prepare enrichment medium: 20 g / L sucrose, 10 g / L yeast extract, 10 g / L peptone, 100 mg / L ampicillin, prepared with distilled water, and adjusted to pH 5.0. Accurately measure 25 mL of sugarcane juice under sterile operation, pour into a conical flask containing 225 mL of enrichment medium, and place it for enrichment culture at room temperature; take 1 mL of the above suspension, slowly inject it into a test tube containing 9 mL of sterile water for gradient dilution, select appropriate dilution gradient liquid and spread it on YPD solid medium plate with 0.1% ampicillin, culture at 30°C for 48 hours, and after the colony grows, pick a single colony and streak it on the YPD medium plate for purification, and store it at 4°C for use.

[0034] 2. Separation of yeast from sugarcane surface:

[0035] (1) Pretreatment of fresh sugarcane: After peeling the fresh sugarcane, cut the sugarcane peel into small pieces, accurately weigh 25 g of the sample under sterile operation, put it into a conical flask filled with 225 mL of enrichment culture medium, and place it for enrichment culture at room temperature;

[0036] (2) Isolation and purification: Take 1 mL of the above bacterial suspension and slowly inject it into a test tube containing 9 mL of sterile water for gradient dilution. Select appropriate dilution gradients and spread them on YPD solid culture medium plates added with 0.1% ampicillin (to inhibit bacterial growth). Incubate at 30°C for 48 h. After colonies grow, pick a single colony and streak it on a YPD culture medium plate for purification, and store it at 4°C for later use.

[0037] A total of 37 yeast strains were isolated from the surface and endophytic parts of fresh sugarcane, of which 11 were from the endophytic part and 26 were from the surface.

[0038] The specific screening steps for yeast strains are as follows:

[0039] The 37 yeast strains isolated and purified were inoculated into seed liquid culture medium, activated and cultured at 30°C for 24 hours, and then inoculated into a triangular flask containing 200 mL of sugarcane juice at a 2.0% inoculum and cultured at 30°C for 3 days.

[0040] Through the olfactory method, four yeast strains with strong fruity and wine aroma were screened out after the fermentation of sugarcane juice, namely YD 11, YD 16, YD 121 and YD 32.

[0041] Example 2: Yeast strain tolerance, β-glucosidase activity and ester production ability detection

[0042] 1. Growth curve

[0043] The activated strains YD11, YD16, YD121 and YD32 were inoculated into the fermentation medium at a 1% inoculum, cultured at 30°C and 180 r / min, and 1 mL of the bacterial solution was taken every 2 h and diluted appropriately, and the OD of the bacterial solution was measured using an ELISA instrument. 600 The growth curve was drawn with fermentation time (t) as the horizontal axis and OD value at a wavelength of 600 nm as the vertical axis.

[0044] Depend on Figure 3 It can be seen that the growth rates of YD 121, YD 32, and YD 16 were all fast. At 8 h, the absorbances reached the maximum values, which were 1.868±0.060, 1.679±0.071, and 1.669±0.042, respectively, and then entered the stable period. However, YD 11 started to grow slowly, and the OD 600 Only 0.488±0.033.

[0045] 2. Ethanol tolerance test

[0046] Place 249mL, 248mL, 247mL, 246mL, and 245mL of fermentation medium in 500mL conical flasks, and add 1mL, 2mL, 3mL, 4mL, and 5mL of anhydrous ethanol respectively after sterilization. Make the ethanol content (V / V) of the medium 2%, 4%, 6%, 8%, and 10%, respectively. Inoculate strains YD 11, YD 16, YD 121, and YD 32 into the fermentation medium at an inoculum of 1%, and culture at 30°C and 180r / min for 24h, and then measure the OD of the bacterial solution. 600 value.

[0047] like Figure 4 As shown, when the ethanol concentration was 2-4%, YD 11, YD 16, YD 121 and YD 32 could still grow, but as the ethanol concentration increased, the growth of the strains was inhibited, among which YD 32 had the best ethanol tolerance.

[0048] 3. pH tolerance test

[0049] The activated YD 11, YD 16, YD 121 and YD 32 bacterial suspensions were inoculated into 50 mL sterilized fermentation medium at a 1% inoculation rate. The pH of the medium was adjusted to 3.0, 4.0, 5.0, 6.0 and 7.0 respectively with 1 mol / L glacial acetic acid solution. After shaking culture at 30°C and 180 r / min for 24 h, the OD of the bacterial suspension was measured. 600 value.

[0050] like Figure 5As shown, YD 11, YD 16, YD 121 and YD 32 all had good pH tolerance, among which the YD32 strain had the highest growth efficiency at both acidic and neutral pH.

[0051] 4. Temperature tolerance test

[0052] The activated YD 11, YD 16, YD 121 and YD 32 bacterial liquids were inoculated into 250 mL sterilized fermentation medium at a 1% inoculation rate, and cultured at 24°C, 27°C, 30°C, 33°C and 36°C with shaking at 180 r / min for 24 h, and the OD values ​​of the bacterial liquids at 600 nm were measured.

[0053] like Figure 6 As shown, YD 11, YD 16 and YD 32 all grew well between 27-30℃, and the OD value began to decrease after 30℃. YD 121 grew well between 27-33℃, and the OD value began to decrease after 33℃. All strains still had a certain amount of growth at 36℃.

[0054] 5. β-glucosidase activity detection

[0055] Determination of the standard curve of p-nitrophenol: Accurately weigh 0.139g of p-nitrophenol, dissolve it in a volumetric flask and dilute to 1000mL to prepare a 1mmol / L p-nitrophenol standard solution. Pipette 1mL, 2mL, 3mL, 4mL, and 5mL of the p-nitrophenol standard solution into 50mL volumetric flasks, numbered 1-5, dilute the five bottles to 50mL with 1mol / L sodium carbonate solution and shake well to prepare p-nitrophenol standard solutions with concentrations of 20, 40, 60, 80, and 100μmol / L for standby use. Then take p-nitrophenol standard solutions of different concentrations and react with 1mol / L sodium carbonate solution, place them at room temperature away from light for 5 minutes, and then determine their absorbance at 400nm. The blank control is an equal amount of deionized water mixed with 1mol / L sodium carbonate solution. Use the blank control to zero, record the absorbance of p-nitrophenol solutions of different concentrations at 400nm, and draw a p-nitrophenol standard curve. The results are shown in the figure below. Figure 7 shown.

[0056] The activated YD 11, YD 16, YD 121 and YD 32 bacterial liquids were inoculated into 250 mL of sterilized fermentation medium at a 2% inoculation rate, and fermented at 30°C and 180 r / min for 72 hours. After the fermentation, the culture liquid was collected and centrifuged in a high-speed refrigerated centrifuge at 4°C and 8000 rpm for 10 minutes. After the centrifugation, the supernatant was collected to obtain a crude enzyme liquid.

[0057] Add 0.1 mL of crude enzyme solution and 0.2 mL of 100 mM citric acid-phosphate buffer solution with a pH of 5.0 and containing 0.75% p-nitrophenol to a centrifuge tube, react for 20 min in a 30°C water bath, add 2 mL of 1 mol / L sodium carbonate to terminate the reaction, and determine the enzyme activity colorimetrically at a wavelength of 400 nm, while making a blank control.

[0058] At 30°C and pH 5.0, the amount of enzyme required to generate 1 μmol of p-nitrophenol per hour is one enzyme activity unit (U). The β-glucosidase activity of each strain was calculated according to the following formula:

[0059]

[0060] Wherein, C is the concentration of p-nitrophenol; V is the reaction volume of the system; t is the reaction time; and N is the dilution multiple.

[0061] The results are shown in Table 1. YD 32 had the highest β-glucosidase activity.

[0062] Table 1 β-glucosidase activity of Y11, Y16, Y121 and Y32

[0063] Strain number β-Glucosidase activity (U / mL) YD 11 86.42±2.06 YD 16 88.15±4.11 YD 121 89.98±3.02 YD 32 90.29±3.35

[0064] 6. Ester production capacity test

[0065] The activated YD 11, YD 16, YD 121 and YD 32 bacterial suspensions were inoculated into the ester production medium at a 1% inoculation rate, and cultured at 30°C, 180 rpm in a shaking incubator for 24 h. The supernatant was collected by centrifugation (8000 rpm, 5 min) and filtered through a 0.22 μm filter membrane to determine the total ester content. The total ester content results are shown in Figure 8 As shown, YD 32 had the strongest ester production ability.

[0066] The ester production medium formula is: glucose 10g / L, yeast extract 10g / L, peptone 20g / L, (NH 4 ) 2 SO 4 0.2g / L, KH 2 PO 4 0.2g / L, MgSO 4 0.2g / L, CaCl 2 0.2g / L.

[0067] In summary, combined with the experimental results of the strain's ethanol, temperature, pH tolerance, β-D-glucosidase activity and ester production ability, strain YD 32 is the optimal yeast strain derived from fresh sugarcane.

[0068] The colonies of strain YD32 on YPD plate medium are as follows Figure 1 As shown, the colonies are convex and round, with smooth surface and neat edges, and are milky white, opaque and creamy. The ITS gene sequence of strain YD32 was identified, and the results showed that it was 100% similar to Hanseniaspora thailandica. The phylogenetic tree was constructed using MEGA software (v11.0). Figure 2 As shown, strain YD 32 was determined to be a strain of Hansenula sporeans from Thailand, and was named as Hansenula sporeans from Thailand YD 32, and deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No: 33320.

[0069] Example 3: Mixed fermentation of Thai Hansenula sporangiophora YD 32 and Saccharomyces cerevisiae CICC 1415 to produce rum 1

[0070] 1. Cultivate Thai Hansen yeast YD 32 and cerevisiae CICC 1415 respectively to obtain a concentration of 1×10 8 CFU / mL of seed culture medium;

[0071] 2. Raw material pretreatment: Wash and squeeze the fresh sugar cane, measure the sugar content of the sugar cane juice, adjust the sugar content of the sugar cane juice to 26±2°Brix with glucose or sucrose, adjust the pH to about 5.0, add nitrogen source, vitamins and other nutrients, heat it at 90℃ for 15min in an induction cooker, and cool it to 30℃ for use;

[0072] 3. Inoculation of yeast: 180 mL of the seed culture solution of Hansenia sporeans YD 32 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and aerobic fermentation was carried out at 28°C for 24 h. Then, 20 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated, and facultative anaerobic fermentation was carried out at 28°C for 5 days to obtain rum fermentation solution. The fermentation process was concluded when the alcohol content of the mash reached 9% Vol.

[0073] Among them, during the facultative anaerobic fermentation process, the upper part of the fermentation liquid directly contacts the air, and the brewer's yeast CICC 1415 mainly performs aerobic fermentation; the middle and lower parts of the fermentation liquid form an anaerobic environment due to the gradual depletion of oxygen, and the brewer's yeast CICC 1415 mainly performs anaerobic fermentation;

[0074] 4. Distillation: The rum fermentation liquid obtained in step 3 is heated to 90±5°C, and distilled in a column distiller to remove the head and tail of the rum to obtain rum with an alcohol content of 44±2% Vol.;

[0075] 5. Aging: The raw liquor obtained by distillation in step 4 is pretreated and then aged in oak barrels. The aging temperature is controlled at 20±2° C., the alcohol content is controlled at 44±2% Vol., and the aging time is 3 months to obtain rum.

[0076] Example 4: Mixed fermentation of Thai Hansenula sporangiophora YD 32 and Saccharomyces cerevisiae CICC 1415 to produce rum 2

[0077] 1. Cultivate Thai Hansen yeast YD 32 and cerevisiae CICC 1415 respectively to obtain a concentration of 1×10 8 CFU / mL of seed culture medium;

[0078] 2. Raw material pretreatment: Wash and squeeze the fresh sugar cane, measure the sugar content of the sugar cane juice, adjust the sugar content of the sugar cane juice to 26±2°Brix with glucose or sucrose, adjust the pH to about 5.0, add nitrogen source, vitamins and other nutrients, heat it at 90℃ for 15min in an induction cooker, and cool it to 30℃ for use;

[0079] 3. Inoculation of yeast: 180 mL of the seed culture solution of Hansenia sporeans YD 32 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and aerobic fermentation was carried out at 28°C for 48 h. Then, 20 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated, and facultative anaerobic fermentation was carried out at 28°C for 5 days to obtain rum fermentation solution. The fermentation process is judged to be over when the alcohol content of the mash reaches 9% Vol.;

[0080] 4. Distillation: The rum fermentation liquid obtained in step 3 is heated to 90±5°C, and distilled in a column distiller to remove the head and tail of the rum to obtain rum with an alcohol content of 44±2% Vol.;

[0081] 5. Aging: The raw liquor obtained by distillation in step 4 is pretreated and then aged in oak barrels. The aging temperature is controlled at 20±2° C. and the alcohol content is controlled at 44±2% Vol. The aging time is 3 months to obtain rum.

[0082] Example 5: Mixed fermentation of Thai Hansenula sporangiophora YD 32 and Saccharomyces cerevisiae CICC 1415 to produce rum 3

[0083] 1. Cultivate Thai Hansen yeast YD 32 and cerevisiae CICC 1415 respectively to obtain a concentration of 1×10 8 CFU / mL of seed culture medium;

[0084] 2. Raw material pretreatment: Wash and squeeze the fresh sugar cane, measure the sugar content of the sugar cane juice, adjust the sugar content of the sugar cane juice to 26±2°Brix with glucose or sucrose, adjust the pH to about 5.0, add nitrogen source, vitamins and other nutrients, heat it at 90℃ for 15min in an induction cooker, and cool it to 30℃ for use;

[0085] 3. Inoculation of yeast: 180 mL of the seed culture solution of Hansenia sporeans YD 32 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and aerobic fermentation was carried out at 28°C for 48 h. Then, 20 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated, and facultative anaerobic fermentation was carried out at 28°C for 5 days to obtain rum fermentation solution. The fermentation process is judged to be over when the alcohol content of the mash reaches 9% Vol.;

[0086] 4. Post-fermentation: After the fermentation in step 4 is completed, the fermentation tank is sealed and subjected to anaerobic fermentation at 28°C for 2 days;

[0087] 5. Distillation: The rum fermentation liquid obtained in step 3 is heated to 90±5°C, and distilled in a column distiller to remove the head and tail of the rum to obtain rum with an alcohol content of 44±2% Vol.;

[0088] 6. Aging: The raw liquor obtained by distillation in step 5 is pretreated and then aged in oak barrels. The aging temperature is controlled at 20±2° C. and the alcohol content is controlled at 44±2% Vol. The aging time is 3 months to obtain rum.

[0089] Comparative Example 1: Mixed fermentation of Thai Hansen yeast YD 32 and brewer's yeast CICC 1415 to produce rum 4

[0090] On the basis of Example 3, in step 3, 100 mL of the seed culture solution of Hansenula sporeensis YD 32 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and aerobic fermentation was carried out at 28° C. for 24 h. Then, 100 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated, and facultative anaerobic fermentation was carried out at 28° C. for 5 days. The other steps remained unchanged to brew rum.

[0091] Comparative Example 2: Mixed fermentation of Thai Hansen yeast YD 32 and brewer's yeast CICC 1415 to produce rum 5

[0092] On the basis of Example 3, in step 3, 150 mL of the seed culture solution of Hansenia sporeans YD 32 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and aerobic fermentation was carried out at 28° C. for 24 h. Then, 50 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated, and facultative anaerobic fermentation was carried out at 28° C. for 5 days. The other steps remained unchanged to brew rum.

[0093] Comparative Example 3: Mixed fermentation of Thai Hansen yeast YD 32 and brewer's yeast CICC 1415 to produce rum 6

[0094] On the basis of Example 4, in step 3, 180 mL of the seed culture solution of Hansenula sporeans YD 32 obtained in step 1 and 20 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 were simultaneously added to 10 L of the sugarcane juice obtained in step 2, and facultative anaerobic fermentation was carried out at 28° C. for 5 days. The other steps remained unchanged to brew rum.

[0095] Comparative Example 4: Mixed fermentation of Thai Hansen yeast YD 32 and brewer's yeast CICC 1415 to produce rum 7

[0096] On the basis of Example 4, in step 3, 20 mL of the seed culture solution of Saccharomyces cerevisiae CICC 1415 obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and facultative anaerobic fermentation was carried out at 28° C. for 48 h. Then, 180 mL of the seed culture solution of Hansenula sporeans YD 32 obtained in step 1 was inoculated, and aerobic fermentation was carried out at 28° C. for 5 days. The other steps remained unchanged to brew rum.

[0097] Comparative Example 5: Fermentation of Rum 8 by Saccharomyces cerevisiae CICC 1415

[0098] 1. Cultivate Saccharomyces cerevisiae CICC 1415 to obtain a concentration of 1×10 8 CFU / mL of seed culture medium;

[0099] 2. Raw material pretreatment: Wash and squeeze the fresh sugar cane, measure the sugar content of the sugar cane juice, adjust the sugar content of the sugar cane juice to 26±2°Brix with glucose or sucrose, adjust the pH to about 5.0, add nitrogen source, vitamins and other nutrients, heat it at 90℃ for 15min in an induction cooker, and cool it to 30℃ for use;

[0100] 3. Inoculation of yeast: 200 mL of the Saccharomyces cerevisiae CICC1415 seed culture solution obtained in step 1 was inoculated into 10 L of the sugarcane juice obtained in step 2, and facultative anaerobic fermentation was performed at 28°C for 5 days to obtain rum fermentation solution. The fermentation process was concluded when the alcohol content of the mash reached 9% Vol.;

[0101] 4. Distillation: The rum fermentation liquid obtained in step 3 is heated to 90±5°C, and distilled in a column distiller to remove the head and tail of the rum to obtain rum with an alcohol content of 44±2% Vol.;

[0102] 5. Aging: The raw liquor obtained by distillation in step 4 is pretreated and then aged in oak barrels. The aging temperature is controlled at 20±2° C. and the alcohol content is controlled at 44±2% Vol. The aging time is 3 months to obtain rum.

[0103] Example 6: Comparison of flavor substance determination and sensory analysis of rum brewed in Examples 3-5 and Comparative Examples 1-5

[0104] The flavor substances of the rum obtained in Examples 3-5 and Comparative Examples 1-5 were determined by GC-MS. The chromatographic conditions are as follows: the chromatographic column is HP-NNOWAX (30m×0.25mm, 0.25μm); the injection port temperature is 250℃; the detector temperature is 300℃; the injection volume is 2L; the carrier gas is helium; the flow rate is 1.0mL / min; there is no split mode; the septum purge gas volume is 20mL / min; the heating program is: 40℃ for 2min, increase to 60℃ at 1℃ / min, then increase to 220℃ at 10℃ / min, maintain for 2min, and the whole process is 40min. The results are shown in Table 2. The rum brewed in Examples 3-4 contains rich key flavor substances such as ethyl caproate, isoamyl acetate, ethyl caprylate, ethyl caprate, ethyl butyrate, and has a strong floral and fruity aroma and wine aroma.

[0105] Table 2 Rum flavor substance determination results

[0106]

[0107] The brewed rum was further evaluated from four aspects: body, mouthfeel, aroma and taste. The results are shown in Table 3. The rum brewed in Example 5 was clear and transparent before aging, and light amber after aging, clear and transparent, with good gloss; it was smooth in the mouth, sweet but not greasy, mellow and full, and had excellent overall balance; the aroma was complex and rich, with the fresh fruity aroma of tropical fruits such as pineapple and banana, mixed with the sweet aroma of vanilla and cinnamon, and a hint of smoky aroma of oak barrels, and the sensory evaluation was the best.

[0108] Table 3 Rum sensory evaluation results

[0109]

Claims

1. A Thai Hansenula sporangial yeast YD 32, whose deposit number is CGMCC No: 33320.

2. A bacterial agent, characterized in that The bacterial agent contains Thai spore Hansenula YD 32.

3. Use of the Thai spore Hansenula YD 32 according to claim 1 or the bacterial agent according to claim 2 in producing substances contained in alcoholic beverages.

4. The use according to claim 3, characterized in that: The substances contained in the alcoholic beverage are one or more of alcohols, esters, ketones, terpenes and aldehyde compounds.

5. The application according to claim 4, characterized in that: The substances contained in the alcoholic beverage are one or more of isobutanol, isopentanol, phenylethyl alcohol, 2-nonanol, ethyl hexanoate, isopentanol, ethyl caprylate, ethyl caprate, ethyl butyrate, phenylethyl acetate, ethyl palmitate, ethyl laurate, damascenone, 2-nonanone, geraniol, nerolidol, and octanal.

6. Use of the Thai spore Hansenula YD 32 according to claim 1 or the bacterial agent according to claim 2 in brewing alcoholic beverages.

7. The use according to claim 6, characterized in that: The alcoholic beverage described is rum.

8. The use according to claim 7, characterized in that: The steps of the application include: (1) inoculating 1.0-2.0% volume fraction of Thai spore Hansenula YD32 bacterial solution into sugarcane juice or sugarcane molasses for fermentation, and aerobically fermenting at 24-34° C. for 24-48 hours, Wherein, the sugarcane juice or sugarcane molasses for fermentation has a sugar content of 24-28° Brix and a pH of 4.5-5.5; (2) The fermentation product obtained in step 1 is inoculated with 0.2-0.5% by volume of a saccharomyces cerevisiae liquid of sugarcane juice or sugarcane molasses for initial fermentation, and subjected to facultative anaerobic fermentation at 24-34° C. for 4-5 days, and then sealed and subjected to anaerobic fermentation for 2-4 days.

9. The use according to claim 8, characterized in that: The bacterial liquid concentrations of the Thai spore Hansenula YD 32 and the cerevisiae yeast were 0.5-1.5×10 8 CFU / mL.

10. The use according to claim 8, characterized in that: In the step 2, the cerevisiae yeast is cerevisiae CICC1415.

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