Pichia kluyveri, mixed leavening agent and method for brewing mulberry fruit wine at low temperature

Abstract

The invention belongs to the technical field of food processing, and particularly relates to pichia kluyveri, mixed fermentation and a method for brewing mulberry fruit wine at low temperature. The invention provides a pichia kluyveri P-MW-4, the Latin name of the pichia kluyveri P-MW-4 is Pichia kluyveri P-MW-4, the pichia kluyveri P-MW-4 is preserved in China General Microbiological Culture Collection Center, the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is July 19, 2023, and the preservation number is CGMCC No.27954. The invention further provides a preparation method of the pichia kluyveri P-MW-4. According to the method, the mulberry fruit wine is brewed at low temperature by using the mixed leavening agent, so that the flavor level of the mulberry fruit wine is increased, the content of volatile acid in the mulberry fruit wine is reduced, and the production effect of the fruit wine is good.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a Pichia kluyveri yeast, a mixed fermentation and a method for brewing mulberry wine at low temperature. Background Art

[0002] Mulberry fruit (Morus alba L.) is rich in nutrients, including flavonoids, terpenoids, vitamins, organic acids and trace elements. It is one of the fruits that are currently regulated as both medicine and food in my country. Mulberry fruit matures seasonally and is not easy to store, which easily leads to a large waste of mulberry resources. With the development of the food industry and the improvement of consumption levels, in order to improve the utilization rate of mulberry resources and reduce resource waste, the categories of mulberry deep-processing products are becoming increasingly rich. Mulberry fruit is a composite fruit composed of a large number of thin-skinned small drupes, so its storage performance is poor. At present, in addition to being eaten fresh, mulberry fruit is widely used in the production of dried fruit products by drying technology, which reduces the nutritional value of the fruit and causes a large loss of flavor.

[0003] As one of the main deep-processed products of fruits, fruit wine is deeply favored by consumers for its unique flavor, high nutritional value and low alcohol content. However, in the process of fruit wine brewing, the conventional production fermentation temperature is high (≥20℃), which can easily lead to the loss of flavor substances in the product and have an adverse effect on the aroma and flavor of the product; in addition, the single production strain also leads to the single flavor and serious homogeneity of mulberry wine.

[0004] In order to solve the above problems, it is urgent to develop a mulberry wine brewing process suitable for production at lower temperatures, so as to better solve the problems faced by mulberry wine production and provide a feasible production basis for low-temperature brewing of mulberry wine. Summary of the invention

[0005] In view of this, in order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a Pichia kluyveri, a mixed fermentation and a method for brewing mulberry wine at low temperature.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Pichia kluyveri P-MW-4, the Latin name of the Pichia kluyveri P-MW-4 is Pichia kluyveri P-MW-4, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a preservation date of July 19, 2023, and a preservation number of CGMCC No. 27954.

[0008] The present invention also provides application of the Pichia kluyveri P-MW-4 in low-temperature brewing.

[0009] The present invention also provides a mixed fermentation agent, comprising the Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1;

[0010] The initial live bacterial concentration of Pichia kluyveri P-MW-4 was 1×10 5 ~1×10 7 cfu / mL; the initial viable bacterial concentration of the Saccharomyces cerevisiae M-FW-LT 1 was 1×10 5 ~1×10 7 cfu / mL.

[0011] The invention also provides application of the mixed fermentation agent in low-temperature winemaking.

[0012] The present invention also provides a method for brewing mulberry wine at low temperature, comprising the following steps:

[0013] (1) crushing mulberry fruits to obtain mulberry juice; mixing the mulberry juice and pectinase, and letting it stand for 20 to 28 hours to obtain a mixture;

[0014] (2) adjusting the sugar content of the mixture to 25-28° Bx and the pH to 3-4 to obtain treated mulberry juice;

[0015] (3) mixing the treated mulberry juice and potassium metabisulfite to obtain mulberry juice to be fermented;

[0016] (4) adding the mixed fermentation agent to the mulberry juice to be fermented, and fermenting at 12 to 16° C. for 15 to 24 days to obtain crude mulberry wine;

[0017] (5) Clarifying and filtering the crude mulberry wine to obtain mulberry wine.

[0018] Preferably, in step (1), the mass ratio of the mulberry juice and pectinase is 1000:0.02-0.04.

[0019] Preferably, in step (2), the method for adjusting the sugar content of the mixture is: adding white sugar to adjust the sugar content of the mixture; the method for adjusting the pH of the mixture is: adding citric acid to adjust the pH of the mixture.

[0020] Preferably, in step (3), the mass ratio of the treated mulberry juice and potassium metabisulfite is 1000:0.07-0.09.

[0021] Preferably, in step (5), the clarification method is: mixing crude mulberry wine and bentonite, and letting it stand for 40 to 56 hours to obtain clarified crude mulberry wine; the mass ratio of the crude mulberry wine and bentonite is 1000:1 to 3.

[0022] Preferably, in step (5), the filtration method is cross-flow filtration.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a Pichia kluyveri P-MW-4, which has a flavor-enhancing effect on mulberry wine; however, Pichia kluyveri P-MW-4 does not produce alcohol when fermented alone, so it is impossible to produce wine alone; if Saccharomyces cerevisiae M-FW-LT 1 is used alone as a fermentation agent, the prepared mulberry wine shows insufficient aroma and lack of flavor. Therefore, the present invention uses Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 to prepare mulberry wine by mixed fermentation, and brews it under low temperature conditions, which not only increases the flavor level of the mulberry wine, but also has a good production effect of the wine.

[0025] The brewing method of the invention gives full play to the aroma production performance of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1, effectively reduces the volatile acid content in mulberry wine, reduces the flavor loss degree of mulberry wine, and thus can improve the quality of mulberry wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the fingerprint of the volatile components of mulberry wine;

[0027] Figure 2 This is the GC-MS spectrum of the mulberry wine described in Example 2;

[0028] Figure 3 This is the GC-MS spectrum of the mulberry wine described in Example 3;

[0029] Figure 4 This is the GC-MS spectrum of the mulberry wine described in Example 4.

[0030] Collection Instructions

[0031] A Pichia kluyveri P-MW-4, the Latin name of the Pichia kluyveri P-MW-4 is Pichiakluyveri P-MW-4, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the address of which is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 19, 2023, and the deposit number is: CGMCC No.27954. DETAILED DESCRIPTION

[0032] The present invention provides a Pichia kluyveri P-MW-4, the Latin name of the Pichia kluyveri P-MW-4 is Pichia kluyveri P-MW-4, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a preservation date of July 19, 2023, and a preservation number of CGMCC No. 27954.

[0033] The present invention also provides application of the Pichia kluyveri P-MW-4 in low-temperature brewing.

[0034] The present invention also provides a mixed fermentation agent, comprising the Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1;

[0035] The initial live bacterial concentration of Pichia kluyveri P-MW-4 was 1×10 5 ~1×10 7 cfu / mL, preferably 1×10 6 cfu / mL; the initial viable bacterial concentration of the Saccharomyces cerevisiae M-FW-LT 1 was 1×10 5 ~1×10 7 The optimal cfu / mL is 1×10 6 cfu / mL.

[0036] In the present invention, the treatment of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 comprises the following steps:

[0037] The Pichia kluyveri P-MW-4 strain and the Saccharomyces cerevisiae M-FW-LT 1 strain were shake-cultured in 100 mL of liquid YPD medium to obtain enriched Pichia kluyveri P-MW-4 and enriched Saccharomyces cerevisiae M-FW-LT 1; the enriched Pichia kluyveri P-MW-4 and the enriched Saccharomyces cerevisiae M-FW-LT 1 were centrifuged at 6000-8000 rpm for 5-15 min, and the supernatant was removed; the precipitate was resuspended in sterile water and washed for 1-3 times to obtain Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1;

[0038] The temperature of the shaking culture is 27-29° C., preferably 28° C.; the rotation speed of the shaking culture is 160-180 rpm, preferably 170 rpm; the time of the shaking culture is 35-45 h, preferably 40 h;

[0039] The centrifugal speed is preferably 7000 rpm; the centrifugal time is preferably 10 min;

[0040] The number of washings is preferably 2 times;

[0041] The liquid YPD culture medium uses water as a solvent and comprises the following components in mass concentrations: 1% yeast extract, 2% peptone, and 2% glucose.

[0042] The invention also provides application of the mixed fermentation agent in low-temperature winemaking.

[0043] The present invention also provides a method for brewing mulberry wine at low temperature, comprising the following steps:

[0044] (1) crushing mulberry fruits to obtain mulberry juice; mixing the mulberry juice and pectinase, and letting it stand for 20 to 28 hours to obtain a mixture;

[0045] (2) adjusting the sugar content of the mixture to 25-28° Bx and the pH to 3-4 to obtain treated mulberry juice;

[0046] (3) mixing the treated mulberry juice and potassium metabisulfite to obtain mulberry juice to be fermented;

[0047] (4) adding the mixed fermentation agent to the mulberry juice to be fermented, and fermenting at 12 to 16° C. for 15 to 24 days to obtain crude mulberry wine;

[0048] (5) Clarifying and filtering the crude mulberry wine to obtain mulberry wine.

[0049] In the present invention, in step (1), the mulberry fruits need to be pretreated before crushing: fresh mulberry fruits that are 80% and / or 90% mature are picked, rotten fruits and moldy fruits are removed, and the fresh mulberry fruits are placed in a 0.4-0.6 mg / L ozone cleaning pool and rinsed at 15-17° C. for 20-30 minutes to obtain washed mulberry fruits; the washed mulberry fruits are sprayed with clean water and blown dry to obtain mulberry fruits;

[0050] Preferably, the method comprises the following steps: picking fresh mulberry fruits that are 90% ripe, removing rotten fruits and moldy fruits, placing the fresh mulberry fruits in a 0.5 mg / L ozone cleaning pool and washing them at 16° C. for 25 minutes to obtain washed mulberry fruits; spraying the washed mulberry fruits with clean water and drying them to obtain mulberry fruits;

[0051] The mass ratio of the mulberry juice and pectinase is 1000:0.02-0.04, preferably 1000:0.03;

[0052] The standing time is preferably 22 to 26 hours, more preferably 24 hours.

[0053] In the present invention, in step (2), the method for adjusting the sugar content of the mixture is: adding white sugar to adjust the sugar content of the mixture;

[0054] The sugar content of the mixture is preferably 26.5°Bx;

[0055] The method of adjusting the pH of the mixture is: adding citric acid to adjust the pH of the mixture;

[0056] The pH of the mixture is preferably 3.5.

[0057] In the present invention, in step (3), the mass ratio of the treated mulberry juice and potassium metabisulfite is 1000:0.07-0.09, preferably 1000:0.08.

[0058] In the present invention, in step (4), the fermentation temperature is preferably 14° C., and the fermentation time is preferably 19.5 days;

[0059] The method for stopping the fermentation is: lowering the temperature to 9-11°C to stop the fermentation, preferably 10°C.

[0060] In the present invention, in step (5), the clarification method is: mixing crude mulberry wine and bentonite, and letting it stand for 40 to 56 hours to obtain clarified crude mulberry wine;

[0061] The standing time is preferably 45 to 51 hours, more preferably 48 hours;

[0062] The mass ratio of the crude mulberry wine and bentonite is 1000:1-3, preferably 1000:2.

[0063] The filtration method is cross-flow filtration.

[0064] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] The Saccharomyces cerevisiae M-FW-LT 1 described in the examples and comparative examples of the present invention was purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration, with the strain collection number: CGMCC No.26749.

[0066] Example 1

[0067] (1) Isolation and purification of strains

[0068] 100 g of healthy and fresh mulberries with stems (picked from the mulberry planting area of ​​Yanbian County, Sichuan Province) were selected and roughly crushed in a clean environment at room temperature of 20±2°C; the pulp and pomace obtained by crushing the fruits were immediately placed in a sterilized fermentation container, and 50 mg / L of potassium metabisulfite was added at the same time. After mixing evenly, low-temperature natural fermentation was carried out at 20°C; the fermentation liquid was naturally fermented for 21 days, and the fermentation liquid was taken every 3 days starting from day 0;

[0069] 1 mL of mulberry fermentation broth samples from 8 periods (0d, 3d, 6d, 9d, 12d, 15d, 18d and 21d) during natural fermentation were diluted to 10 -4 , 10 -5 and 10 -6 ; Take 100 μL of the diluent and spread it evenly on the surface of WL medium (Wallerstein Laboratory Nutrient Agar), and then culture it at a constant temperature of 28°C for 48 hours; After the colonies are formed, pick out potential yeast colonies according to the morphological characteristics of the colonies and streak them on YPD plate culture medium for purification to obtain purified strains;

[0070] (2) Screening of strains

[0071] Primary screening of strains (low temperature resistance screening): The purified strains were activated at 28°C and 180 r / min for 48 h; after activation, gradient dilutions were performed and spread on YPD plate culture medium, and cultured at 12°C for 72 h. The strains with better growth were selected for secondary screening;

[0072] Secondary screening of strains (fermentation capacity screening): strains after primary screening were cultured in YPD liquid medium at 28°C and 180r / min for 48h, and then 50μL of bacterial solution was evenly pipetted into YPD liquid medium with an inverted Duroc tube, and cultured at a constant temperature of 28°C for 72h; during the growth of the strains, the gas production in the Duroc tube was observed every 6h, and the gas volume in the tube was recorded; strains with fast fermentation speed and large gas production were selected for third-level screening;

[0073] The third-level screening of strains (fermentation ability screening): the strains after the second-level screening were inoculated into YPD liquid culture medium and cultured at 28°C for 72 hours; strains with strong aroma and no sulfur odor were screened out;

[0074] (3) The fungal ITS rRNA gene sequence analysis method was used to perform molecular biological identification on the strains that passed the three-level screening, and finally the screened strain was identified as Pichia kluyveri P-MW-4 (Pichia kluyveri P-MW-4).

[0075] Example 2

[0076] Preparation of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1: Pichia kluyveri P-MW-4 strain and Saccharomyces cerevisiae M-FW-LT 1 strain were respectively cultured in 100 mL of liquid YPD medium (water as solvent including the following mass concentrations of components: 1% yeast extract, 2% peptone, and 2% glucose), 28°C, 170 rpm, and shaken for 40 h to obtain enriched Pichia kluyveri P-MW-4 and enriched Saccharomyces cerevisiae M-FW-LT 1; the enriched Pichia kluyveri P-MW-4 and the enriched Saccharomyces cerevisiae M-FW-LT 1 were respectively centrifuged at 7000 rpm for 10 min, and the supernatant was removed; the precipitate was resuspended in sterile water and washed twice to obtain a viable cell count of 1×10 6 cfu / mL of Pichia kluyveri P-MW-4 and the number of viable bacteria was 1×10 6 cfu / mL of Saccharomyces cerevisiae M-FW-LT 1;

[0077] A method for brewing mulberry wine at low temperature:

[0078] (1) Picking fresh mulberry fruits that are eighty to ninety ripe, removing rotten fruits and moldy fruits, placing the fresh mulberry fruits in a 0.5 mg / L ozone cleaning pool at 16° C. for 25 minutes to obtain cleaned mulberry fruits; spraying the cleaned mulberry fruits with clean water and drying them to obtain mulberry fruits; crushing the mulberry fruits to obtain mulberry juice; stirring and mixing the mulberry juice and pectinase at a mass ratio of 1000:0.03, and standing at 16° C. for 24 hours to obtain a mixture;

[0079] (2) adding white sugar to the mixture to adjust the sugar content of the mixture to 27° Bx, and adding citric acid to adjust the pH of the mixture to 3.5 to obtain treated mulberry juice;

[0080] (3) mixing the treated mulberry juice and potassium metabisulfite at a mass ratio of 1000:0.08 to obtain mulberry juice to be fermented;

[0081] (4) Inoculate 1×10 6 cfu / mL Pichia kluyveri P-MW-4 and 1×10 6 cfu / mL Saccharomyces cerevisiae M-FW-LT 1, fermented at 12°C for 18 days, cooled to 10°C to stop fermentation, and obtained crude mulberry wine;

[0082] (5) The crude mulberry wine and bentonite were mixed in a mass ratio of 1000:2 for clarification and cross-flow filtration to obtain mulberry wine.

[0083] Example 3

[0084] Preparation of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 Reference Example 2;

[0085] A method for brewing mulberry wine at low temperature:

[0086] (1) Picking fresh mulberry fruits that are 80% mature, removing rotten fruits and moldy fruits, placing the fresh mulberry fruits in a 0.6 mg / L ozone cleaning pool at 17° C. for 20 minutes to obtain cleaned mulberry fruits; spraying the cleaned mulberry fruits with clean water and drying them to obtain mulberry fruits; crushing the mulberry fruits to obtain mulberry juice; stirring and mixing the mulberry juice and pectinase at a mass ratio of 1000:0.02, and standing at 17° C. for 22 hours to obtain a mixture;

[0087] (2) adding white sugar to the mixture to adjust the sugar content of the mixture to 26° Bx, and adding citric acid to adjust the pH of the mixture to 4.0 to obtain treated mulberry juice;

[0088] (3) mixing the treated mulberry juice and potassium metabisulfite at a mass ratio of 1000:0.07 to obtain mulberry juice to be fermented;

[0089] (4) Inoculate 1×10 6 cfu / mL Pichia kluyveri P-MW-4 and 1×10 6 cfu / mL Saccharomyces cerevisiae M-FW-LT 1, fermented at 16°C for 21 days, cooled to 10°C to stop fermentation, and obtained crude mulberry wine;

[0090] (5) The crude mulberry wine and bentonite were mixed in a mass ratio of 1000:3 for clarification and cross-flow filtration to obtain mulberry wine.

[0091] Example 4

[0092] Preparation of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 Reference Example 2;

[0093] A method for brewing mulberry wine at low temperature:

[0094] (1) Picking fresh mulberry fruits that are 90% ripe, removing rotten fruits and moldy fruits, placing the fresh mulberry fruits in a 0.4 mg / L ozone cleaning pool at 15° C. for 30 minutes to obtain cleaned mulberry fruits; spraying the cleaned mulberry fruits with clean water and drying them to obtain mulberry fruits; crushing the mulberry fruits to obtain mulberry juice; stirring and mixing the mulberry juice and pectinase at a mass ratio of 1000:0.04, and standing at 15° C. for 26 hours to obtain a mixture;

[0095] (2) adding white sugar to the mixture to adjust the sugar content of the mixture to 28° Bx, and adding citric acid to adjust the pH of the mixture to 3.0 to obtain treated mulberry juice;

[0096] (3) mixing the treated mulberry juice and potassium metabisulfite at a mass ratio of 1000:0.09 to obtain mulberry juice to be fermented;

[0097] (4) Inoculate 1×10 6 cfu / mL Pichia kluyveri P-MW-4 and 1×10 6 cfu / mL Saccharomyces cerevisiae M-FW-LT 1, fermented at 16°C for 24 days, cooled to 10°C to stop the fermentation, and obtained crude mulberry wine;

[0098] (5) The crude mulberry wine and bentonite were mixed in a mass ratio of 1000:3 for clarification and cross-flow filtration to obtain mulberry wine.

[0099] Comparative Example 1

[0100] Reference Example 2 is different from Example 2 in that only Pichia kluyveri P-MW-4 is inoculated, and the other steps are the same as Example 2.

[0101] Comparative Example 2

[0102] Reference Example 2 is different from Example 2 in that only Saccharomyces cerevisiae M-FW-LT 1 is inoculated, and other steps are the same as Example 2.

[0103] Experimental Example 1

[0104] The mulberry wine fermented for 6, 12 and 18 days in Example 2 was sent to the Institute of Agricultural Products Storage and Processing of Xinjiang Academy of Agricultural Sciences for detection of volatile components. The volatile component fingerprints were obtained as follows: Figure 1 As shown;

[0105] from Figure 1 It can be seen that the mulberry wine described in Example 2 is rich in lipid substances that constitute the main aroma, especially acetate substances; the rich variety of acetate substances can help the mulberry wine to present a "flower and fruit aroma"; the mulberry wine is also rich in higher alcohol substances, which show a certain accumulation trend with the fermentation process, bringing the mulberry wine a "flower and fruit aroma" and "brandy" characteristic aroma.

[0106] Experimental Example 2

[0107] 4 mL of the mulberry wine described in Examples 2 to 4 was accurately drawn into a clean, dry 20 mL extraction bottle, 0.4 g of sodium chloride (NaCl) and 10 μL of 4-methyl-2-pentanol (mass concentration of 81.1 μg / mL, solvent of water) were added as internal standards to obtain a mixed sample system; the mixed sample system was balanced in a constant temperature water bath at 60° C. for 10 min, and then an SPME (50 / 30 μm DVB / CAR / PDMS, Supelco, Bellefonte, PA) extraction device was inserted into the sample bottle for extraction, the extraction temperature was 60° C., and the headspace extraction was performed for 40 min; the GC-MS chromatogram of the mulberry wine and the alcohol content and residual sugar content of the mulberry wine were obtained, the mulberry wine described in Examples 2 to 4 were classified according to the residual sugar content, and the body, aroma and color of the mulberry wine described in Examples 2 to 4 were observed; the GC-MS chromatogram of the mulberry wine described in Examples 2 to 4 is as follows: Figures 2 to 4 As shown; the alcohol content, residual sugar content, wine type, body, aroma and color of the mulberry wine described in Examples 2 to 4 are shown in Table 1;

[0108] The instruments used were GC-MS coupling instrument (race 1310, GC; TSQ9000, MS) from Thermo Fisher Scientific;

[0109] GC conditions were set as follows: VF-WAXms capillary column (30m×250μm, 0.25μm, Agilent Technologies); injection port temperature 240°C, carrier gas (He) flow rate 1.6mL / min, no split; programmed temperature rise, starting column temperature 40°C, maintained for 2min; increased to 50°C at 4°C / min, maintained for 3min; then increased to 200°C at 5°C / min, maintained for 5min before the measurement was terminated.

[0110] MS condition setting: the standard tuning file was called for determination; electron ionization source, electron energy was set to 70 eV; ion source temperature was 220 °C; mass scan range was 30-400u, full scan; solvent delay time was 1 min.

[0111] Table 1 Quality of mulberry wine

[0112]

[0113] from Figures 2 to 4 It can be seen that the mulberry wines described in Examples 2 to 4 have a high content of phenylethyl acetate, a characteristic flavor substance, and the mulberry wines have a prominent aroma of rose and honey.

[0114] As can be seen from Table 1, basic winemaking parameters such as alcohol content and residual sugar content indicate that the mulberry wines described in Examples 2 to 4 meet the definition of fruit wine types in GB15037-2006.

[0115] Experimental Example 3

[0116] Solid phase microextraction combined with gas chromatography-mass spectrometry was used to perform qualitative and quantitative analysis on the mulberry wine described in Example 2 and Comparative Examples 1 to 2. The test results are shown in Table 2.

[0117] The qualitative and quantitative methods are as follows: the detection results are qualitatively analyzed by the retention index (RI) based on the NIST 14L spectral library search, and the artificial spectra are analyzed. RI uses C6-C30 normal alkanes as internal standards; then the internal standard method is used for quantitative analysis. According to the content of the internal standard (4-methyl-2-pentanol), the content of each volatile component is calculated. The calculation formula is as follows:

[0118] Target substance concentration (μg / mL) = (target substance peak area / internal standard substance peak area)*81.1 (μg / mL).

[0119] Table 2 Aroma component content (μg / mL)

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] As can be seen from Table 2, Pichia kluyveri P-MW-4 brings unique "floral" and "fruity" aromas to mulberry wine, especially the "rose" aroma brought by ester substances; the combination of Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 as a starter changes the aroma type and content of mulberry wine, especially reduces the "fatty taste" brought by caprylic acid, and improves the flower and fruit aromas brought by phenylethyl acetate ("rose" and "floral") and isoamyl alcohol ("whiskey flavor"); therefore, the mixed starter and low-temperature brewing method described in Example 1 reconstruct the composition ratio of the flavor substances in the body of mulberry wine, improve the content level of some key substances, thereby reconstructing the aroma of the wine body and expanding the flavor types of mulberry wine.

[0126] It can be seen from the above embodiments that the present invention provides a Pichia kluyveri, a mixed fermentation agent and a method for brewing mulberry wine at low temperature. The present invention uses a mixed fermentation agent to brew mulberry wine at low temperature, which not only increases the flavor level of the mulberry wine, reduces the volatile acid content in the mulberry wine, but also has a good production effect of the wine.

[0127] Although the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that several improvements and modifications may be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Pichia kluyveri P-MW-4, characterized in that The Latin name of the Pichia kluyveri P-MW-4 is Pichia kluyveri P-MW-4, which is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 19, 2023, and the deposit number is: CGMCC No. 27954.

2. Use of the Pichia kluyveri P-MW-4 described in claim 1 in low-temperature brewing.

3. A mixed fermentation agent, characterized in that: The method comprises the Pichia kluyveri P-MW-4 and Saccharomyces cerevisiae M-FW-LT 1 as described in claim 1; The initial live bacterial concentration of Pichia kluyveri P-MW-4 was 1×10 5 ~1×10 7 cfu / mL; the initial viable bacterial concentration of the Saccharomyces cerevisiae M-FW-LT 1 was 1×10 5 ~1×10 7 cfu / mL.

4. Use of the mixed fermentation agent according to claim 3 in low-temperature winemaking.

5. A method for brewing mulberry wine at low temperature, characterized in that: The steps include: (1) crushing mulberry fruits to obtain mulberry juice; mixing the mulberry juice and pectinase, and letting it stand for 20 to 28 hours to obtain a mixture; (2) adjusting the sugar content of the mixture to 25-28° Bx and the pH to 3-4 to obtain treated mulberry juice; (3) mixing the treated mulberry juice and potassium metabisulfite to obtain mulberry juice to be fermented; (4) adding the mixed fermentation agent of claim 2 to the mulberry juice to be fermented, and fermenting at 12 to 16° C. for 15 to 24 days to obtain crude mulberry wine; (5) Clarifying and filtering the crude mulberry wine to obtain mulberry wine.

6. The method according to claim 5, characterized in that In step (1), the mass ratio of the mulberry juice and pectinase is 1000:0.02-0.

04.

7. The method according to claim 6, characterized in that In step (2), the method for adjusting the sugar content of the mixture is: adding white sugar to adjust the sugar content of the mixture; the method for adjusting the pH of the mixture is: adding citric acid to adjust the pH of the mixture.

8. The method according to claim 7, characterized in that In step (3), the mass ratio of the treated mulberry juice and potassium metabisulfite is 1000:0.07-0.

09.

9. The method according to claim 8, characterized in that In step (5), the clarification method is: mixing crude mulberry wine and bentonite, standing for 40 to 56 hours, and obtaining clarified crude mulberry wine; the mass ratio of the crude mulberry wine and bentonite is 1000:1 to 3.

10. The method according to claim 9, characterized in that In step (5), the filtration method is cross-flow filtration.

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