CN113186052A

CN113186052A - Musalace fermentation brewing process with high caramel aroma yield and high 5-HMF degradation

Info

Publication number: CN113186052A
Application number: CN202110399956.3A
Authority: CN
Inventors: 朱丽霞; 刘真; 马学利; 周芹; 余兆斌; 蒲云峰
Original assignee: Tarim University
Current assignee: Tarim University
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-07-30
Anticipated expiration: 2041-04-14
Also published as: CN113186052B

Abstract

The invention provides a fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation. The invention belongs to the technical field of fermentation industry, and relates to a fermentation brewing process of musalaisi with high caramel aroma yield and high degradation of 5-HMF. The method comprises the following steps: s1: raw material screening and cleaning, S2: raw material stem removal and crushing, S3: squeezing to obtain juice, S4: concentration, S5: primary inoculation, S6: degradation of 5-HMF fermentation, S7: secondary inoculation, S8: producing caramel fragrance and fermenting. The invention has the beneficial effects that: compared with the prior art, firstly, the Musalace is fermented by compounding the saccharomyces cerevisiae F2-P85-3 and the F2-B163-2 strain with high yield of furanone, so that the content of caramel aroma substances in the Musalace can be effectively increased, and the taste of the Musalace is improved; secondly, the process simultaneously adopts C0-Y2-02 bacterial strain for high degradation of 5-HMF for fermentation, so that the content of 5-HMF in the musalaisi can be effectively degraded; finally, the process adopts a fermentation mode, does not produce byproducts, and is safe, environment-friendly and pollution-free.

Description

Musalace fermentation brewing process with high caramel aroma yield and high 5-HMF degradation

Technical Field

The invention belongs to the technical field of fermentation industry, and relates to a fermentation brewing process of musalaisi with high caramel aroma yield and high degradation of 5-HMF.

Background

Musalaisi is a special local product in Xinjiang, has a main local product in Avati county, is a brewed delicious and mellow grape beverage, has high medicinal value, and is rich in amino acids, multiple vitamins, glucose, iron and other nutritional ingredients and trace elements required by a human body. The musalaisi belongs to warm property and is a pure natural green drink. The caramel fragrance endows the wine with unique and aromatic flavor and improves the wine body quality. At present, substances with caramel aroma are mainly furan substances, and the current caramel aroma synthesis method comprises the following steps: natural extraction and chemical synthesis, but the cost of the natural extraction is too high, and the chemical synthesis can generate byproducts, which are not suitable for increasing the content of caramel aroma substances in the musalaisi. The microbial fermentation has the most outstanding advantages, has the advantages of simple synthesis and no pollution, is very suitable for increasing the content of caramel aroma substances in the production of the musalaisi, but the prior art does not adopt the microbial fermentation technology for the production process of the musalaisi to increase the content of the caramel aroma substances in the musalaisi.

5-hydroxymethylfurfural, 5-HMF for short, is an organic substance, has a molecular formula of C6H6O3, a molecular weight of 126.11, is a beige crystalline solid, is a chemical substance generated by dehydrating glucose or fructose, contains a furan ring, an aldehyde group and a hydroxymethyl group in a molecule, and has relatively active chemical properties. The average content of 5-HMF in the food is 1.35mg/100g, and the food can stimulate eye mucosa, upper respiratory tract mucosa and skin at a high dosage. The existing 5-HMF degradation methods comprise a washing method, an overbasing method and a biodegradation method, wherein the washing method is low in degradation efficiency, the overbasing method can generate byproducts, and the biodegradation method is mild in conditions, low in energy consumption and less in waste water. However, the prior art does not provide a production process for degrading the content of 5-HMF in the musalaisi by using a microbial fermentation technology in the production process of the musalaisi.

Disclosure of Invention

In order to solve the above problems, the primary object of the present invention is to provide a fermentation brewing process of mousse with high caramel aroma yield and high 5-HMF degradation, which can increase the content of caramel aroma substances in the mousse.

Another object of the invention is to provide a fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation, which can effectively degrade the content of 5-HMF in the musalaisi.

The last purpose of the invention is to provide a musalaisi fermentation brewing process with high caramel aroma yield and high 5-HMF degradation, which does not produce byproducts, and is safe, environment-friendly and pollution-free.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a musalaisi fermentation brewing process with high caramel aroma yield and high 5-HMF degradation, which comprises the following steps:

s1: screening and cleaning raw materials: ripe Hetian red grapes are picked and washed.

S2: raw material stem removal and crushing: and (3) removing stems of the selected grapes, crushing the grapes, and conveniently squeezing the grapes to obtain juice.

S3: squeezing to obtain juice: the grapes are pressed to obtain grape juice.

S4: concentration: concentrating the grape juice to a sugar degree of 23-35 Brix, cooling to room temperature, and then pumping into a fermentation tank.

S5: primary inoculation: inoculating the activated fresh culture solution of the F2-P85-3 strain and the fresh culture solution of the C0-Y2-02 strain into concentrated grape juice of a fermentation tank for primary inoculation according to the inoculation amount of 2%.

S6: degrading 5-HMF and fermenting: and (2) performing temperature-controlled fermentation on the concentrated grape juice subjected to primary inoculation at 28 ℃ by using a static fermentation method, degrading 5-HMF in the concentrated grape juice, and monitoring the sugar degree, the alcohol content and the nitrogen level every day so that the sugar degree is not more than 196g/L, the nitrogen content is not more than 230mg/L, and the alcohol content is not more than 8%.

S7: secondary inoculation: after the fermentation of the degraded 5-HMF is finished, inoculating the activated fresh culture solution of the F2-B163-2 strain into concentrated grape juice of a fermentation tank for secondary inoculation according to the inoculation amount of 2 percent.

S8: caramel aroma producing fermentation: and (3) fermenting the concentrated grape juice subjected to secondary inoculation to generate caramel aroma at the fermentation temperature of 28 ℃ until the foam is completely disappeared, returning the product temperature to the room temperature, and finishing the fermentation to obtain the finished product of the musalaisi.

Further, the high performance liquid pre-column derivatization method is adopted to detect the concentration content of the total amino acids in the grape juice of the raw material in S1, and the N element level of the red grape selected in S1 is ensured to be not lower than 230 mg/L.

Further, the juice is squeezed in the S3 to ensure that the juice yield is over 75 percent.

Further, the strain F2-P85-3 and the strain C0-Y2-02 in the S5 are both 1%.

Further, in the S5, a YPD culture medium or fresh concentrated grape juice is used for activating the F2-P85-3 strain and the JCY2 strain, the activation temperature is 28 ℃, and the activation time is 24 hours. The F2-P85-3 strain is a saccharomyces cerevisiae strain with excellent caramel aroma and flavor.

Further, when the sugar concentration is 189g/L, the alcoholic strength is 6.9 percent and the nitrogen concentration is 230mg/L in the step S6, performing secondary fermentation until the foam falls back, and cooling the product temperature to room temperature to finish the fermentation.

Further, in the S7, the F2-B163-2 strain is activated by YPD medium at 28 ℃ for 24 h. The F2-B163-2 strain is a strain with high yield of furanone.

Furthermore, in the S8 fermentation process, coating observation is carried out every day, so that the normal growth of the F2-B163-2 fermentation strain inoculated twice is ensured, and the smooth completion of fermentation is ensured.

Further, the YPD medium comprises: 2% of glucose, 2% of peptone, 1% of yeast extract powder and the balance of distilled water, wherein the pH value of the YPD medium is a natural pH value, and the YPD medium is sterilized at high temperature and high pressure by using a high-temperature high-pressure vertical sterilizing pot. The sterilization temperature of the high-temperature and high-pressure sterilization is 121 ℃, the sterilization pressure is 0.12MPa, and the sterilization time is 15 min.

Further, the biological preservation information of the F2-P85-3 strain is as follows:

and (3) classification and naming: saccharomyces cerevisiae, latin name: saccharomyces cerevisiae;

the name of the depository: china general microbiological culture Collection center;

the address of the depository: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North;

the preservation date is as follows: 03 month 10 in 2021;

the preservation number is: CGMCC No. 21886.

Further, the biological preservation information of the F2-B163-2 strain is:

and (3) classification and naming: kluyveromyces pichia, latin name: pichia pastoris;

the preservation date is as follows: 03 month 10 in 2021;

the preservation number is: CGMCC No. 21887.

Further, the biological preservation information of the C0-Y2-02 strain is as follows:

and (3) classification and naming: zygosaccharomyces bailii, latin name: zygosaccharomyces bailii;

the preservation date is as follows: 03 month 10 in 2021;

the preservation number is: CGMCC No. 21885.

The invention has the beneficial effects that: compared with the prior art, firstly, the Musalace is fermented by compounding the saccharomyces cerevisiae F2-P85-3 and the F2-B163-2 strain with high yield of furanone, so that the content of caramel aroma substances in the Musalace can be effectively increased, and the taste of the Musalace is improved; secondly, the process simultaneously adopts C0-Y2-02 bacterial strain for high degradation of 5-HMF for fermentation, so that the content of 5-HMF in the musalaisi can be effectively degraded; finally, the process adopts a fermentation mode, does not produce byproducts, and is safe, environment-friendly and pollution-free.

Drawings

FIG. 1 is a flow diagram of a fermentation process of the present invention;

FIG. 2 is a graph showing the data of the number of fermentation bacteria of mixed bacteria (F2-P85-3, C0-Y2-02 and F2-B163-2) 7 days before fermentation in the fermentation and brewing process of Musalaisi;

FIG. 3 is a graph showing the data of the number of fermentation bacteria of a control strain EC1118 7 days before fermentation in the fermentation brewing process of Musalace of the present invention;

FIG. 4 is a graph showing the data on the change in sugar concentration of mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) and a control group (EC1118 strain) during fermentation in accordance with the present invention;

FIG. 5 is a graph showing the data of the alcohol concentrations produced by the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) and the control group (EC1118 strain) in the fermentation process of the present invention;

FIG. 6 is a graph of sensory score data of the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02)) fermented wine of the present invention versus the control (EC1118) fermented wine;

FIG. 7 is a graph showing the furan compound concentrations of the fermented liquors of the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) of the present invention and the control (EC1118) fermented liquor;

FIG. 8 is a data graph showing the degradation amount and residual amount of 5-hydroxymethylfurfural in the mixed-bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) fermented liquors of the present invention and the control (EC1118) fermented liquor;

FIG. 9 is a plate morphology of the high caramel aroma-producing strain of the present invention (F2-B163-2) WL;

FIG. 10 is a morphology of sensory evaluation strain (F2-P85-3) WL plate of the present invention;

FIG. 11 is a morphology of 5-hydroxymethylfurfural-degrading strain (C0-Y2-02) WL plate of the present invention;

FIG. 12 is a shape chart of mixed WL plates of three bacteria of a high caramel aroma-producing strain (F2-B163-2), a 5-hydroxymethylfurfural-degrading strain (C0-Y2-02) and an organoleptic evaluation strain (F2-P85-3) of the invention;

FIG. 13 is a graph of the data of the tolerance analysis of the caramel highly productive strain (F2-B163-2) of the present invention to OD value variation at different sugar concentrations;

FIG. 14 is a graph of the tolerance analysis of the caramel highly-producing strain of the present invention (F2-B163-2) to the production of a furan species at different sugar concentrations;

FIG. 15 is a graph of the data on the variation of OD values at different N levels for the tolerance analysis of the caramel highly producing strain of the present invention (F2-B163-2);

FIG. 16 is a graph of tolerance analysis of a high caramel aroma-producing strain of the present invention (F2-B163-2) to the production of a furan species at different levels of N;

FIG. 17 is a graph of the tolerance analysis of the caramel highly-producing strain (F2-B163-2) of the present invention with OD value variation data at different alcohol concentrations;

FIG. 18 is a graph of tolerance analysis of a high caramel aroma-producing strain of the present invention (F2-B163-2) to the production of a furan species at different alcohol concentrations;

FIG. 19 is a graph showing the data of the OD value variation at different sugar concentrations in the tolerance analysis of the C0-Y2-02 strain of the present invention;

FIG. 20 is a graph showing the variation of the concentration of degraded 5-hydroxymethylfurfural in the case of the C0-Y2-02 strain tolerance analysis according to the present invention at different concentrations of brix;

FIG. 21 is a graph showing the variation data of OD values at different concentrations of 5-hydroxymethylfurfural in the tolerance analysis of the C0-Y2-02 strain according to the present invention;

FIG. 22 is a graph showing the variation of the concentration of degraded 5-hydroxymethylfurfural at different concentrations of 5-hydroxymethylfurfural in the tolerance analysis of the C0-Y2-02 strain of the present invention;

FIG. 23 is a graph showing the data of the OD value variation at different alcohol concentrations in the tolerance analysis of the C0-Y2-02 strain of the present invention;

FIG. 24 is a graph showing the variation of the concentration of degraded 5-hydroxymethylfurfural in different alcohol concentrations in the tolerance analysis of the C0-Y2-02 strain according to the present invention;

FIG. 25 is a graph showing the data of the OD value changes at different levels of N in the tolerance analysis of the C0-Y2-02 strain of the present invention;

FIG. 26 is a graph of the tolerance assay of the C0-Y2-02 strain of the invention to degraded 5-hydroxymethylfurfural at different levels of N;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

To achieve the above object, the present invention is implemented as follows:

screening high-yield caramel aroma strains:

s21: a total of 65 strains of Saccharomyces cerevisiae and non-Saccharomyces cerevisiae were selected and numbered as in Table 1.

S22: activation of the strain: the 65 types of the culture solution preserved with 25% glycerol were inoculated and activated in a sterilized medium containing 1ml of YPD. The inoculation amount is 2 percent, and the culture is carried out for 24 hours at the constant temperature of (28 +/-1) DEG C. The YPD medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract powder and distilled water, wherein the pH value of the culture medium is a natural pH value, and autoclaving at 121 ℃ for 15 min.

S23: preparing concentrated grape juice:

s231: and selecting raw materials of the red grape: ripe and red grape raw materials, and selecting Chinese olive, foreign matters, rotten fruits and the like. And cleaning the selected grapes.

S232: removing stems: and (3) manually removing stems of the cleaned grapes.

S233: squeezing to obtain juice: and squeezing the grapes subjected to stem removal by using a screw press to obtain juice.

S234: concentrating grape juice: boiling and concentrating the Sucus Vitis viniferae until the sugar degree reaches 28 Brix.

S235: packaging and sterilizing: and (3) filling the concentrated grape juice into triangular bottles, sealing the triangular bottles by 300ml each, placing the triangular bottles into a vertical pressure steam sterilizer for sterilization at the sterilization temperature of 121 ℃ for 15min, and cooling to room temperature for later use.

S24: inoculating and fermenting: inoculating the activated 65 bacterial liquids to be tested into sterilized concentrated grape juice respectively according to the inoculation amount of 2%, putting the grape juice into an incubator at 28 ℃ for fermentation for 14 days, and measuring the generation amount of furanone by HLPC.

S25: detecting the generation amount of caramel fragrant substances: and (3) respectively measuring the concentrations of furanone, acetylfuran and 5-methylfurfural in the fermented concentrated grape juice by using a high performance liquid chromatography, wherein the substances of the furanone, the acetylfuran and the 5-methylfurfural have caramel aroma characteristics and can be used for judging the content of the caramel aroma substances in the product. The data for the concentrations of furanone, acetylfuran and 5 methylfurfural are shown in table 2.

S26: analysis of caramel aroma OVA value results: the OAV value is the aroma activity value of the yeast fermented wine and can be used for representing the caramel aroma content in the musalaisi. The ratio between the content of OAV value in a food product and its aroma threshold in a particular substrate (water, oil, etc.). The threshold value of furanone in water is 0.16mg/L, the threshold value of acetylfuran in water is 10mg/L, and the threshold value of 5-methylfurfural in water is 466.32 mg/L. Data indicating the sum of OAVs of furanone, acetylfuran and 5 methylfurfural among the 65 strains by calculation are shown in table 3, and the strain with the highest sum of OAVs was JFB2 strain with an OAV value of 996.21.

S27: sensory evaluation: seven testers are selected, the caramel aroma is distinguished according to the wine noses, the caramel aroma after 65 strains of bacteria are fermented is graded according to 1-5, and 1 represents that the caramel aroma is light and almost not available; 2-3 represents that the caramel has common fragrance and is not strong; 4-5 represents the strong fragrance of caramel. An alcohol aqueous solution of furanone. Preparing 5 furanone concentration gradients of 200mg/L, 40mg/L, 8mg/L, 1.6mg/L and 0.32mg/L respectively, and reference solution (concentration is obtained by detecting concentration in fermented wine), randomly numbering, presenting to an evaluator, identifying the furanone reference solution (1-5 points in scale), and asking the evaluator to score the wine sample according to the scoring rule and the scoring requirement, wherein the scoring results are shown in Table 5. As can be seen from the scoring Table 5, the fermentation broth of the strain No. F-B18-6 had the highest score, followed by the strain Nos. F2-C507-4 and F2-P85-3, (F-B18-6 and F2-C507-4 were non-Saccharomyces cerevisiae, and the strain F2-P85-3 was Saccharomyces cerevisiae).

Screening of high-degradation 5-HMF strains:

s31: a total of 65 strains of Saccharomyces cerevisiae and non-Saccharomyces cerevisiae were selected and numbered as in Table 1.

S32: activation, streak culture and enrichment culture of the strain:

s321: and (3) activation: the 65 types of the culture solution preserved with 25% glycerol were inoculated and activated in a sterilized medium containing 1ml YPD. The inoculation amount is 2 percent, and the culture is carried out for 24 hours at the constant temperature of (28 +/-1) DEG C. The YPD medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract powder and distilled water, wherein the pH value of the culture medium is a natural pH value, and autoclaving at 121 ℃ for 15 min.

S322: and (3) streak culture: and (3) streaking the activated strain into a solid culture medium by using a sterile inoculating loop, and culturing at the constant temperature of 28 ℃ for 24 hours to grow a single colony.

S323 enrichment culture: and (3) picking out a single colony strain by using a sterile inoculating loop, inoculating the single colony strain into YPD culture solution, and culturing at the constant temperature of 28 ℃ for 24 hours for later use.

S33: preparing concentrated grape juice:

s331: and selecting raw materials of the red grape: ripe and red grape raw materials, and selecting Chinese olive, foreign matters, rotten fruits and the like. And cleaning the selected grapes.

S332: removing stems: and (3) manually removing stems of the cleaned grapes.

S333: squeezing to obtain juice: and squeezing the grapes subjected to stem removal by using a screw press to obtain juice.

S334: concentrating grape juice: boiling and concentrating the Sucus Vitis viniferae until the sugar degree reaches 28 Brix.

S335: packaging and sterilizing: and (3) filling the concentrated grape juice into triangular bottles, sealing the triangular bottles by 300ml each, placing the triangular bottles into a vertical pressure steam sterilizer for sterilization at the sterilization temperature of 121 ℃ for 15min, and cooling to room temperature for later use.

S34: inoculating and fermenting: and respectively inoculating the activated 65 bacterial liquids to be detected into sterilized concentrated grape juice with the inoculation amount of 2%, fermenting in an incubator at 28 ℃ for 14 days, and performing degradation 5-HMF detection analysis.

S35: results and analysis: the detection results of the high-efficiency liquid relative to 5-hydroxymethylfurfural in 65 yeast fermentation liquid are shown in table 4. The number of the strain with the maximum degradation content of 5-hydroxymethylfurfural in 65 yeasts is JCY2, and the degradation content of 5-hydroxymethylfurfural is 0.84 g/L.

By comprehensive analysis, the JFB2 strain is a non-saccharomyces cerevisiae strain with excellent caramel aroma, the C0-Y2-02 strain is a non-saccharomyces cerevisiae strain with high degradation of 5-hydroxymethylfurfural, and the F2-P85-3 strain has the best sensory evaluation on caramel aroma and is a saccharomyces cerevisiae strain. The production process of the musalaisi capable of producing the caramel aroma with high yield and degrading the 5-hydroxymethyl furfural is characterized in that saccharomyces cerevisiae F2-P85-3, an F2-B163-2 strain with high yield of furanone and a C0-Y2-02 strain with high degradation of the 5-hydroxymethyl furfural are subjected to sequential mixed fermentation to produce the musalaisi capable of producing the caramel aroma with high yield and degrading the 5-hydroxymethyl furfural with high yield.

Table 1:

table 2:

table 3:

table 4:

table 5:

strain name	Sensory scoring	Strain name	Sensory scoring
				A3-18d1-02	2.87	F2-16d3-01	2.73
E1-12d2-01	2.87	G0-Y7	2.87
				F1-L193-6	2.73	C0-1d1-05	2.73
G3-12d4-01	2.40	G2-7d1-04	2.73
				B0-Y3-02	3.0	G0-Y9	2.80
F0-B163-2	2.07	A0-Y2-01	2.73
				P-1L1-15	2.07	H0-6d2-03	2.67
F2-M93-20	2.13	E2-21d3-01	2.40
				G3-12d3-03	2.47	C1-7d3-02	2.73
F1-M127-3A	2.60	F2-P85-3	3.13
				F2-P85-1	2.60	F1-7d2-04	2.93
F1-S487-8	2.80	C1-AR3-03	2.87
				G2-9d2-02	2.27	B1-8d1	2.60
F2-B178-11	3.00	H0-6d5-01	2.80
				A0-Y8-02	2.67	G1-3d1-06	2.60
F2-A62-3	2.73	C2-12d1-03	2.73
				F0-T427-1	2.87	F0-Y3-02	2.80
F1-B256-8	2.73	C2-12d2-03	2.33
				F1-X205-4	2.60	H0-6d2-02	2.67
T-344-1	2.67	G2-9d1-05	2.40
				F2-C507-4	3.20	EC1118	2.73
F2-P88-6	2.67	E0-1Y2-004	2.80
				H0-6d3-03	2.80	F0-Y2-02	2.87
F1-X208-4	2.67	B0-Y3-01	2.60
				F0-L368-1	2.53	Blank space	1.67
I0-15d1-07	3.0
				A3-18d3-01	2.67
Beijing	2.40
				G0-Y4	2.67
I0-15d3-03	2.40
				A0-3d1-03	2.87
F2-B163-02	2.93
				I0-15d2-08	2.73
A1-4d5	2.53
				F-B18-6	3.8
B3-23d1-02	3.13
				G2-9d7-01	2.67
A4-25d1-01	2.73
				B0-3d1-06	2.47
G3-12d1-04	2.67

Fermentation and brewing of musalaisi with high caramel aroma yield and high 5-HMF degradation:

S3: squeezing to obtain juice: the grapes are pressed to obtain grape juice.

Detecting the concentration content of total amino acids in the grape juice of the raw material in S1 by using a high performance liquid pre-column derivatization method, and ensuring that the N element level of red grapes selected in S1 is not lower than 230 mg/L.

The juice is squeezed in the S3 to ensure that the juice yield is over 75 percent.

In the S5, the strain F2-P85-3 and the strain C0-Y2-02 are both 1 percent.

In the S5, a YPD culture medium or fresh concentrated grape juice is used for activating the F2-P85-3 strain and the C0-Y2-02 strain, the activation temperature is 28 ℃, and the activation time is 24 hours.

And in the S6, when the sugar concentration reaches 189g/L, the alcoholic strength reaches 6.9 percent and the nitrogen concentration reaches 230mg/L, performing secondary fermentation until foam falls back, and cooling the product to room temperature to finish the fermentation.

In the S7, a YPD culture medium is used for activating the F2-B163-2 strain, the activation temperature is 28 ℃, and the activation time is 24 hours.

In the S8 fermentation process, coating observation is carried out every day, so that the normal growth of the F2-B163-2 fermentation strain inoculated for the second time is ensured, and the smooth completion of fermentation is ensured.

The YPD medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract powder and the balance of distilled water, wherein the pH value of the YPD medium is a natural pH value, and the YPD medium is sterilized at high temperature and high pressure by using a high-temperature high-pressure vertical sterilizing pot. The sterilization temperature of the high-temperature and high-pressure sterilization is 121 ℃, the sterilization pressure is 0.12MPa, and the sterilization time is 15 min.

The biological preservation information of the F2-P85-3 strain is as follows:

the biological preservation information of the F2-B163-2 strain is as follows:

the biological preservation information of the JCY2 strain is as follows:

evaluation of superior strain F2-B163-2, Saccharomyces cerevisiae strain F2-P85-3 and superior strain C0-Y2-02 for mixed fermentation of concentrated grape juice with high caramel aroma yield and high degradation of 5-HMF:

the concentration data of the number of mixed bacteria (F2-P85-3, JCY2 and F2-B163-2) before 7 days of fermentation are shown in FIG. 2. The data of the number of fermentation bacteria of the control strain EC1118 7 days before fermentation are shown in FIG. 3. As can be seen from FIGS. 2 and 3, the concentration of the number of bacteria reached a maximum of 3.24X108(CFU/ml) and 2.8X108(CFU/ml) at 3d by inoculating the strains (F2-P85-3) and (C0-Y2-02) first, and then the concentration of the number of bacteria began to decrease slowly. The strain was then inoculated at 5d (F2-B163-2) and slow growth commenced, with the cell count concentration reaching a maximum of 1.8X108(CFU/ml) at 8 d. The concentration of the number of bacteria in the control group (EC1118) reached a maximum of 3.1X108(CFU/ml) at 3d, and then the concentration of the number of bacteria began to decrease slowly.

The data of the sugar concentration changes of the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) and the control group (EC1118) during the fermentation process are shown in FIG. 4. As can be seen from FIG. 4, the sugar concentration varied within 30 days, and the sugar concentration consumed during the fermentation with the mixed strain was significantly higher than that consumed during the fermentation with the control strain EC1118, and after 30 days of fermentation, the sugar concentrations in the fermented liquors with the mixed strains (F2-P85-3), (F2-B163-2) and (C0-Y2-02) were 113g/L and the sugar concentration in the fermented liquor with the control strain EC1118 was 131 g/L.

The data of the alcohol concentrations generated by the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) and the control group (EC1118) during the fermentation process are shown in FIG. 5. As can be seen from FIG. 5, the alcohol production during the fermentation of the mixed strain was slightly higher than that of the control strain EC1118 due to the variation of the alcohol content within 30 days, and the alcohol contents of the fermented liquors were 17.02% for the mixed strains (F2-P85-3), (F2-B163-2) and (C0-Y2-02) and 15.82% for the control strain EC 1118.

In conclusion, as can be seen from the sugar concentration fermentation graph and the alcohol fermentation graph in the fermentation process of the mixed inoculated strains (F2-P85-3), (F2-B163-2) and (C0-Y2-02) and the control group EC1118, the sugar fermentation capacities of the mixed inoculated strains (F2-P85-3), (F2-B163-2) and (C0-Y2-02) are higher than the sugar fermentation capacity of the control group EC1118, so that the alcohol fermentation capacities of the mixed inoculated strains (F2-P85-3), (F2-B163-2) and (C0-Y2-02) are naturally higher than the alcohol fermentation capacity of the control group EC 1118.

Sensory score data of the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02)) fermented wine and the control (EC1118) fermented wine are shown in FIG. 6.

The furan content of the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) fermented wine and the control (EC1118) fermented wine are shown in FIG. 7, and it can be seen from FIG. 7 that: 172.31mg/L of furanone, 2.54mg/L of acetylfuran and 24.40mg/L of 5-methylfurfural, which are generated by fermenting three strains of F2-P85-3, F2-B163-2 and C0-Y2-02. 56.83mg/L of furanone, 1.83mg/L of acetylfuran and 9.93mg/L of 5-methylfurfural are generated by the fermentation of the control group EC 1118. The furan substances produced by the mixed fermentation of the three strains F2-P85-3, F2-B163-2 and C0-Y2-02 are higher than those produced by the fermentation of the control group EC 1118. As can be seen from the sensory evaluation chart of FIG. 6, the aroma of the caramel fermented mixture of the three strains F2-P85-3, F2-B163-2 and C0-Y2-02 is stronger than that of the caramel fermented by the control EC 1118.

The data of the degradation amount and residual amount of 5-hydroxymethylfurfural in the mixed bacteria (F2-P85-3, F2-B163-2 and C0-Y2-02) fermented wine and the control (EC1118) fermented wine are shown in FIG. 8. As can be seen from FIG. 8, when the strains F2-P85-3, F2-B163-2 and C0-Y2-02 were mixed and fermented for 30 days, the residual 5HMF of 5-hydroxymethylfurfural in the fermented wine was 0.13g/L, the concentration of degraded 5-hydroxymethylfurfural was 0.68g/L, and the degradation rate was 83.9%. The residual 5HMF of the 5-hydroxymethylfurfural in the control group EC1118 fermented wine is 0.16g/L, the concentration of the degraded 5-hydroxymethylfurfural is 0.65g/L, and the degradation rate is 80.2%. The advantages of mixed fermentation and degradation of 5-hydroxymethylfurfural by F2-P85-3, F2-B163-2 and C0-Y2-02 are great.

The high caramel aroma-producing strain (F2-B163-2) WL plate morphology is shown in 9: large volume, rough surface, white color with light green color.

Sensory evaluation of the strain (F2-P85-3) WL plate morphology is shown in FIG. 10: large volume, smooth surface, white creamy.

The morphology of the strain for degrading 5-hydroxymethylfurfural (C0-Y2-02) WL plate is shown in FIG. 11: small volume, smooth surface, white creamy.

The forms of mixed WL plates of a high caramel aroma-producing strain (F2-B163-2), a 5-hydroxymethylfurfural-degrading strain (C0-Y2-02) and a sensory evaluation strain (F2-P85-3) are shown in FIG. 12.

High caramel incense-producing strains (F2-B163-2) tolerance analysis:

1. tolerance analysis for different sugar degrees: the simulated grape juice is used as a fermentation substrate, the sugar concentrations are respectively 4g/L, 68g/L, 132g/L, 196g/L and 260g/L, and the other components are unchanged, and the simulated grape juice is set into 5 treatment groups of 4S, 68S, 132S, 196S and 260S. Each treatment volume is 32mL, the treatment volume is filtered and sterilized through a filter membrane with the aperture of 0.22um, sterilized simulated grape juice is respectively filled into 410 mL sterilized centrifuge tubes, 8mL of each centrifuge tube is respectively connected with F2-B163-2 fresh culture solution, the inoculation amount is 2%, the constant temperature culture is carried out at 28 ℃, the start of 7 days is carried out, the OD value (absorbance OD600nm) is measured every 24 hours, the growth condition of the strain is observed, the fermentation is carried out for 30d, the concentration of furan substances is detected by using HPLC, and the generation amount is calculated. The OD value change data under different sugar concentrations in the fermentation process is shown in figure 13, simulated grape juice is used as a fermentation substrate, the sugar concentrations in the prepared fermentation substrate are respectively 4g/L, 68g/L, 132g/L, 196g/L and 260g/L, the growth OD value of the strain F2-B163-2 is detected by a microplate reader every day 7 days before the constant-temperature fermentation at 28 ℃ by controlling the different changes of the sugar concentrations in the simulated grape juice, and figure 13 shows that the strain F2-B163-2 has different growth capacities under different sugar concentrations and is influenced by the culture time. It is clear that low sugar (4g/L) is the least favorable for bacterial growth, 68g/L is inferior in sugar degree, and the low sugar degree obviously delays a specific growth peak value. The OD values of other sugar degree treatments are not obviously different in the fermentation period, and the OD values of all treatment groups in the 4d fermentation period reach the maximum; among them, the OD value was highest in the 196g/L treatment group and reached approximately 1. The change of the production concentration of the furan substance under different sugar concentrations in the fermentation process is shown in figure 14, after the fermentation liquor is placed at a standstill of 28 ℃ for 30 days, the production amount of the furan substance is detected by HPLC, as shown in figure 14, obviously, when the sugar degree is less than or equal to 196g/L, the production amount of the furan substance is increased along with the increase of the sugar degree until the sugar concentration is 196g/L, the production amount of the furan substance reaches the maximum (the concentration of furanone is 30.48mg/L, the concentration of acetylfuran is 1.088mg/L, and the concentration of 5 methylfurfural is 4.78mg/L), and the production amount of the furan substance is slightly reduced in the following 260S, but has no significant difference with 196S.

2. Tolerance analysis for different nitrogen levels: the simulated grape juice is used as a fermentation substrate, the nitrogen concentrations are respectively 140mg/L, 230mg/L, 330mg/L, 410mg/L and 500mg/L, and other components are unchanged, and the simulated grape juice is set into 140N, 230N, 330N, 410N and 500N 5 treatment groups. The volume of each treatment group is 32mL, the treatment groups are filtered and sterilized through a filter membrane with the aperture of 0.22um, sterilized simulated grape juice is respectively filled into 410 mL sterilized centrifuge tubes, each centrifuge tube is 8mL, fresh culture solution of F2-B163-2 yeast is respectively added, the inoculation amount is 2%, the constant temperature culture is carried out at 28 ℃, the OD value is measured every 24 hours after the start for 7 days, the growth condition of the strain is observed, the strain is fermented for 30d, the concentration of furans is detected by HPLC, the OD value changes under different N concentrations are shown in figure 15, the simulated grape juice is used as a fermentation substrate, different nitrogen concentrations in the fermentation substrate are 140mg/L, 230mg/L, 330mg/L, 410mg/L and 500mg/L, F2-B163-2 bacteria are added, the constant temperature fermentation is carried out at 28 ℃, the previous 7 days, the OD values of the strain are detected by an enzyme labeling instrument every day, the OD value changes of the low nitrogen level N140 and the strain OD value changes in the treatment group with the low nitrogen level N230 are similar, a growth peak value; the OD values for the medium nitrogen level N330 and N410 treatment groups varied approximately, and the OD value for the high nitrogen level treatment group N500 was slightly lower overall than the other treatment groups, indicating that the high nitrogen level was somewhat consistent. All treatment groups, 3d, reached a growth peak with N140 higher than the other treatment groups. The variation of the production concentration of furan species at different concentrations of biotin during fermentation is shown in FIG. 16.

3. Tolerance analysis of different alcohol amounts: the simulated grape juice was used as a fermentation substrate, and the alcohol concentrations of 8%, 10%, 12%, 14% and 16% were prepared, and the other components were unchanged, and the treatment groups were set to 8A, 10A, 12A, 14A and 16A 5. Each treatment volume is 32mL, the treatment volume is filtered and sterilized through a filter membrane with the aperture of 0.22um, sterilized simulated grape juice is respectively filled into 410 mL sterilized centrifuge tubes, 8mL of each centrifuge tube is respectively connected with fresh culture solution of F2-B163-2 yeast, the inoculation amount is 2%, the constant temperature culture is carried out at 28 ℃, the OD value is measured every 24 hours after the beginning of 7 days, the growth condition of the strain is observed, the fermentation is carried out for 30d, the concentration of furan substances is detected by using HPLC, and the generation amount is calculated. The change in OD values at different alcohol concentrations during fermentation is shown in FIG. 17. The variation of the production concentration of the furanic substances at different alcohol concentrations during the fermentation is shown in FIG. 18.

And (3) analyzing the tolerance of the high-degradation 5-hydroxymethylfurfural strain:

1. tolerance characteristics at different sugar concentrations: the simulated grape juice is used as a fermentation substrate, the prepared sugar concentrations are respectively 4g/L, 68g/L, 132g/L, 196g/L and 260g/L, other components are unchanged, and a 5-hydroxymethylfurfural standard is added to prepare the 5-hydroxymethylfurfural with the concentration of 3 g/L. Preparing 32mL of each sugar concentration, filtering and sterilizing through a filter membrane with the aperture of 0.22um, respectively filling sterilized simulated grape juice into 410 mL sterilized centrifuge tubes, respectively adding 8mL of each centrifuge tube into a fresh culture solution of C0-Y2-02 yeast, respectively, inoculating 2 percent of strain, culturing at the constant temperature of 28 ℃, starting for 7 days, measuring an OD value every 24 hours, observing the growth condition of the strain, fermenting for 30 days, detecting the concentration of 5HMF by using HPLC, and calculating the degradation amount. The change in OD values at different sugar concentrations during fermentation is shown in FIG. 19. The variation of the concentration of degraded 5-hydroxymethylfurfural at different sugar concentrations during fermentation is shown in fig. 20. After the fermentation liquids are statically placed at 28 ℃ for 30 days, the degradation amplitude of 5-hydroxymethylfurfural is detected by HPLC, as can be seen from fig. 2-3, the significant difference exists between the treatment groups of 132g/L, 196g/L and 260g/L and the treatment groups of 4g/L and 68g/L, and no significant difference exists in each group, wherein the maximum degradation amount of 5-hydroxymethylfurfural in the treatment group of 196g/L is 2.92 g/L. The change in OD values at different sugar concentrations during fermentation is shown in FIG. 19. The variation of the concentration of degraded 5-hydroxymethylfurfural at different sugar concentrations during fermentation is shown in fig. 20.

2. Different 5-hydroxymethylfurfural concentration tolerance characteristics: the simulated grape juice is used as a fermentation substrate, the prepared 5-hydroxymethylfurfural is 2g/L, 4g/L, 6g/L, 8g/L and 10g/L respectively, and other components are unchanged. The preparation volume of each 5-hydroxymethylfurfural is 32mL, the 5-hydroxymethylfurfural is filtered and sterilized through a filter membrane with the aperture of 0.22um, sterilized simulated grape juice is respectively filled into 410 mL sterilized centrifuge tubes, 8mL of each centrifuge tube is respectively inoculated with a fresh culture solution of C0-Y2-02 yeast, the inoculation amount is 2%, the constant temperature culture is carried out at 28 ℃, 7 days are started, the OD value is measured every 24 hours, the growth condition of a strain is observed, the fermentation is carried out for 30 days, the concentration of the 5-hydroxymethylfurfural is detected by using HPLC, and the degradation amount is calculated. The change of OD values at different concentrations of 5-hydroxymethylfurfural during fermentation is shown in FIG. 21. The change in the concentration of degraded 5-hydroxymethylfurfural at different concentrations of 5-hydroxymethylfurfural during fermentation is shown in fig. 22.

3. Tolerance characteristics for different alcohol contents: taking simulated grape juice as a fermentation substrate, respectively preparing alcohol concentrations of 8%, 10%, 12%, 14% and 16%, keeping other components unchanged, adding a 5-hydroxymethylfurfural standard substance, and preparing the concentration of 5-hydroxymethylfurfural to be 3 g/L. Preparing 32mL of alcohol content, filtering and sterilizing through a filter membrane with the aperture of 0.22um, respectively filling sterilized simulated grape juice into 410 mL sterilized centrifuge tubes, respectively adding 8mL of each centrifuge tube into a fresh culture solution of C0-Y2-02 yeast, respectively, inoculating 2% of yeast, culturing at the constant temperature of 28 ℃, starting for 7 days, measuring an OD value every 24 hours, observing the growth condition of a strain, fermenting for 30 days, detecting the concentration of 5-hydroxymethylfurfural by using HPLC, and calculating the degradation amount. The change of the OD value under different alcohol concentrations in the fermentation process is shown in FIG. 23, and the change of the concentration of the degraded 5-hydroxymethylfurfural under different alcohol concentrations in the fermentation process is shown in FIG. 24.

4. Tolerance characteristics at different nitrogen levels: taking simulated grape juice as a fermentation substrate, preparing nitrogen concentrations of 140mg/L, 230mg/L, 330mg/L, 410mg/L and 500mg/L respectively, keeping other components unchanged, adding a 5-hydroxymethylfurfural standard substance, and preparing the concentration of 5-hydroxymethylfurfural to be 3 g/L. Configuring the volume of each nitrogen concentration to be 32mL, filtering and sterilizing through a filter membrane with the aperture of 0.22um, respectively filling sterilized simulated grape juice into 410 mL sterilized centrifuge tubes, respectively adding 8mL of each centrifuge tube into a fresh culture solution of C0-Y2-02 yeast, respectively inoculating 2% of the yeast, culturing at the constant temperature of 28 ℃, starting for 7 days, measuring an OD value every 24 hours, observing the growth condition of a strain, fermenting for 30 days, detecting the concentration of 5-hydroxymethylfurfural by using HPLC (high performance liquid chromatography), and calculating the degradation amount. The change in OD values at different concentrations of Nmin during fermentation is shown in FIG. 25. The variation of the concentration of degraded 5-hydroxymethylfurfural at different concentrations of biotin during fermentation is shown in fig. 26.

The above embodiments are only for illustrating the present invention, and the scope of the present invention is not limited to the above embodiments. The objectives of the present invention can be achieved by the ordinary skilled person in the art according to the disclosure of the present invention and the ranges of the parameters.

Claims

1. The musalaisi fermentation brewing process with high caramel aroma yield and high 5-HMF degradation is characterized by comprising the following steps of:

s1: screening and cleaning raw materials: selecting and cleaning mature Hetian red grapes;

s3: squeezing to obtain juice: squeezing grapes to obtain grape juice;

s4: concentration: concentrating the grape juice to a sugar degree of 23-35 Brix, cooling to room temperature, and then pumping into a fermentation tank;

s5: primary inoculation: inoculating the activated fresh culture solution of the F2-P85-3 strain and the fresh culture solution of the C0-Y2-02 strain into concentrated grape juice of a fermentation tank for primary inoculation according to the inoculation amount of 2 percent;

s6: degrading 5-HMF and fermenting: performing 28 ℃ temperature-controlled fermentation on the concentrated grape juice subjected to primary inoculation by using a static fermentation method, degrading 5-HMF in the concentrated grape juice, and monitoring the sugar content, the alcohol content and the nitrogen level every day so that the sugar content is not more than 196g/L, the nitrogen content is not more than 230mg/L, and the alcohol content is not more than 8%;

s7: secondary inoculation: after the 5-HMF degradation fermentation is finished, inoculating the activated fresh culture solution of the F2-B163-2 strain into concentrated grape juice of a fermentation tank for secondary inoculation according to the inoculation amount of 2 percent;

2. The process of claim 1, wherein the process further comprises S2: raw material stem removal and crushing: and (3) removing stems of the selected grapes, crushing the grapes, and conveniently squeezing the grapes to obtain juice.

3. The fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein the total amino acid concentration of the grape juice of the raw material is detected by high performance liquid pre-column derivatization in S1, ensuring that the N element level of the red grapes selected in S1 is not lower than 230 mg/L.

4. The process of claim 1, wherein the squeezing of the juice at S3 ensures a grape yield of greater than 75%.

5. The fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein the strains of F2-P85-3 and C0-Y2-02 in S5 are all inoculated in 1%.

6. The fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein the F2-P85-3 strain is activated by YPD medium or fresh concentrated grape juice at 28 ℃ for 24h in S5.

7. The fermentation and brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein in S6, when the sugar concentration reaches 189g/L, the alcoholic strength reaches 6.9%, and the nitrogen concentration reaches 230mg/L, the secondary fermentation is performed until the foam falls back, and the product temperature is reduced to room temperature to finish the fermentation.

8. The fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein the F2-B163-2 strain is activated by YPD medium at 28 ℃ for 24h in S7.

9. The fermentation brewing process of musalaisi with high caramel aroma yield and high 5-HMF degradation of claim 1, wherein during the fermentation process of S8, the fermentation process is performed daily, so as to ensure the normal growth of the secondarily inoculated F2-B163-2 fermentation strain and the smooth completion of the fermentation.

10. The process of claim 1, wherein the YPD medium comprises: 2% of glucose, 2% of peptone, 1% of yeast extract powder and the balance of distilled water, wherein the pH value of the YPD culture medium is a natural pH value, and the YPD culture medium is subjected to high-temperature high-pressure sterilization by using a high-temperature high-pressure vertical sterilization pot, wherein the sterilization temperature of the high-temperature high-pressure sterilization is 121 ℃, the sterilization pressure is 0.12MPa, and the sterilization time is 15 min.