A fermentation strain combination specifically used for Munag grapes and its application

Abstract

The present invention discloses a fermentation strain combination specifically for Munag grapes and its application. The present invention comprises Torulaspora debrueckii and Saccharomyces cerevisiae EC1118, which mainly produce aroma substances and have alcohol reduction functions, to form a specific strain combination specifically for Munag grapes. The strain combination is used to ferment Munag grapes to obtain a semi-dry low-alcohol white wine with rich aroma substances and low ethanol content (alcohol content of 4.13% Vol). The present invention realizes the effective deep processing of Munag grapes, enriches the product variety of Munag grapes, and improves the economic value of Munag grapes.

Description

Technical Field

[0001] The invention belongs to the technical field of food microbial application, and particularly relates to a fermentation strain combination specially used for Munag grapes and application thereof. Background Art

[0002] Wine is an alcoholic beverage made from fresh grapes or grape juice through fermentation. It is a universal beverage in the world. It can be directly absorbed by the human body without prior digestion. Medical research shows that there are about 600 known ingredients in wine that are beneficial to the human body. It is rich in amino acids, minerals and vitamins, which are essential nutrients for the human body. The phenolic substances and oligosaccharides in wine have the function of high oxidants, which can prevent the reactive oxygen produced during the human body's metabolism from harming the human body.

[0003] Munage grape (Vitis vinifera L.cv. Munage) originated in Kashgar, Xinjiang, my country. It belongs to the Eurasian species, also known as winter grape. It is suitable for cultivation in areas with an annual average temperature of 12℃~13℃, an annual frost-free period of more than 220 days, and an annual average rainfall of ≤120mm. Therefore, it is the main distribution area and planting area in southern Xinjiang, my country. Its berries are full, large and sweet, and are deeply loved by consumers. This variety is a high-quality late-ripening fresh food variety with local characteristics, but because of its thin skin, it is easily attacked by fungi and pathogens during post-harvest storage, resulting in water loss, browning, fruit stalk breakage, fruit berry shedding, and even rot and deterioration. Because its maturity period is mostly concentrated in August to October, the storage, transportation and export of fruits have a profound impact on the development of Munage grapes and its surrounding industries.

[0004] At present, there are few studies on the deep processing of Munag grapes. The invention patent with publication number CN105199971B discloses a low-temperature resistant yeast JM33 and its application in Munag wine. Specifically, the alcohol content of Munag wine fermented by strain JM33 is 10.57%, and the alcohol content of Munag wine fermented by strain EC1118 is 10.24%. The alcohol content of Musalaisi wine fermented by Huang Ying et al. is 13.60% and 14.83% (Huang Ying et al. Sugar brewing of Xinjiang Musalaisi wine and wine quality inspection [J]. Food Industry, 2018, 39(7): 89-91). The alcohol content of Bai Munag wine fermented by Wang Haochen et al. is 10.0% vol (Wang Haochen et al. Food Industry Science and Technology [J]. 2020, 41(11): 117-124). However, there is no report on the research on brewing low-alcohol wine (alcohol content ≤ 7%) with Munag grapes. Summary of the invention

[0005] In order to improve the economic value of Muna grapes and develop low-alcohol wine brewed from Mugna grapes, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a fermentation strain combination specifically for Munag grapes, wherein the strain combination is Saccharomyces cerevisiae EC1118 and Torula sporea debrueckii.

[0007] The ability of Dale's spore yeast to produce aroma and reduce alcohol is strong. It is the dominant bacteria in the early fermentation of Munag grapes and can produce rich aroma substances, making the Munag wine fragrant. The ability of brewer's yeast EC1118 to produce ethanol in fermenting Munag grape juice is strong, indicating that it has good adaptability to Munag grape juice. Brewer's yeast EC1118 is the dominant bacteria in the late fermentation, making the ethanol content in Munag wine reach an appropriate level.

[0008] Preferably, the inoculation ratio of Saccharomyces cerevisiae EC1118 and Torulella delgaceae is 4:1 to 1:4, for example, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4.

[0009] Preferably, the effective viable counts of the Saccharomyces cerevisiae EC1118 and the Psoralea coli are both 10 6 ~10 9 CFU / mL or 10 6 ~10 9 CFU / g, e.g. 10 6 CFU / mL, 10 6 CFU / g, 10 7 CFU / mL, 10 7 CFU / g, 10 8 CFU / mL, 10 8 CFU / g, 10 9 CFU / mL, 10 9 CFU / g.

[0010] Furthermore, the effective viable counts of the Saccharomyces cerevisiae EC1118 and the Psoralea delavidensis are both 10 8 CFU / mL.

[0011] In a second aspect, the present invention provides a bacterial agent, which comprises the strain combination and fermentation products thereof described in the first aspect.

[0012] Preferably, the bacterial agent further comprises a yeast cell protectant.

[0013] Furthermore, the yeast cell protective agent includes but is not limited to skimmed milk powder, maltose, sucrose, trehalose, sodium glutamate, lactose, vitamin C and glycine.

[0014] In a third aspect, the present invention provides a composite strain composition, which comprises the strain combination described in the first aspect or the bacterial agent described in the second aspect.

[0015] In a fourth aspect, the present invention provides an application of the strain combination described in the first aspect, the bacterial agent described in the second aspect, or the composite strain composition described in the third aspect 3 in the preparation of Munag wine, wherein the application comprises at least one of the following:

[0016] (1) Application in reducing the alcohol content of Munag wine;

[0017] (2) Application in reducing the acidity of Munag wine;

[0018] (3) Application in reducing residual sugar in Munag wine;

[0019] (4) Application in improving the taste or flavor of Munag wine.

[0020] In a fifth aspect, the present invention provides a method for preparing Munag wine, the preparation method comprising the following steps:

[0021] S1. Select mature, mildew-free and insect-free Munag grapes, wash, remove stems, lightly crush, pasteurize and filter to obtain grape juice;

[0022] S2. After adjusting the pH value of the grape juice, inoculate the strain combination described in the first aspect or the bacterial agent described in the second aspect, and obtain Munag wine after fermentation and precipitation.

[0023] Preferably, the fermentation conditions of the Munag wine include one or more of the following:

[0024] ⑴The initial pH value is 3-5, for example 3, 3.5, 3.61, 4, 4.5, 5.

[0025] (2) Inoculation amount 2-6%, for example 2%, 3%, 4%, 5%, 6%.

[0026] ⑶ The inoculation ratio of Saccharomyces cerevisiae EC1118 and Pseudomonas delavayi is 4:1 to 1:4, for example, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4.

[0027] (4) Saccharomyces cerevisiae EC1118 and Torulella delgaceae were inoculated at the same time;

[0028] ⑸ Fermentation temperature ≤31℃, for example, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 27.83℃, 28℃, 29℃, 30℃, 31℃.

[0029] ⑹ Fermentation time ≤ 144h, for example 48h, 60h, 72h, 84h, 96h, 97h, 108h, 120h, 144h.

[0030] Preferably, the effective viable counts of the Saccharomyces cerevisiae EC1118 and the Psoralea coli are both 10 6 ~10 9 CFU / mL or 10 6 ~10 9 CFU / g, e.g. 10 6 CFU / mL, 10 6 CFU / g, 10 7 CFU / mL, 10 7 CFU / g, 10 8 CFU / mL, 108CFU / g, 10 9 CFU / mL, 10 9 CFU / g.

[0031] In a specific embodiment of the present invention, the method for preparing the Munag wine comprises the following steps:

[0032] S1. Select mature, mildew-free and insect-free Munag grapes, wash, remove stems, lightly crush, pasteurize and filter to obtain grape juice;

[0033] S2. The pH value of the grape juice is adjusted to 3.5-4.0, and then 3-5% of the inoculation amount of Saccharomyces cerevisiae EC1118 and Psoralea coli are inoculated, fermented for 90-100 hours, and the Munag wine is obtained after precipitation.

[0034] Preferably, the effective viable counts of the Saccharomyces cerevisiae EC1118 and the Psoralea coli are both 10 8 CFU / mL.

[0035] Furthermore, the fermentation conditions of the Munag wine are: initial pH 3.6, inoculation amount 4%, inoculation ratio of cerevisiae EC1118 and Delpsacus yeast 1:2, fermentation time 97h, and fermentation temperature 28°C.

[0036] In a sixth aspect, the present invention provides a Munag wine, wherein the Munag wine is prepared according to the preparation method described in the fourth aspect.

[0037] Preferably, the residual sugar content of the Munag wine is ≤6.0 g / L, for example 5.0 g / L, 5.5 g / L, 5.8 g / L, 6.0 g / L.

[0038] Preferably, the total acid content of the Munag wine is ≤5.5 g / L, for example 4.5 g / L, 4.7 g / L, 5.06 g / L, 5.3 g / L, 5.5 g / L.

[0039] Preferably, the alcohol content of the Munag wine is ≤6.0% Vol, for example: 3.3% Vol, 3.5% Vol, 4.0% Vol, 4.13% Vol, 4.5% Vol, 5.0% Vol, 5.5% Vol, 6.0% Vol.

[0040] In a seventh aspect, the present invention provides a food, wherein the food comprises the Munag wine described in the fourth aspect.

[0041] Preferably, the beverage further comprises fruit juice, and the fruit juice includes but is not limited to apple juice, watermelon juice, lemon juice, blueberry juice, papaya juice and raspberry juice.

[0042] Preferably, the beverage further comprises honey and / or granulated sugar.

[0043] Beneficial effects of the present invention:

[0044] The present invention uses Torula sporea debrueckii and Saccharomyces cerevisiae EC1118, which mainly produce aroma substances and have alcohol-reducing functions, to form a specific strain combination dedicated to Munag grapes. The strain combination is used to ferment Munag grapes to obtain a semi-dry low-alcohol white wine with rich aroma substances and low ethanol content (alcohol content of 4.13% Vol). The deep processing of Munag grapes is achieved, the product variety of Munag grapes is enriched, and the economic value of Munag grapes is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shown is the production flow chart of Munag wine;

[0046] Figure 2 Shown is the effect of inoculum size on the amount of volatile components in Munag wine;

[0047] Figure 3 Shown is the effect of inoculation ratio on the amount of volatile components in Munag wine;

[0048] Figure 4 Shown is the effect of fermentation temperature on the ethanol content of Munag wine;

[0049] Figure 5 Shown is the effect of fermentation time on the ethanol content of Munag wine;

[0050] Figure 6 Shown is the effect of initial pH on the ethanol content of Munag wine;

[0051] Figure 7 The electronic nose and sensory evaluation results in Example 8 are shown;

[0052] Figure 8 Shown is the effect of each factor interaction term on ethanol content in the response surface experiment;

[0053] Fig. 9 Shown is the appearance of the finished product of Munag wine;

[0054] In the figure, a. CEG-TD mixed bacteria fermentation group; b. EC1118-TD mixed bacteria fermentation group; different letters represent significant differences (P < 0.05). DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

[0056] Embodiment 1 The production process of Munag wine

[0057] according to Figure 1 The production process of brewing Munag wine is as follows:

[0058] Select mature, mildew-free, and insect-free Munag grapes, thaw them, wash them, remove stems, and slightly crush them. Then heat them to 60°C for pasteurization for 40 minutes. After cooling, filter them with sterile gauze in a clean bench. Subsequently, transfer 600 mL of grape juice to a 1000 mL sterile blue-mouth bottle. Adjust the pH of the grape juice to 3.5 by adding 0.2 M citrate aqueous solution dropwise, and monitor it with a pH meter. Inoculate the grape juice with Saccharomyces cerevisiae (10 8 CFU / mL) and Torulaspora delbrueckii (10 8 CFU / mL), and cultured at 28°C for 96 h, and stored at -20°C in the dark for later use.

[0059] The inoculated yeast combinations were as follows: the mixed fermentation group (EC1118-TD) for the Munag wine inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii; the mixed fermentation group (CEG-TD) for the Munag wine inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii.

[0060] Example 2 Determination of the optimal inoculum size

[0061] The inoculation amount is an important factor affecting the mixed fermentation process. The amount of inoculation will directly affect the growth and metabolism of the fermentation bacteria in the fermentation matrix, thereby affecting the quality characteristics of the fermented product. In this experiment, the changes in the amount of volatile components in the fermentation broth with the fermentation inoculation amount of each bacterial combination were investigated, which is of great significance for clarifying the effect of the inoculation amount of each bacterial combination on the amount of volatile components in Munag wine.

[0062] The specific experimental process is the same as that in Example 1, except that in the grape juice, the CEG-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation amount of 2, 4, 6, 8, and 10% by volume and an inoculation ratio of 1:1; the EC1118-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbruecki at an inoculation amount of 2, 4, 6, 8, and 10% and an inoculation ratio of 1:1, and after culturing at 28°C for 96 hours, the samples are sampled three times in parallel and stored at -20°C in the dark for later use.

[0063] Agilent 6890 gas chromatograph was used to qualitatively analyze the volatile components in the fermentation liquid of Munag wine, and the number of peaks during the operation of the instrument was recorded. The specific measurement conditions are as follows:

[0064] Chromatographic column: polar Agilent HP-5 (50m×250μm×0.25μm) elastic quartz capillary column.

[0065] Inlet temperature: 250℃.

[0066] Split ratio: 15:1.

[0067] Carrier gas: high purity nitrogen (99.9999%).

[0068] Flow rate: 1mL / min.

[0069] Heating program: starting temperature 50°C, hold for 3 min, increase to 200°C at 5°C / min, hold for 8 min, total operation time 41 min.

[0070] like Figure 2As shown in the figure, in the CEG-TD mixed bacteria fermentation group, as the inoculation amount increased to 4%, the number of volatile components in the fermentation broth reached the maximum value, and as the inoculation amount continued to increase, the number of volatile components in the fermentation broth showed a downward trend. This was because the continued increase in the inoculation amount saturated the bacterial density in the matrix, weakened the secondary metabolism in the fermentation system, and reduced the types of volatile components in the fermentation broth.

[0071] In the EC1118-TD fermentation group, as the inoculation amount increased to 6%, the amount of volatile components in the fermentation broth reached the highest value. As the inoculation amount continued to increase, it showed a similar downward trend as the CEG-TD mixed bacteria fermentation group.

[0072] Therefore, inoculation amounts of 2%, 4%, and 6% were selected for the orthogonal optimization experiment of the CEG-TD mixed bacteria fermentation group, and inoculation amounts of 4%, 6%, and 8% were selected for the subsequent orthogonal optimization experiment of the EC1118-TD mixed bacteria fermentation group.

[0073] Example 3 Determination of the Optimal Inoculation Ratio

[0074] The inoculation ratio is an important factor affecting the mixed fermentation process. The inoculation ratio will directly affect the growth and metabolism of non-brewer's yeast in the fermentation bacteria in the fermentation matrix, thereby affecting the quality characteristics of the fermented product. In the initial stage of natural fermentation of wine, the non-brewer's yeast in the fermentation system produces a large amount of aroma substances while producing alcohol. Therefore, the inoculation amount of non-brewer's yeast plays an important role in the number of volatile components in wine. However, due to its poor ethanol tolerance and lack of the ability to ferment into wine alone, a certain amount of brewer's yeast needs to be selected for mixed fermentation. In this experiment, the change of the number of volatile components in the fermentation broth with the inoculation ratio of each bacterial combination was investigated, which is of great significance for clarifying the influence of the inoculation ratio of each bacterial combination on the number of volatile components in Munag wine.

[0075] The specific experimental process is the same as that in Example 1, except that in the grape juice, the CEG-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation ratio of 4:1, 2:1, 1:1, 1:2, and 1:4, respectively, and an inoculation amount of 6%; the EC1118-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii at an inoculation ratio of 4:1, 2:1, 1:1, 1:2, and 1:4, respectively, and an inoculation amount of 6%. After culturing at 28°C for 96 hours, samples are taken in parallel for 3 times, and stored at -20°C in the dark for later use.

[0076] The qualitative analysis of the volatile components in the Munag wine fermentation liquid sample was the same as in Example 2.

[0077] like Figure 3As shown in the figure, in the CEG-TD mixed fermentation group, as the inoculation ratio of Saccharomyces cerevisiae CEG and Torulaspora delbrueckii changed from 4:1 to 1:4, the number of volatile components in the fermentation broth reached the maximum value, while in the EC1118-TD fermentation group, as the inoculation ratio of Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii changed from 4:1 to 1:1, the number of volatile components in the fermentation broth reached the maximum value. At the same time, the inoculation ratio continued to change from 1:1 to 1:4, showing a downward trend, which was due to the different interactions between different Saccharomyces cerevisiae (CEG, EC1118) and Torulaspora delbrueckii.

[0078] Example 4 Orthogonal optimization of the best inoculation method for Munag wine

[0079] Based on the results of the single factor experiment, the levels of each factor in the orthogonal optimization experiment are shown in Tables 1 and 2. After the optimal factor level was obtained, a verification experiment was carried out, and the samples were sampled three times in parallel and stored in the dark at -20°C for future use.

[0080] The qualitative analysis of the volatile components in the Munag wine fermentation liquid sample was the same as in Example 2.

[0081] Table 1. Levels of orthogonal experimental factors in CEG-TD mixed bacteria fermentation group

[0082]

[0083] Table 2. Orthogonal experimental factor level table of EC1118-TD mixed bacteria fermentation group

[0084]

[0085] Table 3 Orthogonal intuitive analysis table of CEG-TD mixed bacteria fermentation group

[0086]

[0087]

[0088] Table 4. ANOVA table of CEG-TD mixed bacteria fermentation group orthogonal experiment

[0089]

[0090] R 2 =0.980

[0091] As shown in Tables 3 and 4, the results of the orthogonal optimization experiment of the CEG-TD mixed fermentation group showed that the best inoculation method for CEG-TD was 6% inoculation amount, 1:3 inoculation ratio, and reverse inoculation. By comparing the R values, it can be seen that A>B=C, indicating that the inoculation amount has a more direct effect on the number of volatile components. This is because the Torulaspora delbrueckii inoculated at the start of fermentation can directly affect the overall fermentation speed and the number of aroma components of Munag wine.

[0092] Table 5 Orthogonal intuitive analysis table of EC1118-TD mixed bacteria fermentation group

[0093]

[0094] Table 6 Variance analysis table of orthogonal experiment of EC1118-TD mixed bacteria fermentation group

[0095]

[0096]

[0097] R 2 =0.955

[0098] As shown in Tables 5 and 6, the results of the orthogonal optimization experiment of the EC1118-TD mixed fermentation group showed that the best inoculation method for EC1118-TD was 4% inoculation amount, 1:2 inoculation ratio, and co-inoculation. By comparing the R values, it can be seen that A>C>B, the inoculation amount and inoculation order have a greater impact on the number of volatile components, while the inoculation ratio has the smallest impact.

[0099] Verification experiments were conducted using two optimal inoculation methods, and the results showed that the number of volatile components in the fermentation broth of the CEG-TD mixed bacteria fermentation group was 41, and the number of volatile components in the fermentation broth of the EC1118-TD mixed bacteria fermentation group was 27, so the model is reliable.

[0100] In summary, by investigating the effects of inoculation size, bacterial strain ratio, inoculation order and other factors on the amount of volatile components in the fermentation broth and optimizing the orthogonal experiment, it was determined that the best inoculation method for CEG-TD was 6% inoculation size, 1:3 inoculation ratio, and reverse inoculation; the best inoculation method for EC1118-TD was 4% inoculation size, 1:2 inoculation ratio, and co-inoculation.

[0101] Example 5 Determination of the Optimum Fermentation Temperature

[0102] Fermentation temperature is an important factor affecting the mixed fermentation process. This experiment examined the changes in ethanol content in the fermentation broth with fermentation temperature of different bacterial strain combinations to clarify the influence of the initial fermentation pH of different bacterial strain combinations on the fermentation of Munag wine.

[0103] The specific experimental process is the same as that of Example 1, except that in the grape juice, the CEG-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%; the EC1118-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%, respectively. After culturing at 19, 22, 25, 28, and 31° C. for 96 h, samples are taken in parallel 3 times, and stored at -20° C. in the dark for later use.

[0104] Agilent 8890 gas chromatograph was used to determine the alcohol content in the fermentation broth samples. The specific conditions were as follows:

[0105] Chromatographic column: polar Agilent HP-5 (30m×320μm×0.25μm) elastic quartz capillary column; detector: FID; injection port temperature: 250℃;

[0106] Split ratio: 30:1; carrier gas: nitrogen (99.999%); flow rate: 1 mL / min; injection volume: 1 μL;

[0107] Heating program: starting temperature 35°C, hold for 5 min, increase to 200°C at 5°C / min, hold for 8 min, total operation time 46 min.

[0108] Column flow rate: 2.9 mL / min.

[0109] Inlet temperature: 230°C; detector temperature: 250°C.

[0110] Hydrogen gas flow rate: 30mL / min; air: 300mL / min.

[0111] The ethanol content of the samples was determined by using a standard curve drawn using different ethanol concentrations (R 2 =0.997,y=642.6x+2.56222).

[0112] like Figure 4As shown in the figure, when the fermentation temperature increased to 25℃, the ethanol content of the CEG-TD mixed bacteria fermentation group reached the highest value. When the fermentation temperature continued to increase to 28℃, the ethanol content of the EC1118-TD mixed bacteria fermentation group reached the highest value, and its ethanol content was much higher than that of the CEG-TD mixed bacteria fermentation group. This is because the optimal growth temperature of yeast is 28℃. Although the ethanol content of the CEG-TD mixed bacteria fermentation group is positively correlated with the fermentation temperature, it is much lower than that of the EC1118-TD mixed bacteria fermentation group. At the same time, considering that the temperature continues to increase, the yeast cannot continue to grow normally. It can be seen that the EC1118-TD mixed bacteria fermentation group has a stronger adaptability to Munag grape juice. Therefore, the EC1118-TD fermentation group was selected to continue the follow-up study, and the fermentation temperatures of 25℃, 28℃, and 31℃ were selected for the response surface optimization experiment.

[0113] Example 6 Determination of the Optimal Fermentation Time

[0114] Fermentation time is an important factor affecting the mixed fermentation process. This experiment examined the changes in ethanol content in the fermentation liquid with the fermentation time of different bacterial strain combinations to clarify the influence of fermentation time of different bacterial strain combinations on the fermentation of Munag wine.

[0115] The specific experimental process is the same as that of Example 1, except that in the grape juice, the CEG-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%; the EC1118-TD mixed fermentation group is inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%, respectively. After culturing at 28°C for 48, 72, 96, 120, and 144 hours, samples are taken in parallel for 3 times, and stored at -20°C in the dark for later use.

[0116] The ethanol content of the fermentation broth sample was detected in the same manner as in Example 5. Figure 5 As shown in the figure, in the EC1118-TD fermentation group, as the fermentation time increased, the ethanol content reached the maximum value at 96h and there was an obvious linear relationship between 24h and 96h. In the CEG-TD mixed bacteria fermentation group, the linear relationship was not obvious and the maximum ethanol content was still lower than that of the EC1118-TD mixed bacteria fermentation group. It can be seen that the EC1118-TD mixed bacteria fermentation group has a stronger adaptability to Munag grape juice. Therefore, the EC1118-TD mixed bacteria fermentation group was selected for subsequent research, and the fermentation time of 3d, 4d, and 5d was selected for response surface optimization experiments.

[0117] Example 7 Determination of the optimal initial pH of fermentation

[0118] The initial pH is an important factor affecting the mixed fermentation process. In this experiment, the ethanol content in the fermentation broth was investigated along with the initial pH of the fermentation of different bacterial species, so as to clarify the influence of the initial pH of the fermentation of different bacterial species on the fermentation of Munag wine.

[0119] The specific experimental process is the same as that of Example 1, except that the pH of the grape juice is adjusted to 3, 4, 5, 6, and 7, respectively, and monitored by a pH meter. In the grape juice, the CEG-TD mixed fermentation group is inoculated with saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%; the EC1118-TD mixed fermentation group is inoculated with saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%, respectively. After culturing at 28°C for 96 hours, samples are taken in parallel for 3 times, and stored at -20°C in the dark for later use.

[0120] The ethanol content of the fermentation broth sample was detected in the same manner as in Example 5. Figure 6 As shown in the figure, the maximum ethanol content in the CEG-TD mixed fermentation group was much lower than that in the EC1118-TD mixed fermentation group; while the EC1118-TD mixed fermentation group had an obvious linear relationship when the pH increased from 3 to 4. It can be seen that the EC1118-TD mixed fermentation group has a stronger adaptability to Munag grape juice. Therefore, the EC1118-TD mixed fermentation group was selected for subsequent research, and the initial fermentation pH was selected as 3, 3.5, and 4 for response surface optimization experiments.

[0121] Example 8 Determination of the optimal mixed bacteria combination for fermentation of munag substrate based on electronic nose and sensory evaluation

[0122] Before response surface optimization, the electronic nose molecular sensory technology combined with sensory evaluation was used to determine the optimal mixed bacteria combinations for two groups of CEG-TD mixed bacteria fermentation and EC1118-TD mixed bacteria fermentation.

[0123] The specific experimental process was the same as that in Example 1, except that the pH of the grape juice was adjusted to 4, the CEG-TD mixed fermentation group was inoculated with Saccharomyces cerevisiae CEG and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%; the EC1118-TD mixed fermentation group was inoculated with Saccharomyces cerevisiae EC1118 and Torulaspora delbrueckii at an inoculation ratio of 1:1 and an inoculation amount of 6%, respectively. After culturing at 28° C. for 96 h, the samples were taken in parallel for 3 times and stored at -20° C. in the dark for later use.

[0124] The fermentation liquid was subjected to electronic nose sensory testing. The instrument used was the Beijing Yinuo C-PEN3 electronic nose instrument. The test process was referred to the instrument manual. The results were as follows: Figure 7As shown in (a): According to the radar chart results of 10 groups of sensor data, the two fermentation combinations have different flavor characteristics under the same fermentation conditions, among which the EC1118-TD group has richer aromatic components (WIC, W3C, W2W) and higher ethanol content (W2S) than CEG-TD, which is consistent with the results of Examples 5, 6, and 7. The EC1118-TD fermentation group has a stronger adaptability to the Munag grape matrix.

[0125] In order to determine the sensory differences of different flavor components of the two groups of fermented wines, 30 professionally trained people were organized to conduct sensory evaluation. The sensory evaluation table (see Table 7) (modified from GB / T 33404-2016) was used to conduct sensory evaluation of the base wine. The specific operation was to select bentonite (0.5g / mL) to clarify the fermented liquid, and then filter it with sterilized medium-speed filter paper to prepare the base wine for sensory evaluation. The results are shown in Table 7. Figure 7 (b) shows that the EC1118-TD mixed fermentation group is superior to the CEG-TD mixed fermentation group in terms of flavor, sweetness, sourness, harmony, color, etc. The total score of the EC1118-TD mixed fermentation group is 83.8, which is higher than the 69.4 of the CEG-TD mixed fermentation group. Therefore, the EC1118-TD mixed fermentation group has better fermentation performance and flavor for Munag grape juice. Subsequently, EC1118-TD was used as the mixed fermentation group to perform response surface optimization to determine the optimal fermentation conditions.

[0126] Table 7 Sensory evaluation table

[0127]

[0128] Example 9 Optimization of the optimal fermentation conditions of Munag wine by response surface method

[0129] Based on the results of the single factor experiment, a response surface experiment was conducted to optimize the fermentation conditions of Munag wine. The response surface experiment design and results are shown in Table 8, and the regression model variance analysis is shown in Table 9.

[0130] After obtaining the optimal factor levels, a verification experiment was carried out, with three parallel samplings to measure the physical, chemical and antioxidant indicators of Munag wine. The results are shown in Table 10.

[0131] GB15038-2006 general analysis method for wine and fruit wine was used to determine the residual sugar content, total acid, pH and alcohol content in Munag wine samples.

[0132] DPPH free radical scavenging ability determination: add 100 μL (centrifuged at 9500 r / min for 5 min) 10-fold diluted supernatant and 100 μL 0.2 mmol / L DPPH-ethanol solution to a 96-well plate, vortex and mix, react at room temperature in the dark for 30 min, measure the absorbance at 517 nm, and calculate the DPPH free radical scavenging rate. The DPPH free radical scavenging ability of the sample was determined by using a standard curve drawn using Trolox standards of different concentrations (R 2 =0.995, y=3x+3.0951).

[0133]

[0134] A 0 :100μL DPPH-ethanol solution + 100μL anhydrous ethanol solution

[0135] A 1 :100μL DPPH-ethanol solution + 100μL sample solution

[0136] A 2 :100μL ethanol solution + 100μL sample solution

[0137] ABTS + Free radical scavenging ability determination: Take 30 μL (centrifuge at 9500 r / min for 5 min) sample solution and 210 μL ABTS + The solution was mixed by vortexing, reacted in the dark for 30 min at room temperature, and its absorbance was measured at 734 nm. The ABTS free radical scavenging ability of the sample was determined by using a standard curve drawn using Trolox standards of different concentrations (R 2 =0.998, y=1.3502x+9.1134).

[0138] ABTS + Solution: 7mmol / LABTS + Prepare working solution with 2.45mmol / L potassium persulfate at a ratio of 1:1, and dilute with anhydrous ethanol to the absorbance value (OD 734 )=0.7±0.02.

[0139]

[0140] Ac: 210 μL ABTS reagent plus 30 μL anhydrous ethanol solution

[0141] As: 210 μL ABTS reagent plus 30 μL sample solution

[0142] Ab: 210 μL of anhydrous ethanol reagent plus 30 μL of sample solution

[0143] Table 8 Response surface experimental design and results

[0144]

[0145]

[0146] Table 9 Variance analysis table of regression model

[0147]

[0148] As shown in Table 9, the lack of fit term is not significant (P=0.0589), and the regression model used in this experiment is at an extremely significant level, so this model is desirable. The extent to which the three factors affect the ethanol content under each experimental condition is: fermentation temperature>fermentation time>initial pH.

[0149] like Figure 8 As shown in the figure, the slope of initial pH is slightly steeper than that of fermentation time. Combined with the contour map, the contour lines on the axis of initial pH are denser. Without changing other conditions, with the increase of initial pH and fermentation time, the ethanol content of Munag wine will first increase to a peak value and then decrease. The slope of initial pH is slightly steeper than that of fermentation temperature. Combined with the contour map, the contour lines on the axis of initial pH are denser, indicating that the effect of initial pH is more significant than that of fermentation time. The more elliptical contour map and steep response surface map indicate that the initial pH and fermentation temperature have an interactive effect. Without changing other conditions, with the increase of initial pH and the increase of fermentation temperature, the ethanol content of Munag wine will first increase to a peak value and then decrease. Without changing other conditions, with the increase of fermentation temperature and the increase of fermentation time, the ethanol content of Munag wine will first increase to a peak value and then decrease.

[0150] The optimal fermentation process of Munag wine was calculated by Design-Expert software as initial pH 3.61, fermentation time 97.34h, fermentation temperature 27.83℃, and the theoretical ethanol content value could reach 4.06% Vol. According to the above conditions, the ethanol content was (4.13±0.13)% Vol through three verification experiments.

[0151] Table 10 Physicochemical and antioxidant indexes of Munag wine

[0152]

[0153] As shown in Table 10, the residual sugar content of Munag wine is 5.80g / L, the total acid content is 5.06g / L, and the alcohol content is 4.13% Vol. Referring to GB15038-2006 General Analysis Method for Grape and Fruit Wine, Munag wine belongs to semi-dry low-alcohol white wine (see Fig. 9 ).

[0154] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A fermentation strain combination specifically for Munag grapes, characterized in that: The strain combination is Saccharomyces cerevisiae EC1118 and Torulaspora debrueckii, the ratio of Saccharomyces cerevisiae EC1118 to Torulaspora debrueckii is 4:1 to 1:4, and the effective viable counts of Saccharomyces cerevisiae EC1118 and Torulaspora debrueckii are both 10 6 ~10 9 CFU / mL or 10 6 ~10 9 CFU / g.

2. A bacterial agent, characterized in that: The bacterial agent comprises the bacterial strain combination and fermentation products thereof according to claim 1.

3. A composite bacterial strain composition, characterized in that: The composite strain composition comprises the strain combination according to claim 1 or the bacterial agent according to claim 2.

4. Use of the strain combination according to claim 1, the bacterial agent according to claim 2, or the composite strain composition according to claim 3 in the preparation of Munag wine, characterized in that: The application includes at least one of the following: (1) Application in reducing the alcohol content of Munag wine; (2) Application in reducing the acidity of Munag wine; (3) Application in reducing residual sugar in Munag wine; (4) Application in improving the taste or flavor of Munag wine.

5. A method for preparing Munag grape wine, characterized in that: The preparation method comprises the following steps: S1. Select mature, mildew-free and insect-free Munag grapes, wash, remove stems, lightly crush, pasteurize and filter to obtain grape juice; S2. After adjusting the pH value of the grape juice, inoculate the strain combination described in claim 1 or the bacterial agent described in claim 2, and obtain Munag wine after fermentation and precipitation.

6. The preparation method according to claim 5, characterized in that: The conditions for fermentation in step S2 are: ⑴The initial pH value is 3-5; ⑵Inoculation amount 2-6%; ⑶ Fermentation temperature ≤ 31°C; ⑷ Fermentation time ≤144h.

7. The preparation method according to claim 5, characterized in that: The inoculation ratio of the brewer's yeast EC1118 and the Delpsacus spores is 4:1 to 1:4; the effective viable counts of the brewer's yeast EC1118 and the Delpsacus spores are both 10 6 ~10 9 CFU / mL or 10 6 ~10 9 CFU / g.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The fermentation conditions of the Munag wine are as follows: initial pH 3.6, inoculation amount 4%, inoculation ratio of brewer's yeast EC1118 and Delpsaccharomyces cerevisiae to 1:2, fermentation time 97h, and fermentation temperature 28°C.

9. A Munag wine, characterized by: The Munag wine is prepared according to the preparation method described in any one of claims 4 to 7; Preferably, the residual sugar content of the Munag wine is ≤6.0 g / L, the total acid content is ≤5.5 g / L, and the alcohol content is ≤6.0% Vol.

10. A food, characterized in that: The food comprises the Munag wine according to claim 8 or 9.

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