Saccharomyces cerevisiae as well as method for preparing roxburgh rose fermented wine and application thereof

Abstract

The invention discloses saccharomyces cerevisiae, a method for preparing roxburgh rose fermented wine and application of the saccharomyces cerevisiae, the saccharomyces cerevisiae GZHM-28 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M20221967, and the preservation date is December 13, 2022. The saccharomyces cerevisiae GZHM-28 disclosed by the invention has relatively high tolerance to high glucose, low pH, SO2 and ethanol, can reduce the acidity of organic acid in food, can remarkably reduce the content of tannin in roxburgh rose juice through a way of adsorbing or generating tannin hydrolase, and is prepared into tasty, slightly sour and low-astringent low-alcohol roxburgh rose wine.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to a brewing yeast and a method and application thereof for preparing roxburghii fermented wine. Background Art

[0002] Rosa roxburghii, also known as Shanwangguo and Ci Li, has a strong and unique aroma, sour, astringent and slightly sweet taste, and is rich in Vc, flavonoids, polysaccharides, polyphenols and SOD. It has important research and application value in anti-oxidation, immune regulation, anti-diabetes, anti-tumor and hypolipidemic aspects. Its planting area in Guizhou Province is as high as 66,700 hectares, with an annual output of more than 100,000 tons. In 2020, it officially became one of the 12 major characteristic and pillar industries in Guizhou Province, with an annual output value of more than 15 billion. The large-scale planting of rosa roxburghii has promoted the development of rosa roxburghii processing enterprises. There are more than 40 rosa roxburghii processing enterprises, including 5 leading enterprises. The related rosa roxburghii products are mainly rosa roxburghii beverages, rosa roxburghii wine, rosa roxburghii dried fruit, preserved fruit, rosa roxburghii jelly, rosa roxburghii yogurt and rosa roxburghii lozenges, with the largest proportion being rosa roxburghii beverages. However, 0.6% to 2.2% of tannin (polyphenols) are contained in roxburghii, which causes the product to have a serious sour taste and affects the taste; too much tannin can easily cause protein or polysaccharide precipitation, causing browning of liquid beverages or alcoholic products, affecting the sensory organs of the product; and tannin itself is an anti-nutritional agent that can chelate trace mineral elements, combine and precipitate free digestive enzymes in the body, and have a certain effect on the human digestive system. Therefore, foods or beverages with too high tannin content may have adverse effects on children, pregnant women and other special groups. This is also the main reason why roxburghii products, although they have high nutritional value and many functions, are still not accepted by the general public, especially by children and young people. Therefore, reducing the tannin content of roxburghii products and improving the taste is a key measure for the roxburghii industry to break through the bottleneck. At present, the methods for processing roxburghii tannins mainly include physical adsorption, chemical precipitation, tannin enzyme hydrolysis and adding chemical astringency inhibitors, but all of them have problems such as long processing cycle, high nutritional loss or high tannin residue, and no substantial increase in product added value, but increase production costs. How to effectively reduce the tannin content of sea buckthorn products, improve the taste, broaden the market audience, and increase product added value is an important way to maintain and develop the sea buckthorn industry.

[0003] Preparation of roxburghii wine is a way to increase the added value of roxburghii products. Traditional roxburghii wine is mainly fruit wine, which is loved by some consumers because of its special flavor and health elements. However, fruit wine is generally made by adding roxburghii fruit or roxburghii juice to ordinary liquor, which will introduce substances such as protein, polysaccharide, and tannin. It is easy to oxidize brown or produce flocculent precipitation with prolonged storage time, and there is still a certain astringency, which affects the sensory perception of the wine. At present, there are some fermented wines in the roxburghii fruit wine market in Guizhou, which are prepared by fermenting roxburghii juice with brewer's yeast. Although it can improve the taste of roxburghii drinks to a certain extent, it provides a new choice for roxburghii lovers. However, most of the brewer's yeast used in roxburghii fermented wine are commercial strains, which have no tannin-reducing performance or limited tannin-reducing ability. The taste of the roxburghii wine produced still cannot meet the taste needs of tannin-sensitive consumers. The main research on tannin-reducing yeast is focused on the field of breeding to improve feed utilization, and the strains with related functions are mainly Candida, which is not suitable for food fermentation. Therefore, finding a brewer's yeast with strong growth performance, acid resistance, rapid fermentation, the ability to improve the flavor of sea buckthorn and significantly reduce sea buckthorn tannins is of great significance for increasing the added value of sea buckthorn products, solving the pain point of the sour and astringent taste of sea buckthorn, and broadening the consumer base. Summary of the invention

[0004] In order to solve the problems of the prior art, the present application provides a method and application of brewing yeast and preparing roxburgh fermented wine. The technical solution is as follows:

[0005] In the first aspect, a brewer's yeast (Saccharomyces cerevisiae) GZHM-28 is provided, and the brewer's yeast GZHM-28 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M20221967 and the preservation date of December 13, 2022.

[0006] In a second aspect, a method for preparing roxburghii fermented wine is provided, comprising the following steps:

[0007] Take sea buckthorn juice and add water, then add white sugar to adjust the sugar content to 10% (m / v), heat to boiling, then cool to room temperature, inoculate brewer's yeast GZHM-28 seed fermentation liquid, and then seal with sterile water. Ferment at room temperature until the escaping bubbles are reduced to 2-3 times / min, transfer the wine into a sterile beverage bottle under sterile conditions, and continue to ferment at room temperature for 7 days.

[0008] Furthermore, after the sterile water is sealed, fermentation is carried out at room temperature for 7-10 days.

[0009] Furthermore, the Saccharomyces cerevisiae GZHM-28 seed fermentation liquid is prepared by the following method:

[0010] Take the slant seeds of Saccharomyces cerevisiae GZHM-28 and streak them on the YEPD solid plate medium, culture them at 28°C for 2 days, pick the milky white, high round and neat-edged colonies, streak them continuously on the YEPD solid plate medium, and activate and culture them 3 times to obtain the activated strain of Saccharomyces cerevisiae GZHM-28;

[0011] The activated Saccharomyces cerevisiae GZHM-28 strain was inoculated into a test tube slant culture medium, and cultured overnight at 28° C. to obtain a Saccharomyces cerevisiae GZHM-28 slant strain;

[0012] The slant strain of Saccharomyces cerevisiae GZHM-28 was transferred to the YEPD test tube liquid culture medium, and statically cultured at 28° C. for 1 day to prepare the test tube seeds of Saccharomyces cerevisiae GZHM-28;

[0013] The test tube seeds of Saccharomyces cerevisiae GZHM-28 were inoculated into YEPD liquid culture medium, cultured overnight at room temperature, and the cells were collected by centrifugation, washed 3 times with saline, and resuspended in 50 mL of saline to obtain the fermentation liquid of Saccharomyces cerevisiae GZHM-28 seeds.

[0014] Furthermore, the centrifugation condition is 6000 rpm and centrifugation for 5 minutes.

[0015] Furthermore, the formula of the YEPD solid plate culture medium includes: 10g yeast powder, 20g peptone, 20g glucose and 2% agar.

[0016] Furthermore, the formula of the test tube slant culture medium is the same as that of the YEPD solid plate.

[0017] Furthermore, the formula of the YEPD liquid culture medium includes: 10 g yeast powder, 20 g peptone, 20 g white sugar, 1000 mL distilled water, pH 6.0, and wet heat sterilization at 115° C. for 20 min.

[0018] Furthermore, the formula of the YEPD test tube liquid culture medium is the same as that of the YEPD liquid culture medium.

[0019] In a third aspect, a method for preparing a fermented roxburghii beverage is provided.

[0020] Furthermore, the roxburghii fermented beverage includes an alcoholic beverage.

[0021] The technical solution provided in the embodiment of the present application brings the following beneficial effects: The brewer's yeast (Saccharomyces cerevisiae) GZHM-28 provided in the embodiment of the present application is characterized in that the brewer's yeast GZHM-28 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M20221967 and a deposit date of December 13, 2022. The brewer's yeast GZHM-28 of the present application is resistant to high sugar, low pH, SO 2 The invention discloses a roxburghii wine prepared by using the saccharomyces cerevisiae GZHM-28 of the present invention, which has a high tolerance to ethanol and can reduce the acidity of organic acids in food. The tannin content in roxburghii juice can be significantly reduced by adsorption or production of tannin hydrolase, and a refreshing, slightly sour, and low-astringent low-alcohol roxburghii wine can be prepared. The roxburghii wine prepared by using the saccharomyces cerevisiae GZHM-28 of the present application has a golden color, high clarification, and does not require filtration. The roxburghii wine fermented by roxburghii and yeast has a rich and pure aroma, rich and lasting foam after pouring, a refreshing taste similar to beer (without malt and hops added), a slightly sour taste, and basically no astringency, and is suitable for public drinking. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The colony and bacterial identification results of Saccharomyces cerevisiae GZHM-28 provided in the examples of the present application;

[0024] Figure 2 It is the 26S rDNA electrophoresis identification result of Saccharomyces cerevisiae GZHM-28 provided in the examples of the present application;

[0025] Figure 3 This is the acid reduction test result of Saccharomyces cerevisiae GZHM-28 provided in the examples of the present application;

[0026] Figure 4 This is the plate tannin reduction test result of the brewer's yeast GZHM-28 provided in the examples of the present application. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below in conjunction with the accompanying drawings.

[0028] Example 1 Screening and Identification of Saccharomyces cerevisiae GZHM-26

[0029] 1. Screening of strains

[0030] 1.1 Sample source

[0031] The strain provided in this application was isolated from black rose grapes purchased from the agricultural product logistics park in Shiban Town, Huaxi District, Guiyang City, Guizhou Province. The grape samples were fresh and uncontaminated, and all rotten fruits were removed.

[0032] 1.2 Separation and purification

[0033] Take the above fresh black rose grapes, remove the stems and crush them, and then put them into a fermentation tank, add white sugar to adjust the sugar content of the grape juice to 30% (m / v), and place it at room temperature for static fermentation. Take 1mL from the fermentation liquid every 3 days for gradient dilution, and then take 200μL of the gradient dilution liquid and evenly spread it on a WL solid plate (yeast extract powder 4.0g, peptone 5.0g, sucrose 50g, potassium dihydrogen phosphate 0.55g, potassium chloride 0.425g, calcium chloride 0.125g, magnesium sulfate 0.125g, ferric chloride 0.0025g, manganese sulfate 0.0025g, bromocresol green 22mg, agar 20g, distilled water 1L, pH 6.5), place it in an anaerobic culture tank (anaerobic gas bag deoxygenation), and culture it at 28℃ for 2d. Pick 100 colonies with high roundness, opaqueness, neat edges, thickness, milky white or slightly green color, etc., similar or consistent with the characteristics of Saccharomyces cerevisiae colonies from the high dilution plate, then streak and separate and purify until the colony morphology is uniform, pick single colonies and transfer them to WL slant medium (same formula as WL solid plate), number them, culture overnight, and store at 4°C for later use (slant seeds).

[0034] 1.3 Preliminary screening of bacterial strains

[0035] The prepared slant seeds were transferred to 10mL YEPD test tube liquid culture medium (10g yeast powder, 20g peptone, 20g white sugar, 1000mL distilled water, pH 6.0, 115℃ moist heat sterilization for 20min), and cultured at 28℃ for 1d to obtain liquid seeds. Take 2% of the liquid seeds, transfer to 20mL simulated grape juice culture medium containing Dulbecco's tubules (sugar content 20%, purchased from Shanghai Hongshun Biotechnology Co., Ltd.), and cultured at 28℃ for 2d. During this period, the gas production start time, Dulbecco's tubule full gas time, agglutination performance and residual sugar in the fermentation liquid of each strain were recorded. The agglutination performance detection method is as shown in Formula 1.

[0036] The specific detection method is as follows: take 5 ml of the above fermentation liquid, collect the bacteria by centrifugation (5000 rpm, 5 min), suspend the bacteria in 10 ml of physiological saline, take 3 ml of the bacterial suspension to detect OD 600 Value, denoted as A o ; Leave the remaining bacterial solution at room temperature for 5 hours, carefully draw 3mL of the upper suspension and measure the OD 600 The value is recorded as A, and the bacterial agglutination rate is calculated as follows.

[0037] Formula 1: Agglutination rate = (A o -A) / A o ×100%

[0038] The preliminary screening results are shown in Table 1. As a result, 31 strains with excellent fermentation performance were screened out. Their initial gas production time and Dulbecco's tubule gasification were within 10h and 12h respectively. The residual sugar content of these strains was less than 15g / L after 2d of fermentation, and the agglutination rate of the bacteria was above 75% within 5h. In general, the earlier the fermentation start time of the strain, the shorter the Dulbecco's tubule gasification time, and the lower the residual sugar content (using a sugar content detector to detect residual sugar), the stronger the fermentation ability of the strain, and the less likely it is to be contaminated by foreign bacteria; a high agglutination rate of the bacteria is conducive to the clarification of the wine in the later stage, which can reduce the clarification time and separation cost of the wine, and is also an important evaluation indicator of excellent brewing yeast. The strains shown in Table 1 all have good fermentation performance and agglutination performance.

[0039] Table 1 Results of preliminary screening test of Saccharomyces cerevisiae strains

[0040]

[0041]

[0042] 1.4 Rescreening of strains for tannin-reducing function

[0043] The prepared slant seeds were transferred to 10 mL YEPD test tube liquid culture medium and cultured at 28 ° C for 1 day to obtain the strain seed solution. The strain seed solution was transferred to 500 mL boiled roxburghii original juice culture medium (15% m / v of white sugar was added to the roxburghii original juice, and the roxburghii original juice was purchased from Guizhou Queji Ecological Agriculture Co., Ltd.) at a ratio of 2%, and cultured at room temperature for 7 days. After the culture, the taste of each roxburghii fermented liquid was tasted, and the strains that could significantly reduce the sour taste of the fermented liquid and significantly improve the taste were screened out, and the tannins (using the Folin-Denis method), Vc (detection reference GB5009.86-2016) and total acid (detection reference GB / T 4927-2008) in the roxburghii juice before and after fermentation were detected. That is, the number of milliliters of 0.1M NaOH consumed for every 100 ml of sample titrated to neutrality, and the sample was heated to exhaust CO before detection. 2 ).

[0044] The results showed that based on the taste, only one strain was screened out that could significantly improve the flavor of the original sea buckthorn juice and reduce its sour taste. The strain number was GZHM-28. This strain can reduce the tannin, Vc and total acid of the original sea buckthorn juice from 752 mg / L, 6.69 g / L and 7.10 before fermentation to 127 mg / L, 2.75 g / L and 2.21 after fermentation. It significantly reduces the sour taste of the original sea buckthorn juice and greatly improves the taste of the sea buckthorn beverage, while retaining more than 40% of Vc. After fermentation with GZHM-28, the acidity of the sea buckthorn beverage is still higher than the acidity upper threshold of high-quality beer in GB / T4927-2008, but the sour taste is not obvious when drinking.

[0045] Table 2 Effect of GZHM-28 on tannin, Vc and total acid of roxburghii juice

[0046] Tannin(mg / L) Vc(g / L) Total acid (mL / 100mL) Before fermentation 752±61 6.69±0.72 21.30±1.30 After fermentation 127±43 2.75±0.25 6.62±0.54

[0047] 2. Identification of strains

[0048] 2.1 Morphological identification

[0049] The GZHM-28 slant seeds were streaked onto a YEPD solid plate (10 g yeast powder, 20 g peptone, 20 g glucose and 2% agar), incubated at 28°C for 48 h, and the colony characteristics were observed. A single colony of GZHM-28 was selected for methylene blue staining and observed under a 40x objective. Figure 1 -A shows that the colony of GZHM-28 in YEPD medium is relatively large, milky white, round, smooth, and has a strong wine aroma. The yeast characteristics are obvious under a 40x objective, as shown in Figure 1. Figure 1 -B, the bacteria are relatively large and oval, and some of them have buds. The colony and bacterial morphological characteristics show that GZHM-28 has typical yeast morphological characteristics, and the colony has a strong wine aroma, which is consistent with the basic characteristics of brewing yeast.

[0050] 2.226S rDNA identification

[0051] The single colony of GZHM-28 isolated by streaking on the plate was used as a template, and the universal primers NL1 and NL4 for fungi were used as primers. A certain amount of 2×TaqPCR mix was added to perform PCR amplification of the D1 / D2 region sequence of 26S rDNA. The reaction conditions were: 98℃5min; 94℃30s, 55℃30s, 72℃35s; 72℃5min, and cycled 30 times. After verification by agarose gel electrophoresis, the sequence size of the D1 / D2 region of 26SrDNA of GZHM-28 was about 600bp, which was in line with expectations. The specific electrophoresis diagram is shown in Figure 2The PCR product was sent to Wuhan Quintiles Biotech Co., Ltd. for sequence analysis. The D1 / D2 region sequence was compared with the BLAST in GenBank (http: / / www.ncbi.nlm.nih.gov / blast / ). After comparison, the homology rate between the 26S rDNA D1 / D2 region sequence of GZHM-28 and Saccharomyces cerevisiaeAA2 was greater than 99%.

[0052] Combining the morphological characteristics and molecular identification results, strain GZHM-28 was identified as Saccharomyces cerevisiae.

[0053] Example 2 Tolerance test of Saccharomyces cerevisiae GZHM-28

[0054] The slant seeds of Saccharomyces cerevisiae GZHM-28 were inoculated into a 10 mL test tube simulated grape juice medium (200 g / L white sugar, 0.05 g / L copper sulfate, 10 g / L peptone, 3 g / L citric acid, 5 g / L yeast extract powder, pH 6.0, and 50 mg / L SO4 was added after sterilization. 2 ) and cultured overnight at 28°C to obtain simulated grape juice liquid seeds. The simulated grape juice liquid seeds were inoculated at an inoculum of 2% into simulated grape juice medium (the formula was the same as that of the test tube simulated grape juice medium) and 30% initial sugar content (adjusted by white sugar, m / v) and initial SO 2 The strains were placed in simulated grape juice liquid medium with 200 mg / L, initial pH 3.0 or 12% ethanol (v / v) (Dulbecco's tubules were inverted in each medium), and cultured at room temperature for 2 days. The growth and fermentation of the strains in each medium were observed, and the initial gas production time (Dulbecco's tubules began to be filled with gas), the time when the Dulbecco's tubules were filled with gas, and the residual sugar in the medium were recorded to evaluate their fermentation capacity under different fermentation pressures. The results are shown in Table 3. The fermentation capacity of Saccharomyces cerevisiae GZHM-28 was 1.3447 W / L, 1.200 mg / L SO 2 , initial pH 3.0 and 12% ethanol. 2 The fermentation start-up speed, gas production performance and sugar metabolism capacity of the fermentation medium were relatively strong, and the tolerance to simulated grape juice with pH 3.0 was strong, and the differences between its fermentation start-up speed, gas production performance and sugar metabolism capacity were small compared with the control medium; it had a certain tolerance to 30% high sugar and 12% ethanol, but the fermentation performance was inhibited, and its fermentation start-up speed, gas production performance and sugar metabolism capacity were seriously hindered, especially the resistance to 12% ethanol was the worst, and its initial gas production and full gas time of fermentation were 39.00h and 58.33h respectively, and the residual sugar content was still 19.20% after 48h of fermentation.

[0055] Table 3 Tolerance test results of Saccharomyces cerevisiae GZHM-28

[0056] Fermentation conditions Initial gas production time (h) Full gas time (h) Residual sugar content (%, m / v) Comparison 7.61±1.33 10.03±1.07 12.01±0.33 30% sugar content 24.72±1.38 29.20±2.30 27.52±0.56 <![CDATA[200mg / LSO 2 ]]> 9.33±0.37 11.51±2.43 15.91±0.31 pH 3.0 9.81±1.22 11.00±0.30 12.50±0.40 12% ethanol (v / v) 39.00±3.75 58.33±4.67 19.20±0.20

[0057] Example 3 Acid reduction performance test of Saccharomyces cerevisiae GZHM-28

[0058] Take the slant seeds of Saccharomyces cerevisiae GZHM-28 and inoculate them in 10mL YEPD liquid medium (10g yeast powder, 20g peptone, 20g white sugar, 1000mL distilled water, pH 6.0, 115℃ wet heat sterilization for 20min), and incubate them at 28℃ overnight to prepare liquid seeds. Take 2% of the liquid seeds and transfer them to 100mL simulated grape juice medium, and incubate them at room temperature for 2d. Collect the cells by centrifugation (6000rpm, 5min), resuspend them in an appropriate volume of physiological saline, and prepare OD 600 ≈1.0 bacterial suspension. Take 5mL of Saccharomyces cerevisiae GZHM-28 bacterial suspension and add equal volumes of 12g / L malic acid, citric acid and tartaric acid solutions respectively, three in each group in parallel, and let stand at room temperature for 3 days. Use 0.1MNaOH to titrate each reaction solution, count the milliliters of 0.1MNaOH required to titrate each acid solution to neutral before and after the reaction, and calculate the degradation rate of Saccharomyces cerevisiae GZHM-28 to various acids. The degradation rate is calculated as shown in Formula 2.

[0059] Formula 2: Degradation rate = (AB) / A×100%

[0060] In the formula, A refers to the milliliters of 0.1M NaOH required to titrate the acidic solution before the reaction; B refers to the milliliters of 0.1M NaOH required to titrate the acidic solution after the reaction. Figure 3 As shown, the degradation rates of the three organic acids by the yeast GZHM-28 under certain conditions are 31.7%, 18.6% and 40.6%, respectively, and the degradation rate of tartaric acid is the highest; the degradation degree of the three organic acids is lower than the degree of acidity reduction after the fermentation of the roxburghii juice in Example 1, which may be related to the growth of the bacteria and the vigorous metabolism during the fermentation process. There is no growth or reproduction of bacteria in this acid reduction test. The test results show that the yeast GZHM-28 has a significant acid reduction ability and can be used for the fermentation of high-acidity food raw materials to reduce their acidity.

[0061] Example 4 Tannin reduction test of Saccharomyces cerevisiae GZHM-28

[0062] 1. Tannin hydrolysis test

[0063] Take the slant seeds of Saccharomyces cerevisiae GZHM-28 and inoculate them on a solid plate containing tannic acid (culture medium formula: 2% peptone, 1% yeast extract powder, 2% sucrose, 0.01% tannic acid, 0.004% bromophenol blue, 1.5% agar) by spot inoculation. Incubate at 28℃ for 5 days and observe the color change around the colony. The results are as follows Figure 4As shown, after 5 days of culture, the colony of Saccharomyces cerevisiae GZHM-28 was larger and had a yellow halo around it, indicating that it could grow normally in the culture medium containing 0.1% tannic acid, had a strong tolerance to tannic acid, and could hydrolyze tannic acid, reduce the pH around the colony, change the color of bromophenol blue, and then cause a yellow halo to appear around the colony.

[0064] 2. Tannic acid degradation test

[0065] Take the slant seeds of Saccharomyces cerevisiae GZHM-28 stored in the refrigerator and streak them on the YEPD solid plate, invert and culture at 28℃ for 48h. Pick a single colony plate with strong growth and streak it on the YEPD solid plate, invert and culture at 28℃ for 48h, repeat 3 times. Pick a single colony with strong growth and inoculate it on the YEPD slant medium (the formula is the same as that of the YEPD solid plate), incubate it at 28℃ overnight, and prepare activated slant seeds.

[0066] Take the activated Saccharomyces cerevisiae GZHM-28 slant seeds and inoculate them in 10mL YEPD test tube liquid culture medium, and let it stand at 28°C overnight to obtain Saccharomyces cerevisiae GZHM-28 test tube liquid seeds. Take 2% of Saccharomyces cerevisiae GZHM-28 test tube liquid seeds and inoculate them in YEPD liquid culture medium containing 10μg / mL tannic acid, and let it stand at 28°C for 24h. Then take 10mL of fermentation broth and centrifuge (6000rpm, 5min), take the supernatant and use Folin-Denis spectrophotometry to determine the tannic acid content of the sample before and after fermentation (for specific detection methods, refer to Geng Nana et al., Folin-Denis spectrophotometric determination of tannic acid content in gallnut [J]. Anhui Agricultural Science, 2013). The results are shown in Table 4. Saccharomyces cerevisiae GZHM-28 can reduce the tannic acid in YEPD culture medium from 7.44μg / mL to 1.66μg / mL within 24h, and the degradation rate is as high as 77.69%. This result confirms that Saccharomyces cerevisiae GZHM-28 has a high tolerance and degradation capacity for tannic acid. The initial value of tannic acid in the sterilized culture medium was 7.44 μg / mL, which was lower than the added amount of 10 μg / mL. This may be due to the co-precipitation of tannic acid with macromolecular substances such as proteins or polypeptides in the culture medium during the sterilization process, resulting in a reduction in free tannic acid.

[0067] Table 4 Results of the degradation test of tannic acid in the culture medium by Saccharomyces cerevisiae GZHM-28

[0068] Before fermentation (μg / mL) After fermentation (μg / mL) Degradation amount (μg / mL) Degradation rate (%) 7.44±0.52 1.66±0.14 5.78±0.38 77.69±5.11

[0069] 3. Tannin adsorption test

[0070] In order to explore the tannin-reducing mechanism of Saccharomyces cerevisiae GZHM-28, tannic acid was used as the test sample to detect the adsorption capacity of the inactivated bacteria on tannic acid and the activity of tannase produced by the fermentation broth. The adsorption capacity test was as follows: the activated Saccharomyces cerevisiae GZHM-28 slant seeds were inoculated in 10mL YEPD test tube liquid culture medium and left to stand at 28℃ overnight to obtain test tube liquid seeds; 2% test tube liquid seeds were inoculated in YEPD liquid culture medium and left to stand at 28℃ for 24h. Then, 5 mL of fermentation liquid was centrifuged (6000 rpm, 5 min), the supernatant was discarded, the bacteria were washed 3 times with an equal volume of saline, and resuspended in 5 mL of saline; the bacterial suspension was inactivated in a boiling water bath for 5 min, and 1 mL of the suspension was mixed and placed in 3 times the volume of 10 μg / mL tannic acid solution, and allowed to stand at room temperature for 30 min, and the supernatant was centrifuged at room temperature (6000 rpm, 5 min), and the tannic acid content before and after the sample solution was treated was detected by Folin-Denis spectrophotometer. The tannic acid adsorption rate meter formula is shown in Formula 3.

[0071] Formula 3: Tannic acid adsorption rate = (AB) / A×100%

[0072] In the formula, A refers to the tannic acid concentration of the sample solution that has not been treated with the inactivated bacterial suspension (μg / mL); B refers to the tannic acid concentration of the solution that has been treated with the inactivated bacterial suspension (μg / mL). The test results are shown in Table 5. Each milliliter of inactivated bacterial suspension can reduce the three-fold volume of tannic acid solution from 8.93μg / mL to 5.56μg / mL within 30 minutes. Inactivated bacteria cannot degrade tannic acid through enzymatic hydrolysis, and physical adsorption and sedimentation are the only mechanism for reducing tannic acid. It can be inferred that Saccharomyces cerevisiae GZHM-28 may reduce the content of tannic acid in the solution by adsorption of cell wall polysaccharides.

[0073] Table 5 Degradation test results of tannic acid inactivated cells of Saccharomyces cerevisiae GZHM-28

[0074]

[0075]

[0076] 4. Tannin enzymatic hydrolysis test

[0077] Take the activated Saccharomyces cerevisiae GZHM-28 slant seeds and inoculate them in 10mL YEPD test tube liquid culture medium, let it stand at 28℃ overnight to obtain test tube liquid seeds; take 2% test tube liquid seeds and inoculate them in YEPD liquid culture medium containing 100μg / mL tannic acid, let it stand at 28℃ for 24h; take 50mL fermentation liquid and centrifuge (6000rpm, 5min), and the obtained supernatant is the crude enzyme liquid. The activity of tannin enzyme is detected by the tannin method (Bao Yuxin, Research on the production of tannin enzyme by solid fermentation of Aspergillus niger, Master's thesis, 2009). Before the enzyme hydrolysis test, the solutions, substrates and crude enzyme liquid involved are placed in a 30℃ water bath for preheating for 5min. The test steps are as follows:

[0078] (1) Prepare 9 test tubes, marked as control group, experimental group and blank group, 3 tubes in each group, add 0.25 mL of propyl gallate solution (prepared by taking out 0.021 g of propyl gallate and dissolving it in 10 mL of citric acid buffer) to each group, add 0.25 mL of crude enzyme solution to the experimental group, and add 0.25 mL of the above citric acid buffer to the blank group, and incubate in a 30°C water bath for 10 min.

[0079] (2) To start the rhodanine reaction, 0.3 mL of methanol rhodanine solution (0.0667 g of rhodanine was diluted to 10 mL with methanol) was added to each test tube and incubated at 30°C for 5 min. To neutralize the reaction, 0.4 mL of KOH solution was added to each test tube, and 0.25 mL of crude enzyme solution was added to the control group. Each group was incubated at 30°C for 5 min. Finally, 4 mL of ddH 2 O, place in a 30℃ water bath for 10 min.

[0080] (3) At a wavelength of 520 nm, the absorbance of the reaction solution in each test tube was measured. The concentration of gallic acid generated in the test group was measured by OD 520 The change in △OD 520 Calculate the △OD with the gallic acid standard curve. 520 =(OD 试验 -OD 空白 )-(OD 对照 -OD 空白 ).

[0081] The tannase activity of the sample was calculated according to Formula 4. The definition of tannase activity was: under the above reaction conditions, the amount of enzyme required to hydrolyze propyl gallate to produce 1 μmol gallic acid per minute was defined as one enzyme activity unit (U).

[0082] Formula 4: E = △X × V × D f / T×Ve

[0083] In the formula, △X represents the change in gallic acid concentration (μmol / L) calculated according to the standard curve; V represents the volume of the reaction solution (mL); D f represents the enzyme dilution multiple; T represents the reaction time (min); Ve represents the enzyme volume (mL). The results are shown in Table 6. The tannin activity of the fermentation broth of Saccharomyces cerevisiae GZHM-28 is about 0.002U / mL, which is weak. However, considering the long fermentation cycle of the brewing process, the strain GZHM-28 still has certain application value in the treatment of high tannin food raw materials.

[0084] Table 6 Results of enzyme activity test of Saccharomyces cerevisiae GZHM-28 fermentation broth

[0085]

[0086] Example 5 Application test of saccharomyces cerevisiae GZHM-28 in roxburghii fermented wine

[0087] Take the slant seeds of Saccharomyces cerevisiae GZHM-28 and streak them on the YEPD solid plate, culture them at 28℃ for 2 days, pick the colonies with strong growth and large colonies, streak them continuously on the YEPD solid plate, and activate and culture them 3 times. Inoculate the activated strains to the test tube slant and culture them overnight at 28℃. Transfer the prepared slant strains to 3 10mL YEPD test tube liquid culture media, and culture them at 28℃ for 1 day to prepare test tube seeds. Take all the liquid seeds and inoculate them into 3 500mL YEPD liquid culture media, culture them at room temperature overnight, collect the bacteria by centrifugation (6000rpm, 5min), wash them with saline 3 times, resuspend them in 50mL saline, and prepare fermentation strains. All the prepared fermentation strains (about 6.25%) were inoculated into 3 jars of 8L boiling sterilized roxburghii juice (the roxburghii original juice was heated and boiled with 2 times the volume of sterile water, and the sugar content was adjusted to 10% (m / v) with white sugar, and immediately poured into a 10L dry heat sterilized glass fermentation jar while hot, and cooled to room temperature), the jar mouth was sealed with pure water, and the gas production was observed, and the fermentation start time (bubble appearance time), peak time (a large amount of gas production time, bubbles appear more than 5 times per minute), and peak duration (duration of bubbles more than 5 times per minute) were recorded. After 7-10 days, the bubbles in each jar were reduced to 2-3 times / min, and the roxburghii wine was aseptically divided into 500mL sterilized beverage bottles and left to stand at room temperature for 7 days. During the process, the time when the wine begins to clarify (the time required for the wine to start to separate after packaging) and the time when the wine is completely clarified (the time required for the wine to have no visible turbidity to the naked eye) are observed, and after the fermentation is completed, the wine's residual sugar, alcohol content (both residual sugar and alcohol content are detected using a handheld refractometer), pH (detected using a pen-based pH meter), Vc, tannin, total acid (the detection methods for Vc, tannin and total acid are the same as those described in 1.4 of Example 1) and sensory indicators (evaluated from the perspectives of color, aroma, foam, sourness, sweetness and astringency, etc.) are detected.

[0088] The test results are shown in Table 7. The seeds of Saccharomyces cerevisiae GZHM-28 were cultured in 33.3% roxburghii juice for 48.0 hours and entered the peak fermentation period, which lasted for about 54.0 hours. The wine was quickly clarified after packaging, and there was no obvious turbidity in the wine at 56.7 hours, indicating that the strain of Saccharomyces cerevisiae GZHM-28 had strong cohesion, which was conducive to the separation of the wine in the later stage. At the end of 7 days of post-fermentation, the residual sugar in the wine was about 3.2%, the alcohol content was about 4.1%, the Vc was about 0.66g / L, the tannin was about 78mg / L, and the total acidity was about 2.75. The above results show that strain GZHM-28 is suitable for fermenting roxburghii juice, and can effectively reduce the acidity and tannin content of roxburghii juice. Although the Vc content of roxburghii juice is significantly reduced, the Vc value of the fermented roxburghii wine is still at a relatively high level in beverages.

[0089] Table 7 Fermentation characteristics of roxburghii juice by Saccharomyces cerevisiae GZHM-28 and test results of some physicochemical characteristics of the liquor

[0090] Fermentation start time (h) 17.0±0.7 Peak time (h) 48.0±1.7 Peak duration (h) 54.0±3.0 Start clarification time (h) 3.0±0.3 Complete clarification time (h) 56.7±4.3 Residual sugar (%) 3.2±0.1 Alcohol content (%) 4.1±0.2 pH 3.4 Vc(g / L) 0.66±0.19 Tannin(mg / L) 78±13 Total acid (ml / 100mL) 2.75±0.21

[0091] After the post-fermentation of the sea buckthorn wine is completed, the wine is taken and placed in a wine glass for tasting. The specific sensory indicators are shown in Table 8. Without adding any malt or hops, the sea buckthorn wine fermented by Saccharomyces cerevisiae GZHM-28 is golden in color, has a fermentation aroma similar to beer, and retains a distinct sea buckthorn aroma. After entering the cup, the foam is rich, delicate, and lasts for a long time. It has a refreshing taste of beer, slightly sour, and basically no astringency, which is suitable for the public to drink. In short, using Saccharomyces cerevisiae GZHM-28 with acid-reducing and tannin-reducing functions as the fermentation strain, using sea buckthorn juice as the raw material, and fermenting at room temperature for a certain period of time, a slightly sour, low-astringent, and low-alcohol sea buckthorn wine with beer flavor can be prepared, which is suitable for the public to drink.

[0092] Table 8 Sensory evaluation results of some rosa roxburghii wine fermented by Saccharomyces cerevisiae GZHM-28

[0093] index Fermentation of roxburghii wine with Saccharomyces cerevisiae GZHM-28 Rosa roxburghii juice control Color Golden Golden aroma It has the aroma of beer fermentation and a distinct aroma of sea buckthorn No fermentation aroma, only a noticeable sea buckthorn aroma Foam Rich, fine foam that lasts long No foam sour Slightly sour taste Strong sour taste Sweetness Slightly sweet Strong sweetness Astringency Basically no astringency, suitable for the general public Strong astringency Refreshing taste It has a refreshing taste similar to beer No refreshing taste

[0094] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A Saccharomyces cerevisiae GZHM-28, characterized in that: The brewer's yeast GZHM-28 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M20221967 and a deposit date of December 13, 2022.

2. A method for preparing roxburghii fermented wine, characterized in that: The following steps are involved: Take sea buckthorn juice and add water, then add white sugar to adjust the sugar content to 10% (m / v), heat to boiling, then cool to room temperature, inoculate brewer's yeast GZHM-28 seed fermentation liquid, and then seal with sterile water. Ferment at room temperature until the escaping bubbles are reduced to 2-3 times / min, transfer the wine into a sterile beverage bottle under sterile conditions, and continue to ferment at room temperature for 7 days.

3. The preparation method according to claim 2, characterized in that: After the sterile water is sealed, fermentation is carried out at room temperature for 7-10 days.

4. The preparation method according to claim 3, characterized in that: The saccharomyces cerevisiae GZHM-28 seed fermentation liquid is prepared by the following method: Take the slant seeds of Saccharomyces cerevisiae GZHM-28 and streak them on the YEPD solid plate medium, culture them at 28°C for 2 days, pick the milky white, high round and neat-edged colonies, streak them continuously on the YEPD solid plate medium, and activate and culture them 3 times to obtain the activated strain of Saccharomyces cerevisiae GZHM-28; The activated Saccharomyces cerevisiae GZHM-28 strain was inoculated into a test tube slant culture medium, and cultured overnight at 28° C. to obtain a Saccharomyces cerevisiae GZHM-28 slant strain; The slant strain of Saccharomyces cerevisiae GZHM-28 was transferred to the YEPD test tube liquid culture medium, and statically cultured at 28° C. for 1 day to prepare the test tube seeds of Saccharomyces cerevisiae GZHM-28; The test tube seeds of Saccharomyces cerevisiae GZHM-28 were inoculated into YEPD liquid culture medium, cultured overnight at room temperature, and the cells were collected by centrifugation, washed 3 times with saline, and resuspended in 50 mL of saline to obtain the fermentation liquid of Saccharomyces cerevisiae GZHM-28 seeds.

5. The preparation method according to claim 4, characterized in that: The formula of the YEPD solid plate culture medium includes: 10g yeast powder, 20g peptone, 20g glucose and 2% agar.

6. The preparation method according to claim 5, characterized in that: The formula of the test tube slant culture medium is the same as that of the YEPD solid plate.

7. The preparation method according to claim 4, characterized in that: The formula of the YEPD liquid culture medium includes: 10g yeast powder, 20g peptone, 20g white sugar, 1000mL distilled water, pH 6.0, and 115°C wet heat sterilization for 20min; the formula of the YEPD test tube liquid culture medium is the same as the YEPD liquid culture medium.

8. Use of the saccharomyces cerevisiae as claimed in claim 1 in preparing a roxburghii fermented beverage.

9. The use according to claim 8, wherein the roxburghii fermented beverage comprises an alcoholic beverage.

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