Rosa roxburghii tratt fruit wine and mixed fermentation process thereof

Abstract

The invention belongs to the field of fruit wine fermentation, and particularly relates to roxburgh rose fruit wine and a mixed fermentation process thereof. The invention firstly discloses novel hansenula polymorpha L31, and the hansenula polymorpha L31 and saccharomyces cerevisiae (BV818) are applied to mixed fermentation of roxburgh rose fruit wine. The obtained rosa roxburghii tratt fruit wine is high in total ester content (0.631 g / L), the alcoholic strength is 10.89% vol, the measured physical and chemical indexes meet the requirements of T / GZSX055.7-2019 Rosa roxburghii tratt fruit wine (fermented wine) of Rosa roxburghii tratt series products, the wine body is clear and transparent, the aroma is rich, the unique style of the rosa roxburghii tratt fruit wine is achieved, and the sensory score is 85.68. Aromatic substance analysis shows that the variety of aromatic substances in the rosa roxburghii tratt fruit wine is enriched through mixed fermentation, the content of the aromatic substances is obviously increased, and a good aroma enhancement effect on the rosa roxburghii tratt fruit wine is shown.

Description

Technical Field

[0001] The invention belongs to the field of fruit wine fermentation, and particularly relates to roxburghii fruit wine and a mixed bacteria fermentation process thereof. Background Art

[0002] Fruit wines in my country include wine, apple wine, plum wine, kiwi wine, peach wine, pomegranate wine, plum wine, dragon fruit wine, mulberry wine, blueberry wine, etc. However, wine is the most produced fruit wine in my country. The income of my country's wine industry accounts for 90% of the income of the entire fruit wine industry, and the income of other fruit wines accounts for 10%, but overall, other fruit wine industries are showing an upward trend. With the continuous promotion of fruit wine, the fruit wine market accounts for about 15% to 20% in the global industry. Wine is the earliest discovered fruit wine with relatively mature production technology.

[0003] Most of the various fruits used to make fruit wine have high nutritional value. During the winemaking process, some of these nutrients will be retained and entered into the wine, giving the wine antioxidant and chronic disease prevention effects.

[0004] Kiwi fruit is also called sheep peach, kiwi fruit, and vine fruit. The texture of mature kiwi fruit is soft and sweet and sour. Kiwi fruit contains a lot of vitamin C, with a content of up to 4.3g / kg. In addition, kiwi fruit also contains organic substances such as kiwi alkaloids, proteolytic enzymes, tannin pectin and sugars, as well as mineral elements such as calcium, potassium, selenium, zinc, and germanium. Kiwi fruit also has health functions such as anti-oxidation, anti-tumor, and improving human immunity. High-quality kiwi fruit wine contains a variety of mineral elements and has a good antioxidant effect. Kiwi fruit can be grown in most parts of my country with a high yield. Kiwi fruit is not resistant to storage. Combined with these characteristics, kiwi fruit wine has received widespread attention.

[0005] Guava, also known as guava and guava, is a tropical fruit that was introduced to my country at the end of the 17th century and is currently mainly distributed in Guangxi, Guangdong, Fujian and other places. Guava has been introduced to Guangdong for more than 200 years and has become a symbolic economic crop in Guangdong. Guava has a thin skin and thick flesh, with a very high edible rate and high nutritional value. Guava contains more protein (crude protein 0.76-1.06g / 100g guava), and also high calcium, phosphorus and potassium content, of which potassium content is as high as 0.29g / 100g. Guava has high vitamin A and vitamin C content and low fat content. Guava has health benefits such as lowering blood sugar, anti-oxidation, improving immunity, and preventing chronic diseases. Regular consumption of guava can play a role in anti-aging, improving physical function, anti-virus, and anti-radiation. Guava fruit contains a variety of aromatic compounds, including 6 ester compounds, 8 alcohol compounds, 4 aldehyde compounds, etc. These substances give guava fruit a unique aroma, which makes guava wine more fragrant and attractive, with outstanding product features and richer nutrition.

[0006] Pitaya, also known as red dragon fruit, dragon fruit, fairy honey fruit, jade dragon fruit, etc., is native to America and later introduced to Asia. It is planted in Guangxi, Guangdong, Hainan, Fujian and other places in China. It is a kind of tropical and subtropical fruit. The nutritional and health value of pitaya is unique. It has the characteristics of clearing away heat and relieving summer heat, quenching thirst and sweetness. There are more vitamins, proteins, cellulose, etc. in the fruit. In addition, the nutritional value of different varieties of pitaya is also different. There are two main types of pitaya commonly seen on the market, one is red pitaya and the other is white pitaya. The red pitaya has a higher content of dietary fiber, polysaccharides, vitamin C and flavonoids, and has stronger effects in promoting intestinal peristalsis, anti-oxidation, lowering blood sugar and anti-cancer. The white pitaya has a higher fatty acid content and better blood lipid lowering effect. Pitaya can be placed for a relatively long time without being squeezed or collided, which is in sharp contrast to other tropical fruits. Combined with its nutritional value, the commercial development value of pitaya wine is relatively high.

[0007] Rosa roxburghii is the fruit of the perennial deciduous shrub Rosa roxburghii of the Rosaceae family. It grows at altitudes of 500m to 2500m and is distributed in Shaanxi, Gansu and Southwest China. The rosa roxburghii produced in Guizhou is the most famous. The flesh of the rosa roxburghii is crisp and the ripe rosa roxburghii is thick and sweet and sour.

[0008] Rosa roxburghii is rich in more than ten kinds of trace elements needed by the human body, such as vitamin C, superoxide dismutase (SOD), carotene, organic acids, etc. In addition, Rosa roxburghii fruit also contains many important biologically active ingredients. The active substances currently isolated from Rosa roxburghii include: protocatechuic acid, roxburghii acid, roxburghii glycosides, etc., which are flavonoids, organic acids, and triterpenoid compounds respectively.

[0009] Seabuckthorn is sweet and refreshing, with a unique aroma. It is also rich in various nutrients needed by the human body. It has many functions such as delaying aging, improving the body's immunity, lowering blood sugar, lowering blood lipids, preventing cancer, and detoxification. Therefore, there is a broad market demand and prospect for the research and utilization of seabuckthorn.

[0010] Sea roxburgh is often processed into preserved fruits, beverages, fruit wine and other products due to its sour and astringent fresh fruit, short picking period and difficult storage. Among them, fruit wine is favored by consumers because of its rich nutrition, strong fruity aroma and low alcohol content. It is reported that the fruit wine brewed from sea roxburgh is stable, clear and has a strong fruity aroma. It better retains the nutritional value and improves the taste of sea roxburgh, making it a high-quality resource for fruit wine brewing.

[0011] The volatile aroma substances in fruit wine are mainly produced by yeast fermentation. The aroma is strong and typical, which is the sign of high-quality fruit wine. In industrial production of fruit wine, multi-strain co-fermentation is often used. The fruit pulp or juice used for fermentation is not sterilized. A certain yeast is artificially inoculated as the dominant bacteria, and fermented with other strains on the fruit skin to produce fruit wine.

[0012] Since grape wine is the earliest developed fruit wine, most of the yeast used in other types of fruit wine in China is wine brewing yeast. Wine brewing yeast is selected for fermenting grape wine. Using it alone to ferment other fruit wines lacks specificity and may reduce the quality of the fruit wine, making the fruit flavor of the fruit wine less prominent, the acidity higher, the typicality of the fruit wine lower, and the methanol content higher.

[0013] Fruit wine fermentation is a complex process involving multiple microorganisms, among which yeast is particularly important. Saccharomyces cerevisiae mainly completes ethanol fermentation with fast fermentation speed and strong fermenting power. Aroma-producing yeast can convert precursor substances in raw materials into flavor substances such as esters, acids, and higher alcohols, and synthesize various enzymes, which play an important role in the formation of fruit wine color and flavor. Among the discovered aroma-producing yeasts, Pichia-like yeasts are the main ester-producing yeasts. Under oxygen-limited conditions, they can also carry out ethanol fermentation, activate key enzymes in the fermentation pathway, and increase the content of metabolites such as ethanol, glycerol, and ethyl acetate. Compared with single-strain fermentation of fruit wine, mixed yeast fermentation can exhibit better characteristics, such as increasing the content of glycerol in the wine body, reducing the content of acetic acid, producing special flavor substances such as esters and volatile phenols, improving the overall quality of the finished wine, and enriching the flavor diversity of fruit wine products. Mixed yeast fermentation can also slow down the fading degree of the wine sample. However, according to the literature, during mixed yeast fermentation, yeast is inhibited by toxic metabolites during its growth process. Metabolites produced by Saccharomyces cerevisiae during fermentation, such as medium- and long-chain fatty acids, oligopeptides, and cyclic higher alcohols, will inhibit the growth of other strains. The influencing factors of the interaction between strains during mixed yeast fermentation are relatively complex, and the interactions between different yeasts also vary greatly. There is no unified conclusion at home and abroad about the nature of their mutual influence so far.

[0014] In order to obtain more fruit wine-specific yeasts, researchers began to screen corresponding fruit wine yeasts from the fruit surface and orchard soil. Through experiments, 3 strains of yeasts suitable for brewing citrus fruit wine were isolated and screened from the citrus peel and citrus orchard soil. ] Wu Zhuofan et al. used loquat pulp fermentation broth, loquat peel, and loquat orchard soil as raw materials for yeast screening. Through primary screening with 2,3,5-triphenyltetrazolium chloride (TTC) selective medium, secondary screening with Durham tubes, and tertiary screening with small-bottle fermentation experiments, the yeast strain GP-34, which is most suitable for fermenting loquat fruit wine, was finally determined from the 209 initially screened yeasts. Luo Jiali et al. isolated 138 yeast strains from fresh sweet orange peel and sweet orange orchard soil using yeast extract peptone dextrose (YPD) medium. Through Durham tube experiments, small-bottle fermentation experiments, sensory evaluation, alcohol tolerance performance evaluation, and sulfur dioxide tolerance performance evaluation at three levels of screening, two species of Hanseniaspora uvarum S017 and F076 that can tolerate 15% vol alcohol and 150 mg / L sulfur dioxide were finally obtained.

[0015] The research on roxburghii wine focuses on yeast screening, compound wine, disease treatment, etc. Yeast is the core of wine brewing, and the important factors that determine the quality of wine are brewing yeast and aroma yeast. The key to brewing high-quality roxburghii wine is to choose the right yeast.

[0016] Aroma yeast has a strong ability to produce esters, which can improve the types and content of aroma of fermented products and increase the unique flavor of fruit wine. However, its wine production ability is weak. Mixed fermentation with brewing yeast has become an important trend, and using mixed fermentation to enhance the aroma of fruit wine has achieved remarkable results in wine.

[0017] In the research on aroma-producing yeast for sea buckthorn wine, Zhao Hubing, Liu Xiaozhu and others screened out two strains of Hanseniaspora uvarum (F119, F13) and one strain of Wickerhamomyces anomalus (C4), which showed certain aroma-enhancing ability. However, the content and types of ester substances that contribute greatly to the aroma of sea buckthorn wine were limited, or slightly decreased. At the same time, there is a lack of research on the mixed-bacteria fermentation process of sea buckthorn wine. Summary of the invention

[0018] The applicant is committed to the research of fruit wine fermentation. After unremitting efforts, a new grape juice spore-bearing Hansen yeast L31 was isolated and applied to the mixed fermentation of sea buckthorn wine with Saccharomyces cerevisiae (BV818). The total ester content of the obtained sea buckthorn wine is relatively high (0.631g / L), and the alcohol content is 10.89%vol. The measured physical and chemical indicators meet the requirements of T / GZSX055.7-2019 "Sea Buckthorn Series Products Sea Buckthorn Wine (Fermented Wine)". The wine is clear and bright, with a strong aroma and a unique style of sea buckthorn wine. The sensory score is 85.68 points. The analysis of aroma substances shows that mixed fermentation enriches the types of aroma substances in sea buckthorn wine, significantly increases the content of aroma substances, and shows a good flavor-enhancing effect on sea buckthorn wine.

[0019] The present application first discloses a grape juice spore-bearing Hanseniaspora uvarum L31, characterized in that the strain was deposited in the China Center for Type Culture Collection on March 20, 2025, with the deposit number: CCTCCNO: M2025536.

[0020] The growth morphology of the Hansenula vitis spores L31 on the YEPD medium is smooth, opaque, and creamy. Microscopic examination of the strain using a microscope shows that the strain is oval in shape and its asexual reproduction method is multi-terminal budding.

[0021] In the fermentation performance experiment, strain L31 was able to produce bubbles within 10 hours, fill the Dulbecco's tubules within 17 hours, and had more flocculated sediments, showing a faster gas production rate and better agglutination.

[0022] The ethanol tolerance test showed that the L31 strain could tolerate an ethanol content of 18%.

[0023] The SO2 tolerance test showed that the upper limit of SO2 concentration that the L31 strain could tolerate was 300 mg / L.

[0024] The glucose tolerance test showed that the upper limit of glucose concentration that the L31 strain could tolerate was 50%.

[0025] The citric acid tolerance test showed that the L31 strain could tolerate citric acid (pH = 2).

[0026] During the growth process of the L31 yeast strain, it showed a relatively short lag phase, lasting about 0 - 4 h, and then quickly entered the logarithmic growth phase, which lasted from 4 h to 8 h. Subsequently, the strain entered the stationary phase at 12 h and lasted from 20 - 32 h. At 16 h, the OD 600nm value reached the peak, marking the entry into the stationary phase of growth. At this time, the strain had the strongest vitality, and the results showed the characteristics of this strain having a short lag phase and quickly entering the logarithmic growth phase.

[0027] The results of molecular sequence identification showed that the phylogenetic tree of the L31 strain showed that the L31 strain and Hanseniaspora uvarum clustered together, so the L31 strain was identified as Hanseniaspora uvarum.

[0028] Secondly, this application discloses a Rosa roxburghii fruit wine, which is characterized by adopting a mixed fermentation process of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818.

[0029] For the said mixed fermentation process, the inoculation volume ratio of the activated cell suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 1:5 to 5:1.

[0030] For the said mixed fermentation process, the inoculation volume ratio of the activated cell suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 5:5 to 5:1.

[0031] For the said mixed fermentation process, the inoculation volume ratio of the activated cell suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 5:3.

[0032] For the said mixed fermentation process, the inoculation amount of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 3 - 7%.

[0033] For the said mixed fermentation process, the inoculation amount of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 4 - 6%.

[0034] The inoculation amount is the percentage of the total volume of the two yeast suspensions in the mass of the fermentation broth.

[0035] The mixed yeast fermentation process includes the following steps:

[0036] Step 1) Adjust the components: Take the original juice of Rosa roxburghii tratt, adjust the initial sugar addition amount with granulated sugar, adjust the pH with citric acid and edible baking soda, and add potassium metabisulfite.

[0037] Step 2) Activate yeast L31: Pick one loop of L31 strain and inoculate it into YEPD liquid medium, and culture it with shaking at 28 °C and 220 r / min for 18 h for standby.

[0038] Step 3) Inoculate the aroma-producing yeast and ferment for 48 h: Pour the adjusted fermentation broth into a sterilized fermentation flask, add the activated L31 yeast solution and shake well, and ferment at a constant temperature and stand still for 48 h.

[0039] Step 4) Activate yeast BV818: Weigh a certain amount of active dry yeast BV818 and add it to 10 mL of 20% sucrose aqueous solution, and culture it at a constant temperature of 30 °C for 30 min.

[0040] Step 5) Inoculate Saccharomyces cerevisiae and ferment: Add the activated BV818 yeast solution to the fermentation flask after the aroma-producing yeast has fermented for 48 h, shake well, and ferment at a constant temperature and stand still.

[0041] Step 6) Terminate fermentation: Add potassium metabisulfite.

[0042] Step 7) Filter: Filter the fermentation broth with four layers of gauze to obtain Rosa roxburghii tratt fruit wine.

[0043] The Rosa roxburghii tratt fruit wine has a relatively high total ester content (0.631 g / L) and an alcohol content of 10.89% vol.

[0044] Advantages of the present invention:

[0045] This application first discloses a new Hanseniaspora uvarum L31 and applies it in the mixed yeast fermentation of Rosa roxburghii tratt fruit wine together with Saccharomyces cerevisiae (BV818). The obtained Rosa roxburghii tratt fruit wine has a relatively high total ester content (0.631 g / L) and an alcohol content of 10.89% vol. The measured physical and chemical indexes meet the requirements of T / GZSX055.7-2019 "Rosa roxburghii tratt Series Products - Rosa roxburghii tratt Wine (Fermented Wine)". The wine body is clear and bright, has a strong aroma, and has a unique style of Rosa roxburghii tratt fruit wine, with a sensory score of 85.68 points. The analysis of flavor substances shows that the mixed yeast fermentation enriches the types of flavor substances in Rosa roxburghii tratt fruit wine, significantly improves the content of flavor substances, and shows a good flavor-enhancing effect on Rosa roxburghii tratt fruit wine. Description of the Drawings

[0046] Appendix Figure 1Process Flow Chart of Mixed Fermentation of Hanseniaspora uvarum (L31) and Saccharomyces cerevisiae (BV818) in Grape Juice

[0047] Appendix Figure 2 Effect of Mixed Fermentation Ratio of L31 Yeast and BV818 Yeast on the Quality of Rosa roxburghii Fruit Wine

[0048] Appendix Figure 3 Effect of Yeast Inoculation Amount on the Quality of Rosa roxburghii Fruit Wine

[0049] Appendix Figure 4 Effect of Fermentation Temperature on the Quality of Rosa roxburghii Fruit Wine

[0050] Appendix Figure 5 Effect of Initial Sugar Addition Amount on the Quality of Rosa roxburghii Fruit Wine

[0051] Appendix Figure 6 Effect of Fermentation Time on the Quality of Rosa roxburghii Fruit Wine Specific Embodiments

[0052] The present invention will be further described in detail below through examples. The following examples are explanations of the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0053] Example 1: Preparation of Rosa roxburghii Fruit Wine

[0054] As shown in the appendix Figure 1 Process Flow Chart of Mixed Fermentation of Hanseniaspora uvarum (L31) and Saccharomyces cerevisiae (BV818) in Grape Juice. The specific process steps are as follows:

[0055] Step 1) Adjust the composition: Take Rosa roxburghii original juice (original juice sugar content 4.7 °Brix), adjust the initial sugar addition amount with granulated sugar, adjust the pH = 4.5 with citric acid and edible baking soda, and add 120 mg / L of potassium metabisulfite;

[0056] Step 2) Activate yeast L31: Pick one loop of L31 strain into YEPD liquid medium, shake culture at 28 °C and 220 r / min for 18 h for standby;

[0057] Step 3) Inoculate aroma-producing yeast and ferment for 48 h: Pour the adjusted fermentation broth into a sterilized fermentation flask, add the activated L31 yeast solution and shake well, and ferment at a constant temperature and stand for 48 h;

[0058] Step 4) Activate yeast BV818: Weigh a certain amount of BV818 active dry yeast into 10 mL of 20% sucrose aqueous solution, and incubate at 30 °C for 30 min;

[0059] Step 5) Inoculation of Saccharomyces cerevisiae and fermentation: After 48 h of fermentation by aroma-producing yeast, add the activated BV818 yeast solution to the fermentation flask, shake well, and ferment at a constant temperature with static placement.

[0060] Step 6) Termination of fermentation: Add 120 mg / L of potassium metabisulfite.

[0061] Step 7) Filtration: Filter the fermentation broth with four layers of gauze to obtain Rosa roxburghii fruit wine.

[0062] YEPD liquid medium: Weigh 1.0 g of peptone, 1.0 g of glucose, and 0.5 g of yeast extract powder into a sterile beaker, add 50 ml of distilled water, boil for 10 min, autoclave at 121 °C for 20 min, and cool to room temperature.

[0063] YPD solid medium: Add 1.0 g of agar powder to the components of YEPD, and the rest is the same. After sterilization and slightly cooling, pour it into a petri dish and cool.

[0064] Counting of yeast: Sequentially dilute the activated yeast to 10 -6 、10 -7 、10 -8 in a laminar flow hood. Take 0.1 mL and evenly spread it on the YPD solid medium, and incubate at 28 °C for 48 h. Count the colonies according to GB 4789.2-2022 "Determination of Total Number of Colonies in Food Microbiology - Colony Counting". Select the plate with no spreading colony growth and a total number of 30 - 300 CFU for counting, and prepare a seed solution with the same bacterial suspension concentration (3×10 7 CFU / mL) for standby.

[0065] Example 2 Single-factor experiment on mixed-bacteria fermentation

[0066] Based on the preparation process of Example 1, the basic fermentation conditions are as follows: The inoculation ratio of L31 yeast and BV818 yeast is 5:5, the yeast inoculation amount is 5%, the fermentation temperature is 26 °C, the initial sugar addition amount is 25%, and the fermentation time is 8 d. Based on the basic conditions, successively investigate the effects of yeast mixing ratio (1:5, 3:5, 5:5, 5:3, 5:1), yeast inoculation amount (3%, 4%, 5%, 6%, 7%), fermentation temperature (22 °C, 24 °C, 26 °C, 28 °C, 30 °C), initial sugar addition amount (15%, 20%, 25%, 30%, 35%), and fermentation time (6 d, 7 d, 8 d, 9 d, 10 d) on the alcohol content and total ester content of Rosa roxburghii fruit wine.

[0067] The obtained results are as follows:

[0068] 2.1 Effect of mixed-bacteria fermentation ratio on the quality of Rosa roxburghii fruit wine

[0069] From the appendix Figure 2It can be seen from (lowercase and uppercase letters represent the significance of total ester content and alcohol content respectively (P < 0.05, Tukey method)) that as the ratio of L31 in mixed yeast fermentation increases within the range of 1:5 to 5:1, the alcohol content of Rosa roxburghii fruit wine gradually decreases and then levels off, while the total ester content first increases and then decreases. Aroma-producing yeast has a strong ability to produce esters, but its ability to produce alcohol is weak, while the opposite is true for Saccharomyces cerevisiae. Therefore, as the ratio of aroma-producing yeast L31 increases, the amount of Saccharomyces cerevisiae BV818 decreases, the contribution of aroma-producing yeast increases, the total ester content increases, and the alcohol content decreases. When the ratio is 5:3, the total ester content is the highest (0.561 g / L). When the ratio further increases (5:1), the total ester content decreases, possibly because the excessive content of L31 yeast causes yeast to respire and reproduce too quickly, resulting in the oxidation of some ester substances. Through comprehensive analysis, the mixed yeast ratios of 5:5, 5:3, and 5:1 are selected for the orthogonal experiment. At this time, the total ester content is relatively high and the alcohol content is appropriate.

[0070] 2.2 Effect of inoculation amount of mixed yeast on the quality of Rosa roxburghii fruit wine

[0071] It can be seen from the appendix Figure 3 that as the inoculation amount of mixed yeast increases within the range of 3% to 7%, the alcohol content of Rosa roxburghii fruit wine first increases and then levels off, while the total ester content first increases and then decreases. The amount of yeast inoculation is limited by the strain and the fermentation mother liquor, and at the same time will affect the fermentation efficiency of fruit wine. When the yeast inoculation amount is 6%, the alcohol content reaches the maximum of 13.47% vol. At this time, Saccharomyces cerevisiae BV818 has a relatively high sugar conversion rate. When the yeast inoculation amount is 4% to 7%, the change in alcohol content is small. When the inoculation amount is 5%, the total ester content is the highest at 0.378 g / L, and the most ester substances are produced by yeast L31. When the inoculation amount continues to increase, the total ester content decreases. It may be that excessive yeast reproduction consumes nutrients excessively, and at the same time, the accumulation of metabolites further inhibits the metabolic activities of yeast, thereby reducing the formation of ester substances. Through comprehensive analysis, considering the aroma-enhancing effect of yeast L31, the yeast inoculation amounts of 4%, 5%, and 6% are selected for the orthogonal experiment.

[0072] 2.3 Effect of mixed yeast fermentation temperature on the quality of Rosa roxburghii fruit wine

[0073] It can be seen from the appendix Figure 4 that as the mixed yeast fermentation temperature increases within the range of 22°C to 30°C, the alcohol content of Rosa roxburghii fruit wine first increases and then levels off, while the total ester content first increases and then decreases. Fermentation temperature has a great influence on the growth and metabolism of yeast. Both too high and too low temperatures are not conducive to yeast fermentation and the formation of metabolites. When the temperature is 26°C, the total ester content is the highest (0.366 g / L), and the alcohol content reaches a relatively large value (13.06% vol). When the temperature is 30°C, too high a temperature is not conducive to the growth and metabolism of L31. At the same time, the activities of small molecules in the fermentation broth increase, affecting the retention of aroma components, and the formation of ester substances decreases. Through comprehensive analysis, the fermentation temperatures of 24°C, 26°C, and 28°C are selected for the orthogonal experiment.

[0074] Effect of Initial Sugar Addition Amount in Mixed-Strain Fermentation on the Quality of Rosa roxburghii Fruit Wine

[0075] As can be seen from the appendix Figure 5 As the initial sugar addition amount in mixed-strain fermentation increases within the range of 15 - 35%, the alcohol content and total ester content of Rosa roxburghii fruit wine show a trend of first increasing and then decreasing. Sugar is the fermentation substrate for yeast. When the sugar content is too low, the fermentation substrate is insufficient, while when the sugar content is too high, the osmotic pressure increases, which will inhibit yeast metabolism, thus reducing the production of alcohol and ester substances. When the initial sugar addition amount is 25%, the alcohol content and total ester content of Rosa roxburghii fruit wine reach the maximum values, which are 13.42% vol and 0.514 g / L respectively. As the addition amount further increases (35%), the increase in the alcohol content and total ester content of Rosa roxburghii fruit wine is accompanied by an overly sweet taste of the wine body. Through comprehensive analysis, an initial sugar addition amount of 25% is selected for the orthogonal experiment.

[0076] Effect of Mixed-Strain Fermentation Time on the Quality of Rosa roxburghii Fruit Wine

[0077] As can be seen from the appendix Figure 6 As the mixed-strain fermentation time increases within the range of 6 - 10 d, the alcohol content of Rosa roxburghii fruit wine first increases and then levels off, while the total ester content first increases and then decreases. In the early stage of fermentation, the sugar source is sufficient, and yeast grows rapidly. As the fermentation time increases, the alcohol content and total ester content increase rapidly. In the later stage, the sugar source decreases, and yeast begins to decline, resulting in a slow or stagnant increase in the alcohol content. When the fermentation time is 6 - 7 d, the fermentation time is too short, and the accumulation of yeast metabolites is insufficient. When the fermentation time is 8 d, the total ester content reaches the maximum value (0.599 g / L), and the alcohol content is relatively high (12.31% vol). When the fermentation time further increases, the alcohol content stabilizes while the total ester content decreases. This is mainly because the metabolites accumulated during the growth of Saccharomyces cerevisiae (BV818) inhibit the growth or cause the death of aroma-producing yeast (L31), and at the same time, ester substances may be further metabolized or transformed. Through comprehensive analysis, a mixed-strain fermentation time of 8 d is selected for the orthogonal experiment.

[0078] Example 3 Orthogonal Experiment Design of Mixed-Strain Fermentation

[0079] Based on the preparation process of Example 1 and the single-factor experiment of Example 2, three factors with greater influence on the fermentation of Rosa roxburghii fruit wine are selected for the orthogonal experiment, namely the yeast compound ratio (A), fermentation temperature (B), and yeast inoculation amount (C). Three levels are selected, and the alcohol content (Y1) and total ester content (Y2) are used as evaluation indicators. The L9(3 4 ) orthogonal experiment design is used to determine the optimal process, as shown in Table 1 specifically.

[0080] Table 1 Factors and Levels of Orthogonal Experiment for Optimizing Mixed-Strain Fermentation Process

[0081]

[0082] The Rosa roxburghii Tratt fruit wine was prepared according to the fermentation process parameters in Table 1, and the results are shown in Table 2.

[0083] Table 2 Results of the orthogonal test on mixed - strain fermentation of Rosa roxburghii Tratt fruit wine

[0084]

[0085]

[0086] As can be seen from Table 2, when the alcohol content was used as the evaluation index, according to the range of extreme differences, the order of the influence of the three factors on the Rosa roxburghii Tratt fruit wine was: B (fermentation temperature) > A (yeast compound ratio) > C (yeast inoculation amount). At this time, the optimal process combination was A2B3C2, that is, the yeast compound ratio was 5:3, the fermentation temperature was 28 °C, and the yeast inoculation amount was 5%. After experimental verification, the alcohol content of the Rosa roxburghii Tratt fruit wine was 12.76% vol, and the total ester content was 0.560 g / L. When the total ester content was used as the evaluation index, the order of the influence of the three factors was: A > C > B. At this time, the optimal process combination was A2B2C3, that is, the yeast compound ratio was 5:3, the fermentation temperature was 26 °C, and the yeast inoculation amount was 6%. At this time, the alcohol content of the Rosa roxburghii Tratt fruit wine was 10.89% vol, and the total ester content was relatively high at 0.631 g / L. As can be seen from Table 3, factors A, B, and C had a significant influence on the alcohol content of the Rosa roxburghii Tratt fruit wine (P < 0.05); A and C had a significant influence on the total ester content of the Rosa roxburghii Tratt fruit wine (P < 0.05), and factor B had no significant influence on the total ester content of the Rosa roxburghii Tratt fruit wine (P > 0.05). To make the best use of the aroma - enhancing potential of mixed - strain fermentation on Rosa roxburghii Tratt fruit wine, the optimal process conditions were determined as A2B2C3: yeast compound ratio 5:3, fermentation temperature 26 °C, yeast inoculation amount 6%, initial sugar addition amount 25%, and fermentation time 8 d.

[0087] Table 3 Analysis of variance table of factors in the orthogonal test of Rosa roxburghii Tratt fruit wine fermentation (alcohol content / total ester)

[0088]

[0089] Note: "*" indicates (P < 0.05).

[0090] Analysis of the physical and chemical indexes of Rosa roxburghii Tratt fruit wine by mixed - strain fermentation in Example 4

[0091] After determining the optimal process conditions A2B2C3 for the mixed - strain (BV818 + L31, hereinafter referred to as the mixed - strain group) fermentation of Rosa roxburghii Tratt fruit wine through the orthogonal test in Example 3, the obtained Rosa roxburghii Tratt fruit wine was compared with the Rosa roxburghii Tratt fruit wine fermented by a single strain (BV818, hereinafter referred to as the single - strain group) under the same process conditions (the preparation process was based on Example 1, and the L31 bacterial liquid was replaced with the same volume of BV818 bacterial liquid), and the physical and chemical indexes were detected. The detection methods are as follows:

[0092] Alcohol content: Refer to the density bottle method in GB 5009.225-2023 National Food Safety Standard - Determination of Ethanol Concentration in Wine and Edible Alcohol;

[0093] Total sugar, titratable acid, volatile acid, and dry extract content: Refer to the direct titration method, potentiometric titration method, titration method, and density bottle method in GB / T 15038-2006 General Analytical Methods for Wines and Fruit Wines;

[0094] Total ester and solid content: Refer to GB / T 10345-2022 Methods for Analysis of Chinese Liquor;

[0095] Vitamin C content: Refer to the 2,6-dichlorophenolindophenol titration method in GB 5009.86-2016 National Food Safety Standard - Determination of Ascorbic Acid in Foods;

[0096] Sensory evaluation: The scoring criteria are shown in Table 4:

[0097] Table 4 Sensory Scoring Criteria for Rosa roxburghii Fruit Wine

[0098]

[0099] Determination of aroma components:

[0100] (1) HS-SPME extraction conditions: Add 7 mL of the sample, 1.5 g of NaCl, and 10 μL of 2-octanol (56 mg / L, internal standard) to the headspace vial. Shake at 45 °C for 5 min, extract the headspace with a 120 μm DVB / CWR / PDMS extraction head for 40 min, and desorb at 250 °C for 5 min before GC-MS determination.

[0101] (2) GC-MS conditions and analysis: DB-FFAP capillary column (60 m × 0.25 mm × 0.25 μm), carrier gas (He) flow rate 1.2 mL / min, injection port temperature 250 °C. The column temperature is raised from 40 °C to 50 °C at a rate of 10 °C / min, held for 2 min, then raised to 80 °C at a rate of 3 °C / min, held for 2 min, and then raised to 245 °C at a rate of 5 °C / min, held for 3 min. The quadrupole temperature is 150 °C, the mass spectrometry interface temperature is 250 °C, and the mass spectrometry scanning range is 10 amu - 550 amu. The ion chromatogram is retrieved through the Wiley spectral library and the National Institute of Standards and Technology (NIST) 2.0 spectral library, and substances with a matching degree ≥ 80 are retained for quantification by the internal standard method.

[0102] Data processing: Use Graph Pad Prism and Excel for data processing and plotting, and SPSS Statistics 27.0 for significance analysis. All experiments are determined in parallel three times, and the results are expressed as

[0103] The results are shown in Table 5.

[0104] Table 5 Results of Physicochemical Indexes of Rosa roxburghii Fruit Wine in Single-Strain Group and Mixed-Strain Group (n = 3)

[0105]

[0106] As can be seen from Table 5, the total ester content in the mixed-strain group is relatively high (0.631 g / L), which is 1.6 times that of the single-strain group; the alcohol content in the mixed-strain group (10.89% vol) is lower than that of the single-strain group, which is consistent with the relatively high total sugar content (10.62 g / L); the solid content in the mixed-strain group (32.24 g / L) is lower, and the wine body is more transparent and clear; the sensory score of the mixed-strain group (85.68 points) is relatively high, the wine body is clear and bright yellow, the flavors of Rosa roxburghii and wine are coordinated, the taste is clean, and it has a unique style of Rosa roxburghii fruit wine; the vitamin C content in the mixed-strain group (1189.18 mg / 100 mL) is relatively high, and the antioxidant active substances are well retained. In addition, the alcohol content, total sugar, titratable acid, volatile acid, dry extract, and vitamin C content in the mixed-strain group meet the requirements specified in T / GZSX055.7-2019 "Rosa roxburghii Series Products - Rosa roxburghii Wine (Fermented Wine)" (alcohol content ≥ 10% vol, total sugar ≤ 45 g / L, titratable acid ≥ 6.0 g / L, volatile acid ≤ 1.6 g / L, dry extract ≥ 16.0 g / L, vitamin C ≥ 120 mg / 100 mL).

[0107] Analysis of Flavor Substances in Rosa roxburghii Fruit Wine by Mixed-Strain Fermentation in Example 5

[0108] Take the Rosa roxburghii fruit wine prepared by the mixed-strain fermentation process and the single-strain fermentation process in Example 4 for flavor substance detection, and the results are shown in Table 6 as follows:

[0109] Table 6 Analysis of Aroma Components of Rosa roxburghii Fruit Wine Fermented by Single Saccharomyces cerevisiae and Composite Yeast

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Note: "a", the aroma characteristics refer to http: / / www.Thegoodscentscompany.com / and http: / / www.chemicalbook.com / ; "-" indicates not detected.

[0116] The results are shown in Table 6. 60 flavor substances were detected in the single-strain group, including 2 kinds of C6 alcohols, 9 kinds of higher alcohols, 6 kinds of acetate esters, 15 kinds of ethyl fatty acid esters, 8 kinds of other esters, 7 kinds of acids, 4 kinds of carbonyl compounds and 9 kinds of other substances, with a total content of 666.30 μg / L. 67 flavor substances were detected in the mixed-strain group. Compared with the single-strain group, it had 1 more kind of acetate ester, 2 more kinds of ethyl fatty acid esters, acids and other substances, and the content was 1688.81 μg / L. In terms of the type and content of flavor substances, the mixed-strain group was higher than the single-strain group, and its content was 2.5 times that of the single-strain group.

[0117] Alcohols are important components of fruit wine, and higher alcohols are mainly produced during fermentation. The main alcohol substances in the single-strain group and the mixed-strain group are trans-3-hexenol, isoamyl alcohol and phenethyl alcohol, accounting for 96.3% and 95.2% of the total alcohol content respectively. Among them, the content of isoamyl alcohol is the highest. Isoamyl alcohol can endow fruit wine with the aroma of apple brandy. Phenethyl alcohol is mainly synthesized from phenylalanine in glycolysis and has the fragrance of roses. Trans-3-hexenol is produced by the degradation of unsaturated fatty acids and has rich floral fragrance. The total amount of alcohol substances in the mixed-strain group (304.71 μg / L) was significantly higher than that in the single-strain group (127.50 μg / L), which was 2.4 times that of the single-strain group. The fruit wine of Rosa roxburghii tratt fermented by mixed strains had a richer fragrance of alcohol substances.

[0118] Esters are the most important aroma-contributing substances in the fruit wine of Rosa roxburghii tratt, and are mainly synthesized through esterification reaction and the metabolism of higher alcohols. The ester substances in the fruit wine of Rosa roxburghii tratt mainly include acetate esters and ethyl fatty acid esters, which make the fruit wine of Rosa roxburghii tratt present rich floral and fruity aromas. The content of acetate esters in the single-strain group and the mixed-strain group accounted for 31.9% and 47.1% of the total esters respectively; ethyl fatty acid esters accounted for 64.7% and 50.9% of the total esters respectively. Among them, the top 4 kinds of acetate esters and the top 4 kinds of ethyl fatty acid esters mainly endow the fruit wine of Rosa roxburghii tratt with banana flavor, sweet fruit flavor, grass flavor and floral flavor, etc. The total content of 32 ester substances in the mixed-strain group (1033.14 μg / L) was significantly higher than that of 29 ester substances in the single-strain group (446.43 μg / L), which was 2.3 times that of the single-strain group, indicating that Saccharomyces cerevisiae L31 had a significant aroma-enhancing effect on the ester substances in the fruit wine of Rosa roxburghii tratt. This was consistent with the conclusion that Hanseniaspora uvarum in grape juice had a specific regulatory effect on the formation of acetate esters and ethyl fatty acid esters during mixed-strain fermentation.

[0119] Acids can balance the aroma of Rosa roxburghii Tratt fruit wine. When the concentration is low, it produces a special aroma flavor, and when the concentration is high, it produces an unpleasant pungent smell. Among the acid substances in the single-strain group and the mixed-strain group, the first 4 types have relatively high contents. The total content of 9 acid substances in the mixed-strain group (229.37 μg / L) is higher than that of 7 acid substances in the single-strain group (66.44 μg / L), which is 3.5 times that of the single-strain group. The mixed-strain group shows a more harmonious aroma. Carbonyl compounds mainly include ketones and aldehydes. The total content of carbonyl compounds in the mixed-strain group (22.18 μg / L) is higher than that in the single-strain group (7.72 μg / L), which is 2.9 times that of the single-strain group. Among them, the first 2 substances in the mixed-strain group account for 78.8% of the total content of carbonyl compounds, making the Rosa roxburghii Tratt fruit wine present a buttery flavor, fruity flavor and brandy smell, indicating that yeast L31 has an obvious aroma-enhancing effect on carbonyl compounds. In terms of other substances, the total content in the mixed-strain group (91.45 μg / L) is higher than that in the single-strain group (15.56 μg / L), which is 4.1 times that of the single-strain group. The unique citronellol and 4-vinyl-2-methoxyphenol in the mixed-strain group endow the Rosa roxburghii Tratt fruit wine with a richer flower and fruit fragrance.

[0120] Comprehensively analyzing, mixed-strain fermentation enriches the types of flavor substances in Rosa roxburghii Tratt fruit wine, significantly improves the content of flavor substances, especially alcohols, esters and acid substances, showing a good aroma-enhancing effect on Rosa roxburghii Tratt fruit wine.

[0121] This application uses mixed-strain fermentation of Rosa roxburghii Tratt fruit wine with Hanseniaspora uvarum (L31) and Saccharomyces cerevisiae (BV818). The obtained Rosa roxburghii Tratt fruit wine has a relatively high total ester content (0.631 g / L), an alcohol content of 10.89% vol. The measured physical and chemical indexes meet the requirements of T / GZSX055.7-2019 "Rosa roxburghii Tratt Wine (Fermented Wine) in Rosa roxburghii Tratt Series Products". The wine body is clear and bright, has a strong aroma, and has a unique style of Rosa roxburghii Tratt fruit wine, with a sensory score of 85.68 points. The analysis of flavor substances shows that mixed-strain fermentation enriches the types of flavor substances in Rosa roxburghii Tratt fruit wine, significantly improves the content of flavor substances, and shows a good aroma-enhancing effect on Rosa roxburghii Tratt fruit wine.

[0122] Example 6: Rosa roxburghii Tratt fruit wine prepared by optimizing the mixed-strain fermentation process

[0123] Step 1) Adjust the ingredients: Take Rosa roxburghii Tratt original juice (original juice sugar degree 4.7 °Brix), adjust the initial sugar addition amount to 25% with white granulated sugar, adjust the pH = 4.5 with citric acid and edible baking soda, and add 120 mg / L of potassium metabisulfite;

[0124] Step 2) Activate yeast L31: Pick one loop of L31 strain into YEPD liquid medium, shake culture at 28 °C and 220 r / min for 18 h for standby;

[0125] Step 3) Inoculate aroma-producing yeast and ferment for 48 h: Pour the fermented liquid after adjusting the ingredients into a sterilized fermentation bottle, inoculate 3.75% of the activated L31 yeast liquid, shake well, and ferment at a constant temperature of 26 °C for 48 h;

[0126] Step 4) Activation of yeast BV818: Weigh a certain amount of active dry yeast BV818 into 10 mL of 20% sucrose aqueous solution and incubate at 30 °C for 30 min.

[0127] Step 5) Inoculation of Saccharomyces cerevisiae and fermentation: Add 2.25% of the activated BV818 yeast solution to the fermentation flask after 48 h of fermentation of the aroma-producing yeast, shake well, and ferment statically at a constant temperature for 8 days.

[0128] Step 6) Termination of fermentation: Add 120 mg / L of potassium metabisulfite.

[0129] Step 7) Filtration: Filter the fermentation broth with four layers of gauze to obtain Rosa roxburghii fruit wine.

[0130] The above invention content and embodiments describe the basic principles, main features and advantages of this invention patent application. Those skilled in the art should understand that this invention patent application is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only the optimal technical solutions of this invention patent application. Without departing from the spirit and scope of this invention patent application, this invention patent application will have various changes and improvements, all of which fall within the scope of this invention patent application claimed. The scope of protection claimed by this invention patent application is defined by the appended claims and their equivalents.

Claims

1. Hanseniaspora uvarum L31, characterized in that, The strain was deposited at the China Center for Type Culture Collection on March 20, 2025, with the deposit number: CCTCC NO: M2025536.

2. A Rosa roxburghii fruit wine, characterized in that, The mixed fermentation process of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 is adopted.

3. The rosa roxburghii tratt fruit wine according to claim 2, characterized in that, In the mixed fermentation process, the inoculation volume ratio of the activated bacterial suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 1:5 to 5:

1.

4. The Rosa roxburghii tratt fruit wine according to claim 2, characterized in that In the mixed fermentation process, the inoculation volume ratio of the activated bacterial suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 5:5 to 5:

1.

5. The Rosa roxburghii tratt fruit wine according to claim 2, characterized in that, In the mixed fermentation process, the inoculation volume ratio of the activated bacterial suspensions of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 5:

3.

6. The rosa roxburghii tratt fruit wine according to claim 2, wherein In the mixed fermentation process, the inoculation amount of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 3% to 7%.

7. The Rosa roxburghii Tratt fruit wine according to claim 2, wherein In the mixed fermentation process, the inoculation amount of Hanseniaspora uvarum L31 and Saccharomyces cerevisiae BV818 used is 4 to 6%.

8. The Rosa roxburghii tratt fruit wine according to any one of claims 6-7, characterized in that The inoculation amount is the percentage of the total volume of the two yeast suspensions in the mass of the fermentation broth.

9. The Rosa roxburghii tratt fruit wine according to any one of claims 2-7, characterized in that, The steps of the mixed fermentation process are as follows: Step 1) Adjust the components: Take the original juice of Rosa roxburghii tratt, adjust the initial sugar addition amount with granulated sugar, adjust the pH with citric acid and edible baking soda, and add potassium metabisulfite; Step 2) Activate yeast L31: Pick one loop of L31 strain and inoculate it into YEPD liquid medium, shake culture at 28°C and 220 r / min for 18 h for standby; Step 3) Inoculate the aroma-producing yeast and ferment for 48 h: Put the adjusted fermentation broth into a sterilized fermentation bottle, add the activated L31 yeast liquid and shake well, and ferment at a constant temperature and stand still for 48 h; Step 4) Activate yeast BV818: Weigh a certain amount of active dry yeast BV818 and add it to 10 mL of 20% sucrose aqueous solution, and culture at a constant temperature of 30°C for 30 min; Step 5) Inoculate Saccharomyces cerevisiae and ferment: Add the activated BV818 yeast liquid to the fermentation bottle after the aroma-producing yeast has fermented for 48 h, shake well, and ferment at a constant temperature and stand still; Step 6) Terminate fermentation: Add potassium metabisulfite; Step 7) Filter: Filter the fermentation broth with four layers of gauze to obtain Rosa roxburghii tratt fruit wine.

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