Rhodotorula mucilaginosa and application thereof in kiwi fruit wine

Abstract

The invention discloses rhodotorula mucilaginosa and application of the rhodotorula mucilaginosa in kiwi fruit wine, and belongs to the technical field of microorganisms. The rhodotorula mucilaginosa M07 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center on October 24, 2024, and the preservation number is CGMCC No.32331. The rhodotorula mucilaginosa M07 disclosed by the invention has the advantages that the rhodotorula mucilaginosa In a strain tolerance determination result, the strain M07 can tolerate the pH value of 3.0, the sugar concentration of 450 g / L and the alcohol concentration of 4%. The concentration of ethyl acetate, ethyl hexanoate, ethyl caprylate, phenethyl alcohol and ethyl caprate in kiwi fruit wine brewed by the rhodotorula mucilaginosa strain is improved compared with that of Angel fruit wine yeast, and the relative content of ethyl acetate can reach 20.033%; in the kiwi fruit wine fermented by the composite yeast, the two yeasts can interact with each other to adjust the content of each substance in the wine; the rhodotorula mucilaginosa fermented kiwi fruit wine has a certain positive effect on the flavor of the kiwi fruit wine.

Description

Technical Field

[0001] This invention relates to a type of Rhodotorula glutinis and its application in kiwifruit wine, belonging to the field of microbial fermentation. Background Technology

[0002] Kiwifruit, also known as Chinese gooseberry, has a brown fuzzy exterior and bright green flesh with small black seeds inside. It has a sweet and sour taste and is rich in vitamin C, vitamin K, dietary fiber, and antioxidants.

[0003] Kiwi fruit wine is an alcoholic beverage made from kiwifruit. This wine is produced by fermenting fresh kiwifruit or its juice with sugar and yeast. The process aims to infuse the wine with the unique flavor and nutrients of kiwifruit, resulting in a refreshing and flavorful drink. Kiwi fruit wine has a delicate color and a rich kiwifruit aroma. In terms of taste, it retains the sweetness of kiwifruit while also possessing a slightly tart flavor, making it a delightful beverage. Kiwi fruit wine is suitable as a celebratory drink or a pleasant treat during leisure time. Its unique flavor makes it one of the most popular products in the fruit wine market.

[0004] In fruit wine brewing, yeast, the fermenting agent, plays a crucial role in the quality of the wine, directly affecting its taste, flavor, and sensory and physicochemical properties such as alcohol content. Currently, commercial yeasts such as wine yeast and Angel yeast are commonly used in fruit wine brewing. However, due to the lack of specialized yeasts with excellent fermentation performance, the yield and quality of kiwifruit wine cannot be guaranteed. Furthermore, traditional fermentation methods involve inoculating activated yeast seed liquid into the fruit juice for fermentation without replenishing the yeast. This limits the sugar content that the yeast can utilize, resulting in generally low alcohol content in the fruit wine. In recent years, researchers have successively used commercial yeasts or isolated and purified yeasts to brew fruit wine from different varieties of kiwifruit. Among them, the article "Screening of Kiwi Fruit Wine Yeast and Optimization of Fermentation Process" discloses the isolation and screening of yeast from Shaanxi kiwi fruit peel for kiwi fruit wine fermentation, with an inoculation amount of 8%, a sugar content of 240 g / kg, and an alcohol content of 14.14% vol; the article "Research on the Brewing Process of Soft-fleshed Kiwi Fruit Wine" uses Angel active dry yeast to brew soft-fleshed kiwi fruit wine, with an inoculation amount of 1.4%, a sugar content of 20.7%, and an alcohol content of 10.4% vol; the article "Exploration of the Brewing Process of Red-fleshed Kiwi Fruit Wine" uses red-fleshed kiwi fruit wine with an alcohol content of 12.54% vol. Patent CN104312832A discloses the brewing of kiwi fruit wine using honey, white sugar, and yeast; patent CN104130904B discloses a brewing method for kiwi fruit wine using honey, yeast, and sulfur dioxide. All of the above methods have drawbacks such as low alcohol content and insufficient flavor compounds.

[0005] With the improvement of kiwifruit cultivation technology, my country's kiwifruit supply is showing an oversupply trend. Developing new technologies for fruit wine brewing and screening and applying special strains of bacteria are worth paying attention to in order to solve the problem of spoilage caused by the oversupply of kiwifruit. Using kiwifruit to brew fruit wine can not only alleviate the problem of resource waste, but also drive the economic development of mountainous areas. Summary of the Invention

[0006] To address the above problems, this invention provides a type of yeast (Rhodotorula glutinis). Rhodotorula mucilaginosa M07, the aforementioned Rhodotorula glutinis M07, was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32331.

[0007] The present invention also provides a probiotic preparation, wherein the probiotic preparation contains the above-mentioned Rhodotorula glutinis ( Rhodotorula mucilaginosa )M07.

[0008] In one embodiment of the present invention, the probiotic preparation contains the Rhodotorula glutinis (…). Rhodotorula mucilaginosa M07 live cells and / or cell metabolites, and / or inactivated cells, and / or lyophilized cells.

[0009] In one embodiment of the present invention, the probiotic preparation contains Rhodotorula glutinis (Rhodotorula glutinis). Rhodotorula mucilaginosa The quantity of M07 is ≥ 1 × 10 6 CFU / mL or 1×10 6 CFU / g.

[0010] In one embodiment of the present invention, the probiotic preparation also contains Angel yeast.

[0011] In one embodiment of the present invention, the Red Yeast M07 and Angel Yeast were obtained by mixing them in a ratio of (1~2): (1~2) of inoculum concentration.

[0012] In one embodiment of the present invention, the Red Yeast M07 and Angel Yeast were obtained by mixing them at a ratio of 1:1.

[0013] The present invention also provides a product containing the above-mentioned red yeast ( Rhodotorula mucilaginosa M07 or the above-mentioned probiotic preparations.

[0014] In one embodiment of the present invention, the product is food, functional food, food for special medical purposes, health product, feed, feed additive, or food additive.

[0015] In one embodiment of the present invention, the food is a grain product, vegetable product, fruit product, meat product, seafood, egg product, dairy product, soy product, or beverage. In one embodiment of the present invention, the food is a special dietary food; In one embodiment of the present invention, the health product also contains acceptable excipients.

[0016] The present invention also provides the above-mentioned red yeast ( Rhodotorula mucilaginosa M07, or the application of the above-mentioned microbial agents in the preparation of fermented products, wherein the fermented products are fermented alcoholic beverages or fermented vinegar.

[0017] In one embodiment of the present invention, the fermented alcoholic beverage is fruit wine, and the fermented vinegar is fruit vinegar.

[0018] In one embodiment of the present invention, the fermented alcoholic beverage is: yellow wine, cooking wine, rice wine, sweet rice wine, wine, beer, or baijiu; the fermented vinegar is edible vinegar; In one embodiment of the present invention, the fruit wine is, but is not limited to, pineapple wine, kiwi wine, dragon fruit wine, or lychee wine.

[0019] This invention also provides a method for brewing kiwifruit wine, comprising the following steps: enzymatic hydrolysis to obtain kiwifruit puree, first step of adding sugar to obtain kiwifruit pulp, inoculating with bacterial strain to obtain kiwifruit fermentation mash, and second step of adding sugar to obtain kiwifruit wine.

[0020] The present invention also provides an application of the aforementioned Rhodotorula glutinis strain in the brewing of kiwifruit wine.

[0021] This invention also provides a method for brewing kiwi wine, the method comprising the following steps: (1) Enzymatic hydrolysis of fruit puree: Peel and pulp kiwifruit, add pectinase, and hydrolyze to obtain kiwifruit puree; (2) First step of sugar addition: Add white sugar to the kiwi fruit puree in step (1); stir thoroughly to dissolve, adjust the pH to 3.6~4.0, heat at 55~65℃ for 15~25 min to obtain kiwi fruit pulp; (3) Inoculation of strains: After the kiwifruit pulp is cooled to 15~25℃, inoculate it with the Rhodotorula glutinis M07 as described in claim 1 or the probiotic preparation as described in any one of claims 2~4 at an inoculation amount of 0.1~0.2 g / L, let it stand at 26~30℃ for 2~4 days to ferment and obtain kiwifruit fermentation mash. (4) Second step of sugar supplementation: Add 8-12% (w / w) of honey to the kiwi fermentation mash obtained in step (3), shake well, let stand at 26-30℃ for 1-3 days to continue fermentation, and obtain kiwi fruit wine; In one embodiment of the present invention, the amount of pectinase added is 0.08~0.12% (w / w). In one embodiment of the present invention, the enzymatic hydrolysis method after adding pectinase is as follows: enzymatic hydrolysis at 30~35℃ for 3~4 h; In one embodiment of the present invention, the amount of white sugar added is 5~6% (w / w).

[0022] Beneficial effects (1) This invention provides a red yeast, which is taxonomically named red yeast ( Rhodotorula mucilaginosa The strain M07, with accession number CGMCC No. 32331, was found to be tolerant of pH 3.0, sugar concentration of 450 g / L, and alcohol concentration of 4% in strain tolerance tests. The *Rhodotorula glutinis* strain screened in this invention can be used in the fermentation of kiwifruit wine and has a positive effect on its flavor.

[0023] (2) The concentrations of ethyl acetate, ethyl hexanoate, ethyl octanoate, phenylethanol and ethyl decanoate in the kiwi wine brewed using the Rhodotorula glutinis strain of the present invention are higher than those of Angel Fruit Wine Yeast, with the relative content of ethyl acetate reaching 20.033%; in the kiwi wine fermented by the compound yeast, the two yeasts can interact and regulate the content of various substances in the wine.

[0024] Preservation of biological materials A strain of red yeast ( Rhodotorula mucilaginosa M07, taxonomically named Rhodotorula glutinis Rhodotorula mucilaginosa It was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32331. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 For red yeast ( Rhodotorula mucilaginosa Colony morphology of M07; Figure 2 For red yeast ( Rhodotorula mucilaginosa Microscopic morphological features of M07; Figure 3 For red yeast ( Rhodotorula mucilaginosa Phylogenetic tree of 26S rDNA from M07; Figure 4 For red yeast ( Rhodotorula mucilaginosa Growth curve of M07; Figure 5 For red yeast ( Rhodotorula mucilaginosa Acid tolerance curve of M07; Figure 6 For red yeast ( Rhodotorula mucilaginosa Glucose tolerance curve of M07; Figure 7 For red yeast ( Rhodotorula mucilaginosa Ethanol tolerance curve of M07; Figure 8 A flowchart of the kiwi wine fermentation process; Figure 9 To determine the quality of kiwi wine under different fermentation temperatures; Figure 10 To assess the quality of kiwi wine under different initial pH conditions; Figure 11 To assess the quality of kiwi wine under different amounts of secondary sugar supplementation; Figure 12 To evaluate the quality of kiwi wine under different inoculation amounts. Detailed Implementation

[0027] The culture media involved in the following examples are as follows: YPD liquid culture medium: Weigh 50.0 g of YPD liquid culture medium powder (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), heat and dissolve it in 1000 mL of distilled water, and autoclave at 121℃ for 15 minutes.

[0028] PDA medium: Weigh 40.1 g of PDA medium powder (purchased from Guangdong Huankai Microbial Technology Co., Ltd.), add 1 L of distilled water or deionized water, stir and heat to boiling until completely dissolved, dispense into test tubes or Erlenmeyer flasks, and autoclave at 121℃ for 15 minutes.

[0029] The sources of raw materials involved in the following examples Wild kiwifruit, purchased from the farmers' market in Suining County, Shaoyang City; kiwifruit (80% ripe, reducing sugar content 6.2~7.0g / 100g), honey (produced from Nanshan Ranch, Chengbu, Shaoyang, reducing sugar content 30.0~32.1g / 100g).

[0030] The experimental methods involved in the following embodiments (1) Methods for detecting alcohol content Add 100 mL of distilled water and heat to distill. Collect 100 mL of distillate and determine its alcohol content using a density meter. For specific alcohol detection methods, refer to the literature "Comparison of mud characteristics and fermentation performance of aged and conventional fermentation pits [J]. Food and Machinery, 2015, 31(01): 2-5."

[0031] (2) Sensory evaluation methods The sensory evaluation team consisted of 10 people. They were trained before the sensory evaluation and scored the color, aroma and taste of the fruit wine products. The scoring criteria are shown in Table 1.

[0032] Table 1 Sensory Evaluation Criteria

[0033] (3) Culture of strains Solid culture: Inoculate Rhodotorula glutinis M07 into PDA medium and incubate statically at 28°C for 3-5 days.

[0034] Liquid culture: Rhodotorula glutinis M07 was inoculated into YPD liquid medium and cultured with shaking at 28℃ and 100 rpm for 2 days.

[0035] (4) Flavor detection methods The flavor compounds in kiwifruit wine were determined using gas chromatography-mass spectrometry (GC-MS). The testing was conducted by Nanjing Yanke Testing Technology Co., Ltd.

[0036] Sample preparation: Transfer 5 mL of sample to a 15 mL extraction bottle, add a magnetic stir bar, and then seal quickly. Age the SPME extraction fiber tip at 250°C in the GC-MS inlet until no impurity peaks are observed. Place the sample bottle on a solid-phase microextraction apparatus, set the temperature to 40°C and the stirring speed to 1000 rpm; preheat the sample bottle in the extraction apparatus for 15 min; insert the SPME extraction tip into the headspace of the sample through the bottle cap, extend the fiber tip, ensuring the extraction tip is approximately 1.0 cm above the sample surface, and perform headspace extraction for 40 min; retract the fiber tip and remove it from the sample bottle; then insert the extraction tip into the GC-MS inlet, extend the fiber tip, and desorb at 250°C for 3 min before injection analysis.

[0037] Instrument conditions: chromatographic column: HP-INNOWAX (60.0 m × 250 μm, 0.25 μm); initial temperature 40℃, held for 2 min, ramped up to 120℃ at 5℃ / min, ramped up to 230℃ at 10℃ / min, held for 10 min; vaporization chamber temperature 250℃; transfer line temperature 240℃; carrier gas: He; carrier gas flow rate 1.0 mL / min; splitless. Mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 230℃; quadrupole 150℃; scan mode: Scan; scan mass range: 20–500 u.

[0038] Qualitative analysis: The detected components were qualitatively analyzed using the MS database NIST23 and retention time. Quantitative analysis: The area normalization method uses the percentage of the peak area of ​​the identified component to the sum of the areas of all identified components as the quantitative result. The formula is as follows:

[0039] Where: C i The content of a certain identified component, % A i The peak area corresponding to a specific identified component; n represents the total number of identified components.

[0040] Example 1: Strain Screening and Identification The specific steps are as follows: 1. Screening of strains Take 25 g of kiwifruit pulp, crush it, add 225 mL of sterile water, and prepare 10 -2 10 -3 10 -4 10 -5 10 -6 The sample was diluted. Each dilution was plated on three plates and incubated at 28°C for 2 days. Colony growth was observed, and strains exhibiting yeast morphology were selected and streaked three times to obtain pure cultures.

[0041] The results showed that yeast was screened from wild kiwifruit pulp and named M07.

[0042] 2. Identify the colony morphology and molecular biology of the preserved strains.

[0043] (1) Colony morphology characteristics: When grown on PDA medium, the colonies are round or oval, pink, smooth and moist, easy to pick up, and have a uniform texture. When observed under an optical microscope, the cells of the strain are spherical or oval.

[0044] (2) Molecular biological identification: 26S rDNA D1 / D2 sequencing of the strain was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with the GenBank nucleic acid sequence database using sequence homology comparison (BLAST) to construct a phylogenetic tree.

[0045] The 26S rDNA sequence (SEQ ID NO.1) is as follows: .

[0046] The results showed that strain M07 and Rhodotorula sp. Highest similarity, identified as Rhodotorula (Red yeast genus), specifically Rhodotorula mucilaginosa (Rhodotorula glutinis); and on October 24, 2024, it was deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 32331.

[0047] Example 2: Performance testing of Rhodotorula glutinis strain The specific steps are as follows: Preparation of seed culture: The yeast strain was inoculated into YPD liquid medium and cultured with shaking at 28℃ and 100 rpm for 2 days to obtain the seed culture.

[0048] 1. Growth curve determination The seed culture was inoculated into 200 mL of YPD medium at a rate of 2% (v / v) and cultured at 28℃. The OD600 value was measured every 2 h. The growth curve of Rhodotorula glutinis was plotted with time on the x-axis and OD value on the y-axis.

[0049] The results are as follows Figure 4 As shown, Rhodotorula glutinis M07 is in a lag phase from 0 to 4 hours, enters the logarithmic growth phase from 8 to 40 hours, and the number of yeasts increases rapidly. Subsequently, the growth rate decreases and it enters a stationary phase.

[0050] 2. Acid tolerance The seed culture was inoculated into 100 mL of YPD medium at an inoculation rate of 2% (v / v), and the initial pH was adjusted to 2.0, 3.0, 4.0, 5.0 and 6.0 respectively. The culture was carried out at 28℃, and samples were taken every 8 h to measure OD600.

[0051] The results are as follows Figure 5 As shown, the OD value of Rhodotorula glutinis M07 decreases with decreasing pH, and the strain basically does not grow at pH 2. Between pH 3 and 6, the OD value of the strain increases continuously over time, and strain M07 can still grow at pH 3.

[0052] Therefore, the highest tolerance pH for Rhodotorula glutinis M07 is pH 3.0.

[0053] 3. Glucose tolerance The seed culture was inoculated into 100 mL of YPD medium at an inoculation rate of 2% (v / v), and the initial sugar concentration was adjusted to 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, and 450 g / L, respectively. The culture was carried out at 28℃, and samples were taken every 8 h to measure OD600.

[0054] The results are as follows Figure 6 As shown, the OD value of strain M07 decreased with increasing sugar concentration. The OD value of the strain increased continuously over time, and strain M07 could still grow slowly at sugar concentrations of 400 g / L and 450 g / L.

[0055] Therefore, the highest tolerable sugar concentration for Rhodotorula glutinis M07 is 450 g / L.

[0056] 4. Ethanol tolerance The seed culture was inoculated into 100 mL of YPD medium at an inoculation rate of 2% (v / v), and the initial alcohol volume fraction was adjusted to 2%, 3%, 4%, 5%, and 6% (v / v), respectively. The culture was carried out at 28°C, and samples were taken every 8 h to measure OD600.

[0057] The results are as follows Figure 7 As shown, strain M07 can grow at alcohol concentrations of 2% and 3%, but growth is partially inhibited at 3%. Growth is relatively slow at 4% alcohol concentration, but accelerates after 40 hours. Strain M07 barely grows at alcohol concentrations of 5% and 6%.

[0058] Therefore, Rhodotorula glutinis M07 can tolerate up to 4% (v / v) of ethanol.

[0059] Example 3: Two-step sugar supplementation method for fermenting kiwi fruit wine The process of fermenting kiwi fruit wine using the two-step sugar-supplementing method is as follows: Figure 8 As shown, the steps are as follows: 1. Preparation of Rhodotorula glutinis M07 bacterial culture The yeast strain was inoculated into YPD liquid medium and cultured with shaking at 28℃ and 100 rpm for 2 days; the bacterial concentration after culture was 4.6 × 10⁻⁶. 7 CFU / mL.

[0060] 2. Preparation of Fruit Wine (1) Enzymatic hydrolysis of fruit puree: Take 80% ripe and soft kiwifruit, peel and pulp it, add 0.1% (w / w) pectinase (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number: M6346), and enzymatically hydrolyze at 35℃ for 4 h to obtain kiwifruit puree; (2) First step of sugar addition: Add 5% (w / w) white sugar to the kiwi fruit puree in step (1); stir thoroughly to dissolve, adjust the pH to 3.8, heat at 55~65℃ for 20 min to obtain kiwi fruit pulp; (3) Inoculation with strains: After the kiwifruit pulp has cooled to 15~25℃ (room temperature), inoculate at a rate of 0.15 g / L (0.04 g of bacterial concentration is 5×10⁻⁶). 7 (CFU / mL), inoculated with Rhodotorula glutinis M07, and fermented at 28℃ for 3 days to obtain kiwifruit fermentation mash; (4) Second step of sugar addition: Add 10% (w / w) sterilized honey cooled to 15~25℃ (room temperature) to the kiwi fermentation mash obtained in step (3), shake well to dissolve completely, let stand at 28℃ to continue fermentation for 2 days to obtain kiwi fruit wine.

[0061] Example 4: Two-step sugar supplementation method for fermenting kiwi fruit wine The two-step sugar-supplementation method for fermenting kiwi fruit wine is as follows: 1. Preparation of Rhodotorula glutinis M07 bacterial culture The yeast strain was inoculated into YPD liquid medium and cultured with shaking at 28℃ and 100 rpm for 2 days; the bacterial concentration after culture was 4.6 × 10⁻⁶. 7 CFU / mL.

[0062] 2. Preparation of Fruit Wine (1) Enzymatic hydrolysis of fruit puree: Take 80% ripe and soft kiwifruit, peel and pulp it, add 0.1% (w / w) pectinase (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number: M6346), and enzymatically hydrolyze at 35℃ for 4 h to obtain kiwifruit puree; (2) First step of sugar addition: Add 5% (w / w) white sugar to the kiwi fruit puree in step (1); stir thoroughly to dissolve, adjust the pH to 3.8, heat at 55~65℃ for 20 min to obtain kiwi fruit pulp; (3) Inoculation with strains: After the kiwifruit pulp has cooled to 15~25℃ (room temperature), inoculate at a rate of 0.15 g / L (0.04 g of bacterial concentration is 5×10⁻⁶). 7 (CFU / mL), inoculated with Rhodotorula glutinis M07, and fermented at 24℃ for 3 days to obtain kiwifruit fermentation mash; (4) Second step of sugar addition: Add 4% (w / w) sterilized honey cooled to 15~25℃ (room temperature) to the kiwi fermentation mash obtained in step (3), shake well to dissolve completely, let stand at 28℃ to continue fermentation for 2 days to obtain kiwi fruit wine.

[0063] Example 5: Optimization of fermentation conditions for kiwi fruit wine (1) Effect of fermentation temperature on the quality of kiwifruit wine Based on Example 4, the fermentation temperatures in step (3) were changed to 4℃, 20℃, 24℃, 28℃, 32℃ and 36℃ respectively to prepare kiwi fruit wine. Its alcohol content was tested and sensory evaluation was carried out.

[0064] The results are as follows Figure 9 As shown, as the temperature rises, the growth and metabolism of yeast become more and more vigorous. When the temperature is 28℃, the alcohol content of kiwi fruit wine is as high as 15.2% vol. When the temperature is as high as 36℃, yeast has difficulty surviving, resulting in a lower alcohol content of kiwi fruit wine. When the temperature is at a low temperature of 4℃, the fermentation cycle of yeast is prolonged, and the alcohol content is only 5.1% vol.

[0065] Sensory evaluation results showed that 24℃~28℃ is the suitable fermentation temperature for kiwifruit wine. Especially at 28℃, the wine has a pale yellow color, a slightly sweet and sour taste, and a rich aroma of alcohol and kiwifruit. Therefore, 28℃ was selected as the optimal fermentation temperature.

[0066] (2) Effect of initial pH on the quality of kiwifruit wine Based on Example 4, the fermentation pH in step (2) was changed to 3.2, 3.6, 3.8, 4.0, 4.4, and 4.8 to prepare kiwi fruit wine. Its alcohol content was tested and sensory evaluation was performed.

[0067] The results are as follows Figure 10 As shown, the alcohol content of kiwifruit wine initially increases and then decreases with rising pH. The highest alcohol content (14.2% vol) is achieved at an initial pH of 3.8, which also corresponds to the optimal sensory score. While the alcohol content can still reach 8.2% vol at a pH as low as 3.2, the wine becomes less clear, has a stronger acidic and astringent taste, and a less desirable flavor. Therefore, a pH of 3.6–4.0 is the optimal fermentation pH for kiwifruit wine, with the best sensory score observed at pH 3.8. Thus, 3.8 is determined as the optimal initial pH.

[0068] (3) The effect of the second sugar supplement on the quality of kiwifruit wine Based on Example 4, the amount of honey added in step (4) was changed to 4, 7, 10, 13, 16, and 19% (w / w) to prepare kiwi fruit wine. Its alcohol content was tested and sensory evaluation was performed.

[0069] The results are as follows Figure 11 As shown, the alcohol content of kiwifruit wine increases with the increase of sugar addition. When the second step of sugar addition is 10% (w / w), the alcohol content of kiwifruit wine is 15.4% vol; when the sugar addition is 13%, the alcohol content is 14.9% vol. Therefore, 7% (w / w) to 10% (w / w) is the suitable sugar addition amount for kiwifruit wine, especially 10% (w / w) as the most ideal second step sugar addition amount.

[0070] (4) Effect of yeast inoculation amount on the quality of kiwifruit wine Based on Example 4, the amount of yeast inoculated in step (3) was changed to 0.05, 0.10, 0.15, 0.20, and 0.25 g / L to prepare kiwi fruit wine. Its alcohol content was tested and sensory evaluation was performed.

[0071] The results are as follows Figure 12As shown, without yeast inoculation, the alcohol content of kiwifruit wine is only 1.6% vol. With increasing yeast inoculation, the alcohol content rises steadily and then levels off. At an inoculation amount of 0.15 g / L, the alcohol content reaches 16.0% vol. At this level, the wine is golden in color, clear and transparent, with a rich alcoholic aroma, a balanced sweet and sour taste, and optimal overall sensory qualities. Therefore, 0.1 g / L to 0.2 g / L is a suitable inoculation amount, with 0.15 g / L being the most ideal.

[0072] In summary, the optimal fermentation conditions for kiwifruit wine are a fermentation temperature of 28℃, an initial fermentation pH of 3.8, a second sugar supplement of 10% (w / w) honey, and a yeast inoculation amount of 0.15 g / L.

[0073] Example 6: Detection of flavor substances in kiwifruit wine Kiwifruit wine was prepared under the conditions described in Example 3, and its flavor compounds were analyzed. A total of 69 volatile flavor compounds were detected, including 27 esters, 12 alcohols, 7 acids, and 8 aldehydes and ketones. Ethyl acetate, ethyl decanoate, and ethyl octanoate were the main esters in the kiwifruit wine, while phenethyl alcohol, isoamyl alcohol, and isobutanol were the main alcohols. Table 2 shows the main flavor compounds and their relative contents in some of the kiwifruit wines.

[0074] Table 2: Flavor Compounds

[0075] Ethyl acetate imparts a fruity and wine-like aroma; ethyl hexanoate gives the wine a pineapple-banana similar scent; ethyl octanoate contributes apricot, floral, and fruity aromas; phenylethyl alcohol has a unique aroma with a mellow and lasting rose and other diverse flavors; caprylic acid offers a variety of flavors, including fruity, candy, and cheese notes; and ethyl decanoate mainly contributes a waxy and fruity flavor. These esters, alcohols, and acids all play a role in the overall flavor of the wine. In kiwi wine fermented with *Rhododendron simsii*, ethyl acetate has the highest relative content.

[0076] Example 7: Effects of different yeasts on kiwifruit wine brewing Based on Example 3, the inoculation of Angel Yeast (purchased from Angel Yeast Co., Ltd.) in step (3) was changed to prepare kiwifruit wine, and the flavor substances were tested. The main flavor substances and their relative contents in some kiwifruit wines are shown in Table 3; at the same time, Table 3 also gives the test results of the fruit wine of Example 3 for easy comparison.

[0077] Table 3: Flavor Compounds

[0078] Note: "-" indicates not detected.

[0079] A total of 56 volatile flavor compounds were detected, including 23 esters, 11 alcohols, 6 acids, and 4 aldehydes and ketones, which is fewer than that of *Rhodotorula glutinis*. Ethyl acetate, ethyl decanoate, and ethyl palmitate are the main esters in kiwifruit wine, while phenylethanol, isoamyl alcohol, and isobutanol are the main alcohols.

[0080] As shown in Table 2, ethyl hexanoate and ethyl decanoate were not detected in the kiwi wine fermented by Angel Fruit Wine Yeast, indicating that Red Yeast can increase the content of ethyl hexanoate and ethyl decanoate in kiwi wine. The relative contents of ethyl acetate and ethyl octanoate decreased compared with the test results of Example 6. The content of octanoic acid increased. The content of phenylethanol in the kiwi wine fermented by Red Yeast was higher than that in the kiwi wine fermented by Angel Fruit Wine Yeast, indicating that the kiwi wine of Example 3 has a richer rose and rose aroma.

[0081] Example 8: The effect of compound yeast on kiwi fruit wine brewing Based on Example 3, the inoculation of *Rhodotorula glutinis* and *Saccharomyces cerevisiae* in step (3) was changed (added at a ratio of 1:1) to prepare kiwifruit wine, and the flavor compounds were tested. Table 4 shows the main flavor compounds and their relative contents in some of the kiwifruit wines; Table 4 also provides the test results of the fruit wine from Example 3 for easy comparison.

[0082] Table 4: Flavor Compounds

[0083] A total of 63 volatile flavor compounds were detected, including 26 esters, 12 alcohols, 6 acids, and 4 aldehydes and ketones. The total number of compounds was between that of *Rhododendron simsii* and *Angelica wine yeast*. Ethyl acetate, ethyl decanoate, and ethyl octanoate were the main esters in kiwifruit wine, while phenylethanol, isoamyl alcohol, and butanediol were the main alcohols.

[0084] As shown in Table 3, the relative contents of ethyl acetate, ethyl hexanoate, ethyl octanoate, phenylethanol, octanoic acid, and ethyl decanoate in the kiwi wine fermented with compound yeast are between the test results of Example 6 and Example 7, indicating that the two yeasts can mutually regulate the contents of each substance in the wine.

[0085] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of red yeast ( Rhodotorula mucilaginosa M07, characterized in that, The aforementioned Rhodotorula glutinis was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32331.

2. A probiotic preparation, characterized in that, It contains the red yeast M07 as described in claim 1.

3. The probiotic preparation according to claim 2, characterized in that, The probiotic preparation contains live cells and / or cell metabolites of the Rhodotorula glutinis M07, and / or inactivated cells, and / or freeze-dried cells. Preferably, the number of Rhodotorula glutinis M07 in the probiotic preparation is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. The probiotic preparation according to claim 2 or 3, characterized in that, The probiotic preparation also contains Angel yeast; Preferably, the Rhodotorula glutinis M07 and Angel yeast are mixed in a ratio of (1~2): (1~2) to obtain the yeast. Preferably, the Rhodotorula glutinis M07 and Angel Yeast are mixed at a concentration ratio of 1:

1.

5. A product characterized in that, The product contains the Rhodotorula glutinis M07 as described in claim 1 or the probiotic preparation as described in any one of claims 2 to 4.

6. The product according to claim 5, characterized in that, The products mentioned are food, functional food, special medical purpose formula food, health products, feed, feed additives or food additives; Preferably, the food is a grain product, vegetable product, fruit product, meat product, seafood product, egg product, dairy product, soy product, or beverage. Preferably, the food is a special dietary food; Preferably, the health product also contains acceptable excipients.

7. The use of Rhodotorula glutinis M07 according to claim 1, or the probiotic preparation according to any one of claims 2 to 4, in the preparation of fermented products, characterized in that, The fermented products are fermented alcoholic beverages and fermented vinegar.

8. The application according to claim 7, characterized in that, The fermented alcoholic beverage is fruit wine, and the fermented vinegar is fruit vinegar.

9. The application according to claim 7, characterized in that, The fermented alcoholic beverages are: yellow rice wine, cooking wine, rice wine, sweet rice wine, wine, beer, and baijiu; the fermented vinegar is edible vinegar; Preferably, the fruit wine is, but is not limited to, pineapple wine, kiwi wine, dragon fruit wine, or lychee wine.

10. A method for brewing kiwi wine, characterized in that, Including the following steps: (1) Enzymatic hydrolysis of fruit puree: Peel and pulp kiwifruit, add pectinase, and hydrolyze to obtain kiwifruit puree; (2) First step of adding sugar: Add white sugar to the kiwi puree from step (1); Stir thoroughly to dissolve, adjust pH to 3.6-4.0, and heat at 55-65℃ for 15-25 minutes to obtain kiwi fruit pulp; (3) Inoculation of strains: After the kiwifruit pulp is cooled to 15~25℃, inoculate it with the Rhodotorula glutinis M07 as described in claim 1 or the probiotic preparation as described in any one of claims 2~4 at an inoculation amount of 0.1~0.2 g / L, let it stand at 26~30℃ for 2~4 days to ferment and obtain kiwifruit fermentation mash. (4) Second step of sugar supplementation: Add 8-12% (w / w) of honey to the kiwi fermentation mash obtained in step (3), shake well, let stand at 26-30℃ for 1-3 days to continue fermentation, and obtain kiwi fruit wine; Preferably, the amount of pectinase added is 0.08~0.12% (w / w). Preferably, the enzymatic hydrolysis method after adding pectinase is: enzymatic hydrolysis at 30~35℃ for 3~4 h; Preferably, the amount of white sugar added is 5~6% (w / w).

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