Kluyveromyces and its applications

Abstract

The present application relates to the field of microbial technology, and particularly relates to a Kluyveromyces and application thereof. The Kluyveromyces has a preservation number of CGMCC NO:34842. The Kluyveromyces is used for preparing a fermented wine beverage, and has the beneficial effects of high acid tolerance, high polyphenol tolerance, effective acid reduction and high aroma preservation when bayberry and / or juice thereof is used as a fermentation raw material.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Kluyveromyces yeast and its applications. Background Technology

[0002] Yangmei ( Myrica rubra It is a subtropical fruit with Chinese characteristics. The fruit is rich in active ingredients such as anthocyanins, vitamin C, organic acids and minerals, and has both edible and medicinal value.

[0003] Waxberries lack a protective peel and ripen during the hot and rainy season, making them prone to rotting and spoilage after harvesting, resulting in a high loss rate. To reduce losses and increase added value, deep processing has become crucial, and fermented waxberry wine, with its moderate alcohol content and rich fruit aroma, has become one of the mainstream deep processing directions.

[0004] However, existing bayberry wine production methods and technologies have significant shortcomings. Most current bayberry wines are made by soaking bayberries in high-purity alcohol, resulting in high alcohol content. When using fermentation methods to prepare bayberry wine, relying on surface bacteria on the bayberries leads to complex microbial communities, poor fermentation control, significant batch-to-batch flavor variations, and susceptibility to contamination and spoilage. Commercially available general-purpose yeasts are mostly used in wine and fruit wine production, lacking sufficient tolerance to the high acidity and polyphenol content of bayberries, resulting in slow fermentation, limited aroma production, and difficulty in highlighting the unique fruity aroma of bayberries. Furthermore, existing methods produce bayberry wines with high levels of organic acids (such as citric acid and malic acid). Traditional chemical deacidification methods easily damage the flavor and color, while physical deacidification (such as ion exchange and electrodialysis) is costly and depletes active ingredients. Biological deacidification relies on yeast or lactic acid bacteria, but suitable strains are scarce.

[0005] Therefore, it is necessary to breed a brewing yeast that is specific to bayberries, resistant to high acidity, high in polyphenols, and can effectively reduce acidity, and to construct a matching method for the fermentation and preparation of bayberry wine. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a Kluyveromyces yeast and its applications to address the lack of a specific, acid-tolerant, high-polyphenol-rich brewing yeast for bayberries that can effectively reduce acidity. The Kluyveromyces yeast provided by this invention has the advantages of high acid tolerance, high polyphenol content, and effective acid reduction when used in the preparation of fermented products (especially wine), making it particularly suitable for brewing bayberry wine using bayberries or their juice as fermentation raw materials. Furthermore, this invention also provides a fermentation method based on the aforementioned Kluyveromyces yeast, resulting in a bayberry wine with low fusel alcohol content and high fruit aroma.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions: The first aspect of this invention provides a Kluyveromycin ( Kluyveromyces sp.The Kluyveromyces species described herein has the accession number CGMCC NO: 34842.

[0008] The present invention also provides a microbial agent comprising Kluyveromyces as described in the first aspect. Kluyveromyces sp. ).

[0009] In some implementations, the Kluyveromyces yeast exists in the form of sludge, liquid, powder, dried cells, immobilized cells, extract, or any combination of the above forms.

[0010] The present invention also provides a method for preparing a fermented alcoholic beverage, the method comprising: incorporating the aforementioned Kluyveromycin ( Kluyveromyces sp. The microbial preparations described herein are brought into contact with the fermentation raw materials for fermentation.

[0011] In some implementations, the fermentation reaction system contains a carbon source.

[0012] In some implementations, the carbon source is glucose.

[0013] In some implementations, when the carbon source is glucose, the mass ratio of glucose added at the start of fermentation to the fermentation feedstock is 1:(30-80), preferably 1:(40-60).

[0014] In some implementations, the fermented alcoholic beverage is fruit wine, rice wine, wine, beer, or spirits.

[0015] Preferably, when the fermented alcoholic beverage is fruit wine, the fermentation raw material is bayberry ( Myrica rubra ) and / or its juice.

[0016] In some embodiments, the fermentation temperature is 4°C-15°C, preferably 4°C-8°C, for example 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C.

[0017] In some embodiments, the pH of the fermentation reaction system is 2-5, preferably 3-4.

[0018] In some implementations, seed culture is also included prior to fermentation.

[0019] In this invention, the seed culture includes inoculating the Kluyveromyces yeast or the inoculum agent into a seed culture medium for activation, thereby obtaining a seed liquid or solid seed containing the Kluyveromyces yeast for use in the fermentation.

[0020] In some embodiments, the temperature for seed culture is 22°C-30°C, preferably 25°C-30°C, for example 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0021] In some implementations, the seed culture time is 2-3 days.

[0022] In some embodiments, the seed culture medium is YPD medium.

[0023] The present invention also provides the application of the Kluyveromyces or the inoculum described herein in the preparation of fermented products.

[0024] In some implementations, the fermented product is a fermented food or a fermented beverage.

[0025] Preferably, the fermented beverage is a fermented alcoholic beverage or fermented vinegar.

[0026] In this invention, the fermented alcoholic beverage is fruit wine, rice wine, rice wine, grape wine, beer, or baijiu (Chinese liquor).

[0027] In this invention, the fermented vinegar is fruit vinegar or edible vinegar.

[0028] The present invention also provides a bayberry wine, wherein the acidity of the bayberry wine is less than 12 mg / mL.

[0029] In some embodiments, the bayberry wine is made with Kluyveromycin as described in this invention. Kluyveromyces sp. It is prepared as described in the bacterial agent or by the method described.

[0030] This invention also provides a method for reducing the acidity of fruit wine, the method comprising adding the Kluyveromycin described in this invention during the fermentation and preparation of the fruit wine. Kluyveromyces sp. (or the bacterial agent described in this invention).

[0031] In this invention, "methanol purity" or "ethanol purity" refers to the proportion of methanol or ethanol in alcohols as detected by chromatography.

[0032] In this invention, "ethanol content" and "ethanol concentration" have the same meaning, both referring to the percentage of ethanol in the total volume of the wine (or fermentation products).

[0033] The present invention relates to the isolation and preservation of biological materials, specifically: The Kluyveromyces 9521 isolated in this invention was deposited on June 10, 2025, 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: 34842, culture name 9521, and classification name Kluyveromyces 9521. Kluyveromyces sp. .

[0034] The beneficial effects of this invention are achieved through the following technical solutions: 1. The strain was selected from the natural fermentation of bayberries in Xianju and identified as Kluyveromyces oryzae, named 9521. It is currently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO:34842.

[0035] 2. The prepared bayberry wine has a bright color and clear liquid. In particular, its aroma is more intense than that of ordinary yeast and has the fragrance of passion fruit.

[0036] 3. During low-temperature fermentation, the strain has a shorter delay period, longer stability, and is more resistant to ethanol.

[0037] 4. The acidity of bayberry wine brewed using Kluwer Pichia pastoris is reduced, and the taste is enhanced.

[0038] 5. The alcohol content of fermented bayberry dry red wine is 12-13 degrees.

[0039] 6. Acidity of dried bayberry: 11.86 mg / ml, acidity of commercial yeast: 16.75 mg / ml (sodium hydroxide titration method).

[0040] The beneficial effects of this invention are as follows: the Kluyveromyces yeast provided by this invention exhibits multiple excellent characteristics in the preparation of fermented products: low methanol content, excellent aroma preservation performance, strong resistance to ethanol and acid, and good acid-reducing ability. Furthermore, this invention has also developed a fermentation process highly adapted to the strain, resulting in a bayberry wine with low methanol content, rich fruit aroma, and low acidity, possessing broad market prospects.

[0041] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0042] Figure 1 The image shows the HPLC chromatogram of bayberry wine prepared by fermentation with commercial yeast; the retention time of the methanol peak is 1.559 min and the retention time of the ethanol peak is 1.743 min.

[0043] Figure 2 The image shows the HPLC chromatogram of bayberry wine prepared by fermentation with Kluyveromyces 9521; the retention time of the methanol peak is 1.572 min and the retention time of the ethanol peak is 1.767 min. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0045] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0046] Example 1: Strain screening and fermentation (1) Select fresh, ripe, and unrotten bayberry fruits, crush them, and extract the juice and incubate them at 28℃ for 2-3 days.

[0047] (2) After culturing, the juice is divided into 10... -4 10 -5 10 -6 10 -7 10 -8 The culture was diluted and streaked onto sterilized YPD medium plates, then incubated at 28°C. After screening, a strain with excellent acid-lowering and fermentation capabilities was obtained and named 9521. Based on the strain identification results, 9521 is *Kluyveromyces kluyveromyces*.

[0048] (3) Pick a single colony and streak it onto a sterilized 500 mL eggplant-shaped YPD medium for secondary activation culture to obtain the bacterial strain (solid medium about 2 / 3; inoculum suspension concentration: ≥5.0×10). 8 CFU / mL, inoculation volume 20 mL per eggplant bottle; strain grows only on the surface, with mycelia / colony covering 2 / 3 of the surface area as mature seeds).

[0049] (4) Crush 100 kg of bayberry fruit and put it into a fermentation tank. Use bayberry juice to wash off a bottle of bacteria from an eggplant bottle and inoculate it into the bayberry wine fermentation tank.

[0050] (5) Add 2 kg of glucose to the fermenter, adjust the solid-liquid ratio in the fermenter, ferment at 4-15℃, and check the sugar content and pH change every 24 hours. Control the pH to maintain 3.2-3.4 until the glucose content drops below 4%, stop fermentation, and check the ethanol content.

[0051] Table 1 Residual sugar content After fermentation, the bayberry pits and residue were filtered out, and the ethanol concentration of the bayberry wine was determined using the general analytical method for wine and fruit wine (GB / T-15038-2006). The results showed that the ethanol content of the bayberry dry red wine obtained from fermentation tanks 3 and 2 was 12%-13% (alcohol by volume, ABV).

[0052] The acidity of the prepared bayberry wine was determined by sodium hydroxide titration, and the purity (percentage of alcohols) of methanol and ethanol in the wine was determined by chromatography. The test results are as follows: Figure 1 , Figure 2 And as shown in Table 2.

[0053] Table 2 When fermenting with 9521, the acidity was significantly lower than that under the same conditions using commercial yeast (Zymaflore). ® VL1 fermentation also has a lower methanol content than commercial yeast.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A type of Kluyveromyces ( Kluyveromyces sp. ), characterized in that, The Kluyveromyces species described herein has the accession number CGMCC NO: 34842.

2. A microbial agent, characterized in that, The inoculum contains Kluyveromyces as described in claim 1. Kluyveromyces sp. ); Preferably, the Kluyveromyces yeast exists in the form of sludge, liquid, powder, dried cells, immobilized cells, extract, or any combination of the above forms.

3. A method for preparing a fermented alcoholic beverage, characterized in that, The method includes administering the Kluyveromycin (as described in claim 1) Kluyveromyces sp. Fermentation can be carried out by contacting the inoculum as described in claim 2 with the fermentation raw materials.

4. The method as described in claim 3, characterized in that, The fermentation reaction system contains a carbon source; the preferred carbon source is glucose. Preferably, when the carbon source is glucose, the mass ratio of glucose added at the start of fermentation to the fermentation feedstock is 1:(30-80), more preferably 1:(40-60).

5. The method as described in claim 3 or 4, characterized in that, The fermented alcoholic beverage is fruit wine, rice wine, wine, beer, or baijiu (Chinese liquor). Preferably, when the fermented alcoholic beverage is fruit wine, the fermentation raw material is bayberry ( Myrica rubra ) and / or its juice.

6. The method according to any one of claims 3-5, characterized in that, The fermentation conditions are: a temperature of 4℃-15℃, preferably 4℃-8℃; and / or, a pH of 2-5, preferably 3-4, in the fermentation reaction system.

7. The method according to any one of claims 3-6, characterized in that, The fermentation process includes seed culture, which involves inoculating the Kluyveromyces as described in claim 1 or the inoculum as described in claim 2 into a seed culture medium for activation, to obtain a seed liquid or solid seed containing the Kluyveromyces for use in the fermentation. Preferably, the seed culture meets one or more of the following conditions: (1) The temperature is 22℃-30℃, preferably 25℃-30℃; (2) The incubation period is 2-3 days; (3) The seed culture medium is YPD medium.

8. The Kluyveromyces yeast as described in claim 1 ( Kluyveromyces sp. (or the application of the microbial agent as described in claim 2 in the preparation of fermented products;) Preferably, the fermented product is a fermented food or a fermented beverage; the fermented beverage is preferably a fermented alcoholic beverage or fermented vinegar. Furthermore, the fermented alcoholic beverage is fruit wine, rice wine, wine, beer, or spirits; and / or, the fermented vinegar is fruit vinegar or edible vinegar.

9. A type of bayberry wine, characterized in that, The acidity of the bayberry wine is less than 12 mg / mL; Preferably, the bayberry wine is made with Kluyveromycin as described in claim 1. Kluyveromyces sp. It can be prepared by the microbial agent as described in claim 2 or by the method described in any one of claims 3-7.

10. A method for reducing the acidity of fruit wine, characterized in that, The method includes adding Kluyveromycin as described in claim 1 during the fermentation and preparation of the fruit wine. Kluyveromyces sp. (or the bacterial agent as described in claim 2).

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