Pichia pastoris and its application

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a Pichia pastoris and application thereof. The preservation number of the Pichia pastoris is CGMCC NO:34841. The Pichia pastoris is used for preparing a fermented wine beverage, and has the beneficial effects of low methanol production, high aroma preservation, acid resistance and anthocyanin resistance when using waxberry and / or juice thereof as a fermentation raw material.

Description

Technical Field

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

[0002] Yangmei ( Red myrtle Myrica is a small tree or shrub belonging to the Myricaceae family and the Myrica genus. It is a berry native to my country. It is rich in various free amino acids, anthocyanins, organic acids, and a variety of volatile aromatic substances that are beneficial to the human body, making it an ideal raw material for brewing low-alcohol fruit wines.

[0003] However, existing bayberry wine production methods and technologies have significant shortcomings. Current bayberry wine brewing processes generally use glucose as the primary carbon source for fermentation, resulting in wines that fail to retain the original fruit aroma. Furthermore, most commercially available bayberry wines are infused bayberry wines made by soaking bayberries in high-purity spirits, resulting in high alcohol content and a predominantly spirits flavor, lacking the sweet and sour taste of bayberries and their unique aroma. On the other hand, commercially available wine yeasts are designed for fermentation raw materials such as grapes and apples, exhibiting poor tolerance to the high acidity (pH 2.8-3.2) and high anthocyanin content of bayberries, leading to slow or halted fermentation. This results in poor compatibility, producing bayberry wines with high methanol content, sharp acidity, and a thin flavor profile, making stable and controllable large-scale production difficult. Therefore, developing a specific bayberry-specific brewing yeast with low methanol production, high aroma retention, acid resistance, and anthocyanin tolerance, and constructing a corresponding bayberry wine fermentation method, has become a critical bottleneck that the industry urgently needs to overcome. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a Pichia pastoris and its applications to address the shortcomings of existing brewing yeasts that are specifically designed for bayberries, have low methanol production, high aroma retention, acid resistance, and anthocyanin tolerance. The Pichia pastoris provided by this invention, when used in the preparation of fermented products (especially wine), has the advantages of low methanol production, high aroma retention, acid resistance, and anthocyanin tolerance, and is particularly suitable for brewing bayberry wine using bayberries or their juice as fermentation raw materials. Furthermore, this invention also provides a fermentation method adapted to the aforementioned Pichia pastoris, resulting in a bayberry wine with low methanol content and a high bayberry aroma.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions: The first aspect of this invention provides a Pichia pastoris ( Shepherd's pie The preservation number of the Pichia pastoris is CGMCC NO: 34841.

[0006] The present invention also provides a microbial preparation comprising Pichia pastoris as described in the first aspect. Shepherd's pie ).

[0007] In some implementations, the Pichia pastoris exists in the form of mycelial sludge, mycelial liquid, immobilized cells, mycelial powder, dried mycelium, extract, or any combination of the above forms.

[0008] The present invention also provides a method for preparing a fermented alcoholic beverage, the method comprising: [the method is described in the original text, but the provided text is incomplete and cannot be accurately translated without further context.] Shepherd's pie The microbial preparations described herein are brought into contact with the fermentation raw materials for fermentation.

[0009] In some embodiments, the content of Pichia pastoris in the fermentation reaction system is one bottle of mature Pichia pastoris seed added per 50 L of fermentation system; one bottle of mature seed is obtained by the following preparation method: adding solid culture medium to a 500 mL culture flask, filling the flask to 2 / 3 of its volume; inoculating the solid culture medium with a suspension of Pichia pastoris, wherein the concentration of the bacterial suspension is ≥5.0 × 10⁻⁶. 8 The inoculum volume is 20 mL, and the Pichia pastoris is cultured until it grows to cover 2 / 3 of the surface area of ​​the solid culture medium with hyphae or colonies.

[0010] In some implementations, the fermentation reaction system contains a carbon source.

[0011] In some implementations, the carbon source is honey, glucose, and / or white sugar.

[0012] In some implementations, when the carbon source is honey, the ratio of the volume of honey added at the start of fermentation to the mass of the fermentation feedstock is 2-10 mL / kg, preferably 3-8 mL / kg, for example 5 mL / kg.

[0013] Preferably, the fermentation process includes a step of replenishing the honey.

[0014] Preferably, the amount of honey added to the feed is 1-3 times the volume of honey added at the start of fermentation, for example, 2 times.

[0015] Preferably, the feeding is carried out when the sugar concentration is below 5%.

[0016] Preferably, the replenishment is done in a single step.

[0017] 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), more preferably 1:(40-60), and even more preferably 1:50.

[0018] In some implementations, the fermented alcoholic beverage is fruit wine, rice wine, wine, beer, or spirits.

[0019] Preferably, when the fermented alcoholic beverage is fruit wine, the fermentation raw material is bayberry ( Red myrtle ) and / or its juice.

[0020] In some embodiments, the fermentation temperature is 10°C-25°C, preferably 10°C-18°C, more preferably 12°C-15°C, for example 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C.

[0021] In some implementations, the fermentation time is 36 h-384 h, preferably 288-360 h.

[0022] In some embodiments, the pH of the fermentation reaction system is 2-5, preferably 3-4.

[0023] In some implementations, seed culture is also included prior to fermentation.

[0024] In this invention, the seed culture includes inoculating the Pichia pastoris or the microbial preparation into a seed culture medium for activation, thereby obtaining a seed liquid or solid seed containing the Pichia pastoris for use in the fermentation.

[0025] In this invention, the fermentation process further includes a filtration and / or clarification step.

[0026] 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.

[0027] In some implementations, the seed culture time is 2-3 days.

[0028] In some embodiments, the seed culture medium is YPD medium.

[0029] The present invention also provides the aforementioned Pichia pastoris ( Shepherd's pie The application of the microbial preparations mentioned above in the preparation of fermented products.

[0030] In some implementations, the fermented product is a fermented food or a fermented beverage.

[0031] Preferably, the fermented beverage is a fermented alcoholic beverage or fermented vinegar.

[0032] In this invention, the fermented alcoholic beverage is fruit wine, rice wine, rice wine, grape wine, beer, or baijiu (Chinese liquor).

[0033] In this invention, the fermented vinegar is fruit vinegar or edible vinegar.

[0034] In this invention, "methanol purity" or "ethanol purity" refers to the proportion of methanol or ethanol in alcohols as detected by chromatography.

[0035] 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).

[0036] The present invention relates to the isolation and preservation of biological materials, specifically: The *Pichia pastoris* 9520 isolated in this invention was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO:34841, culture name 9520, and classification name *Pichia pastoris*. Pichia sp. .

[0037] The beneficial effects of this invention are achieved through the following technical solutions: 1. Fermentation was carried out using a strain of bacteria isolated from the natural fermentation of Xianju bayberries. The strain was identified as Pichia pastoris and named Pichia pastoris 9520, CGMCC NO.34841.

[0038] 2. Pichia pastoris can reduce the methanol content in wine.

[0039] 3. Honey is added in stages: It is added twice during the brewing process. The first addition is one-third of the total amount. When the sugar concentration is below 5%, the remaining honey is added a second time. The purpose is twofold: 1. To utilize the existing sugars in the bayberries first, while maintaining the alcohol concentration in the fermentation tank and extending the fermentation cycle of the bacteria. 2. To increase the ethanol content and aroma of the wine after the second addition of honey.

[0040] 4. Using honey as a carbon source: Honey is a complex mixture, mainly composed of glucose and fructose, as well as some minerals, enzymes, vitamins, organic acids, etc. Honey itself has a certain aroma, which can be retained in the wine during the brewing process of bayberry wine.

[0041] 5. The viscosity of honey itself can increase the density of wine and make it feel fuller.

[0042] 6. The honey used is local Xianju honey.

[0043] The beneficial effects of this invention are as follows: the Pichia pastoris provided by this invention exhibits multiple excellent characteristics in the preparation of fermented products: low methanol yield, excellent aroma retention, strong acid resistance, and good tolerance to anthocyanins. Furthermore, this invention has developed a fermentation process highly compatible with the strain, resulting in a bayberry wine with low methanol content and a rich fruity aroma, demonstrating feasibility for industrial production and possessing significant application value and broad market prospects.

[0044] 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

[0045] Figure 1 The HPLC chromatogram of bayberry wine prepared by fermentation with commercial yeast is shown. Figure 2 The HPLC chromatogram of bayberry wine prepared by fermentation with Pichia pastoris 9520. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] Example 1: Method for Isolating Bacterial Strains (1) Take 10 test tubes, add 9 mL of water to each test tube, wrap them up, and put them in an autoclave at 121℃ for 30 min.

[0049] (2) After wrapping the petri dish, inoculation loop and dropper, put them in the sterilizer and sterilize at 121°C for 30 min.

[0050] (3) After preparing the YPD medium, add agar powder at a ratio of 3%, wrap it up, put it in an autoclave, and sterilize it at 121°C for 30 minutes.

[0051] (4) Turn on the ultraviolet lamp of the clean bench and sterilize for 60 minutes. After sterilization, turn on the ventilation.

[0052] (5) Cool the sterilized YPD medium to about 60°C, pour the medium into a petri dish, and let it stand overnight to solidify.

[0053] (6) After crushing the bayberry fruit, take 1 mL with a dropper and add it to the first test tube. Then take 1 mL from the first test tube and add it to the second test tube, and so on, diluting to 10 mL. -10 .

[0054] (7) Take 10 -6 -10 -10 Dilute the solution, use an inoculation loop to add the solution to a petri dish, and then transfer it to the petri dish using the streak method.

[0055] (8) Place the streaked petri dish upside down in an incubator at 28°C and incubate for 2-3 days.

[0056] (9) The cultured petri dishes are used as the first generation. Single colonies are selected and expanded into test tubes as the second generation. Then, the same operation method is used to expand them into eggplant bottles for brewing fermentation.

[0057] After screening, a strain with excellent methanol degradation and fermentation capabilities was obtained and named 9520. According to the strain identification results, 9520 is Pichia pastoris.

[0058] The Pichia pastoris 9520 isolated in this embodiment 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:34841, culture name 9520, and classification name Pichia pastoris. Pichia sp. .

[0059] Example 2 (1) Select 100 kg of fresh, ripe, and unrotten bayberry fruit, crush them, and put them into a fermentation tank. Use the juice to extract the selected inoculum. Take one bottle (500 mL eggplant-shaped bottle, about 2 / 3 of solid culture medium; inoculation suspension concentration: ≥5.0×10) 8 CFU / mL, inoculation volume 20 mL per eggplant bottle; strain grows only on the surface, mycelium / colony covering 2 / 3 of the surface area is mature seed) After the culture is mature, the whole bottle is added to a 50 L fermenter.

[0060] (2) Control the fermentation temperature between 12-15℃, and detect the sugar change every 24 hours. The initial sugar content is between 9.5%-10.3% (natural sugar content of bayberry).

[0061] (3) Weigh out 1.5 L of honey (density calculated as 1.36 g / mL) and set aside. Add 500 mL of honey to fermentation tank 1 and 2 kg of glucose to fermentation tank 2 to start fermentation.

[0062] (4) After fermentation for 168 hours, add the remaining 1 L of honey to fermentation tank 1 and continue fermentation, then test the residual sugar content. During natural fermentation, the pH is 3.2-3.4, and the pH is controlled to be maintained at 3.2-3.4.

[0063] (5) When the glucose content is below 5%, stop fermentation and test the ethanol content.

[0064] The residual sugar data from fermenting bayberries using the isolated Pichia pastoris 9520 are shown in Table 1. After 15 days of fermentation, the residual sugar content in fermentation tank 1 was 3.8%, and in fermentation tank 2 it was 4%. After fermentation, the mixture was filtered, and the aroma of fermentation tank 1 was more mellow than that of fermentation tank 2.

[0065] 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 fermentation tank 1 had an ethanol content of 12.5% ​​(alcohol by volume, ABV), while fermentation tank 2 had an ABV of 9.8%. In summary, fermenting bayberries using the isolated Pichia pastoris 9520 and adding honey in stages resulted in a bayberry wine with a higher alcohol concentration than that obtained using glucose as the main carbon source, and the wine also possessed a subtle honey aroma.

[0066] Example 3 Using commercial yeast (ZYMAFLORE) ® VL1) 1000 g, Pichia pastoris 9520, were used to ferment bayberry wine using the method described in Example 2, which uses honey as the main carbon source.

[0067] After fermentation, 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 prepared bayberry wine was determined by chromatography.

[0068] Test results as follows Figure 1 and Figure 2 And as shown in Table 2.

[0069] Table 2 9520 is Pichia pastoris. When fermentation is carried out using 9520, the methanol content in the resulting bayberry wine is significantly lower than that when commercial yeast is used.

[0070] 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 Pichia pastoris ( Pichia pastoris ), characterized in that, The preservation number of the Pichia pastoris is CGMCCNO: 34841.

2. A microbial preparation, characterized in that, The microbial preparation comprises Pichia pastoris as described in claim 1. Pichia pastoris ); Preferably, the Pichia pastoris exists in the form of mycelial sludge, mycelial liquid, immobilized cells, mycelial powder, dried mycelium, 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 Pichia pastoris as described in claim 1 (… Pichia pastoris Fermentation is carried out by contacting the microbial preparation as described in claim 2 with the fermentation raw material.

4. The method as described in claim 3, characterized in that, The fermentation reaction system contains a carbon source; the carbon source is preferably honey, glucose, and / or white sugar. Preferably, the method satisfies the conditions selected from (1) and (2): (1) When the carbon source is honey, the ratio of the volume of honey added at the start of fermentation to the mass of the fermentation raw material is 2-10 mL / kg, preferably 3-8 mL / kg, for example 5 mL / kg; The fermentation process preferably includes a step of adding honey as feed; more preferably, the amount of honey added as feed is 1-3 times the volume of honey added at the beginning of fermentation, for example, 2 times, and / or, the feeding is carried out when the sugar concentration is below 5%, and / or, the feeding is added all at once; (2) When the carbon source is glucose, the mass ratio of glucose added at the start of fermentation to the fermentation raw material is 1: (30-80), preferably 1: (40-60), and even more preferably 1:

50.

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 10℃-25℃, preferably 10℃-18℃, and more preferably 12℃-15℃; and / or, The fermentation time is 36 h-384 h, with an optimal time of 288-360 h.

7. The method according to any one of claims 3-6, characterized in that, The pH of the fermentation reaction system is 2-5, preferably 3-4.

8. The method according to any one of claims 3-7, characterized in that, The fermentation process includes seed culture prior to the fermentation; the seed culture includes inoculating a seed culture medium with Pichia pastoris as described in claim 1 or a microbial preparation as described in claim 2 for activation, to obtain a seed liquid or solid culture containing the Pichia pastoris for use in the fermentation; and / or, The fermentation process also includes a filtration and / or clarification step; Preferably, the seed culture conditions are: a temperature of 22℃-30℃, more preferably 25℃-30℃; and / or, a culture time of 2-3 days; and / or, The seed culture medium is YPD medium.

9. The Pichia pastoris as described in claim 1 ( Pichia pastoris (or the application of the microbial preparation as described in claim 2 in the preparation of fermented products.) 10. The application as described in claim 9, characterized in that, The fermented product is a fermented food or a fermented beverage. Preferably, the fermented beverage is 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.

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