A method for isolating Pichia kudrica zwichi.

Abstract

This invention relates to a method for isolating Pichia pastoris. The method includes sample preparation: grinding a medium- and high-temperature fermented koji sample and then serially diluting it to obtain a bacterial suspension; selective plating: plating the bacterial suspension onto a selective culture medium; and high-temperature isothermal incubation: culturing the plated selective culture medium at 38℃-42℃ until single colonies grow; wherein the selective culture medium is YPD basal medium supplemented with chloramphenicol and acetic acid. This method effectively inactivates background bacteria, selectively enriches the target strain, and significantly increases the ethyl acetate yield of the obtained strain.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a method for isolating Kudria zwibich yeast. Background Technology

[0002] Baijiu, a traditional Chinese distilled spirit, relies on the Daqu (a type of starter culture) as its core saccharification and fermentation agent. The complex microbial community within Daqu forms the key foundation for the flavor compounds in baijiu. Yeasts are crucial functional microorganisms in Daqu, participating in alcoholic fermentation and aroma synthesis. Pichiakudriavzevii, a unique and advantageous yeast strain in medium- and high-temperature Daqu, possesses physiological characteristics such as tolerance to high sugars, acids, and temperatures. During the fermentation process of medium- and high-temperature Daqu, it not only efficiently completes alcoholic fermentation but also metabolizes and produces various flavor precursors such as esters and alcohols, playing a vital role in enhancing the body and unique flavor of baijiu. However, in the complex fermentation environment of Daqu, this strain often coexists closely with a wide variety and large number of molds, bacteria, and other wild yeasts, posing a significant challenge to its efficient and specific isolation and purification. Therefore, efficient isolation and purification of this strain is a prerequisite for conducting functional research, targeted cultivation of superior strains, and optimization of the Daqu fermentation process.

[0003] The prevalence, functional importance, and reproducibility of *Pichia kudriavzevii* in the Daqu (fermentation starter) system for Baijiu (especially at medium and high temperatures) have become a scientific consensus in this field. Numerous systematic studies have repeatedly confirmed that *Pichia kudriavzevii* is a core member of the fungal community constituting Daqu for Baijiu, and is not an accidental or randomly occurring species. Specifically, in light-aroma Daqu, this fungus is clearly listed as a "major yeast group"; during the fermentation process of high-temperature Daqu, especially in the early stages, *Pichia kudriavzevii* is the dominant microorganism in different parts; it is also one of the "key fungi" involved in the formation of the crucial "starter skin" structure of Daqu. Furthermore, as a core representative of the *Pichia* genus, it is classified as an "interacting microorganism" driving the ester production function of Daqu, directly contributing to the generation and accumulation of flavor substances such as esters. These findings are not limited to theoretical research, but have also been verified in practical applications: researchers have successfully screened out several strains of highly tolerant Kudriazvichi yeast from the mash of Maotai-flavor liquor, and confirmed that they can still maintain excellent fermentation characteristics under high acid and high temperature stress, effectively improving raw material utilization and ethanol production, while regulating the content of higher alcohols.

[0004] Existing microbial isolation techniques have significant limitations in practical applications. Traditional YPD or PDA media lack targeted selection pressure. During cultivation, rapidly growing mold hyphae easily spread and cover the entire plate, obscuring or even inhibiting the target yeast colonies, making it difficult to obtain independent single colonies. To improve this situation, some existing techniques attempt to add antibiotics to the culture medium to inhibit bacterial growth or use organic acids to adjust the pH to inhibit mold. However, using antibiotics alone cannot solve the problem of mold contamination, and simply adding organic acids often indiscriminately inhibits the growth activity of the target yeast while inhibiting other microorganisms, resulting in low isolation efficiency. More importantly, existing methods often rely on conventional temperatures of 28°C to 30°C for cultivation, failing to fully utilize the unique thermotolerant physiological properties of *Pichia pastoris*. These mild cultivation conditions allow a large number of non-target thermotolerant microorganisms to survive and multiply, not only reducing the specificity of screening but also resulting in strains that often lack sufficient high-temperature adaptability, making them difficult to directly apply to the high-temperature fermentation process of Daqu (a type of Chinese koji).

[0005] Therefore, developing an isolation method that is simple to operate, highly selective, and capable of targeted enrichment of strains with superior traits has become a technical bottleneck that urgently needs to be overcome in the current research field of microorganisms in Baijiu brewing. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of existing technologies in the separation of Kudria zwibichi yeast from medium- and high-temperature Daqu (a type of starter culture), such as poor inhibition of contaminating bacteria, low separation efficiency of target strains, high purification difficulty, and insufficient identification accuracy. This invention provides a method for efficiently separating Kudria zwibichi yeast from medium- and high-temperature Daqu.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution: A method for isolating Pichia kudrica zwichi yeast, comprising the following steps: (1) Sample preparation: The medium- and high-temperature Daqu sample was ground and then serially diluted to obtain a bacterial suspension; (2) Selective coating: The bacterial suspension is coated onto a selective culture medium; (3) High temperature constant temperature culture: The selective culture medium after coating is placed in a 38℃-42℃ environment for culture until a single colony grows; The selective culture medium is YPD basal medium supplemented with chloramphenicol and acetic acid; The final concentration of the chloramphenicol is 150-250 mg / L, used to inhibit bacterial growth; The final concentration of the acetic acid is 0.2%-0.5% (v / v), which is used to create an acidic environment to inhibit the growth of mold and non-acid-resistant bacteria. The aforementioned medium-high temperature Daqu is a Daqu in which the content of Kudria zweibichi yeast in the Daqu fungus accounts for more than 10% in the early stage of fermentation.

[0008] The culture temperature of 38℃-42℃ and the acetic acid concentration of 0.2%-0.5% create a synergistic selection pressure that specifically promotes the growth of Pichia kudrica and inhibits other yeasts and contaminating microorganisms.

[0009] Further, in step (1), a differential heat shock pretreatment is performed on the sample before processing: the Daqu bacteria suspension is placed in a water bath at 48℃-50℃ for 12-18 minutes, and then quickly transferred to an ice bath at 4℃ for 3-5 minutes for shock.

[0010] Furthermore, the method for preparing the selective culture medium includes: After sterilizing the components of YPD basal culture medium, cool it to 50-60℃, then add sterile filtered chloramphenicol solution and acetic acid, mix well and pour into plates; The final concentration of chloramphenicol is 200 mg / L, and the final concentration of acetic acid is 0.3%.

[0011] Furthermore, the basic components of the YPD culture medium are: 10 g / L yeast extract, 20 g / L glucose, 20 g / L peptone, and 15-20 g / L agar.

[0012] Furthermore, the medium-high temperature Daqu sample is Daqu from the middle to late stage of fermentation, preferably Daqu from the 2nd to the 10th day of fermentation.

[0013] Furthermore, in step (3), the culture time is 48-72 hours; before coating culture, the selective culture medium plate is left to stand and dry at room temperature for 0.5-24 hours after preparation, until there are no free water droplets on the surface.

[0014] Furthermore, the method also includes step (4): picking out the morphologically typical single colonies that grew in step (3), inoculating them into ordinary YPD medium for purification culture, and obtaining pure strains of Pichia kudrica.

[0015] Furthermore, the method also includes functional screening of the obtained pure strains, selecting strains with at least one of the following characteristics: high ester production capacity, high ethanol tolerance, or high acid tolerance for use in baijiu brewing.

[0016] Compared with the prior art, the present invention has significant progress and beneficial effects: This invention creatively constructs a three-dimensional synergistic selection pressure system of "high temperature-acidity-antibiotics," overcoming the dilemma of traditional isolation methods that struggle to simultaneously inhibit bacteria and promote growth. Through a clever combination of specific high-temperature culture conditions and low-concentration acetic acid, the germination pathway of fungal spores is effectively blocked and bacterial growth is completely inhibited, achieving specific enrichment and efficient isolation of the target bacteria.

[0017] Another key highlight of this method is the introduction of a differentiated heat shock pretreatment step. Utilizing the inherent differences in cell membrane stability and stress resistance mechanisms between the target strain and associated bacteria, a precisely controlled heat shock and cold shock cycle selectively induces background bacteria into a dormant or inactive state, while preserving the high activity of the target bacteria. This physical treatment significantly reduces background interference on the plates, greatly improves the colony purity of the initial screening plates, and reduces the workload of subsequent purification and identification.

[0018] Furthermore, this method is highly valuable for optimizing culture medium preparation and culture conditions. By strictly controlling the plate drying time, suitable humidity on the surface of the culture medium is ensured, and the effective concentration of acetic acid in the microenvironment of the culture medium is effectively maintained, avoiding selective pressure failure caused by volatilization. The entire process does not require expensive reagents or complex equipment, is simple to operate, and has good repeatability, making it particularly suitable for widespread application in Daqu (a type of Chinese liquor) production lines.

[0019] Most importantly, the *Pichia pastoris* strains obtained through this method naturally inherit excellent heat and acid resistance. When subsequently applied to inoculate for high-temperature fermentation of baijiu (Chinese liquor), these strains exhibit strong environmental adaptability and colonization advantages, significantly increasing the content of ester flavor compounds in the mash and improving the quality of the liquor, demonstrating extremely high industrial application value and economic benefits. Attached Figure Description

[0020] Figure 1 Colony growth on acidic YPD medium at 40°C; Figure 2 Colony growth in acidic YPD medium at 30°C; Figure 3 Colony growth on conventional YPD medium at 40°C. Detailed Implementation

[0021] The present invention will be further described below through specific embodiments, but the scope of protection of this application is not limited to the embodiments.

[0022] The experimental materials and instruments used in this embodiment are all commercially available products. Among them, yeast extract, peptone, and agar are special reagents for microbial culture; glucose is analytical grade; chloramphenicol and acetic acid are biochemical reagents; medium- and high-temperature koji is a sample of koji used for fermentation in baijiu production; high-speed tissue homogenizer, pressure steam sterilizer, 40℃ constant temperature incubator, sterile operating table, PCR instrument, and agarose gel electrophoresis instrument are routine instruments for microbial experiments.

[0023] In the koji-making workshop of baijiu enterprises, it is necessary to rapidly and with high purity separate Pichia pastoris, a yeast with excellent ester-producing properties, from the medium- and high-temperature koji during the mid-fermentation stage, for use in the preparation of subsequent inoculants for enhanced fermentation. Traditional separation methods cannot meet the needs of industrial-scale production due to severe mold contamination, significant interference from other microorganisms, and long screening cycles. This embodiment aims to establish a standardized engineering separation process using the technology of this patent application. Example 1:

[0024] The Daqu used in this embodiment is a high-temperature Daqu fermented on the 5th day, and its Kudriazwibich yeast content accounts for 10% of the Daqu fungal content.

[0025] I. Sample Collection and Pretreatment Sampling: On the 5th day of fermentation, 500g of Daqu sample was collected from the area in the center of the fermentation room where the temperature reached 45℃, sealed and refrigerated and transported back to the laboratory.

[0026] Suspension preparation: Weigh 50g of Daqu sample, crush it through a 40-mesh sieve, add 450mL of sterile physiological saline, and shake on a shaker at 200rpm for 30 minutes to prepare the initial suspension.

[0027] Differentiated heat shock treatment: Take 10 mL of the initial suspension and put it into a sterile test tube.

[0028] Heat shock: Place in a 49℃ constant temperature water bath and process precisely for 15 minutes.

[0029] Cold shock: Immediately transfer to a 4°C ice-water mixture and incubate for 4 minutes.

[0030] II. Preparation of Acidic YPD Selective Medium 1. Preparation of basic solution: Prepare YPD liquid culture medium according to the ratio of 10g / L yeast extract, 20g / L glucose, 20g / L peptone and 18g / L agar.

[0031] 2. Sterilization and cooling: Sterilize with high-pressure steam at 121℃ for 20 minutes, then cool to 55℃.

[0032] 3. Additive injection: Add filtered and sterilized chloramphenicol stock solution to bring the final concentration to 200 mg / L; Add sterile glacial acetic acid to bring the final concentration to 0.3% (v / v).

[0033] Note: Acetic acid must be added at low temperature and mixed quickly to reduce evaporation.

[0034] 4. Pouring and Drying: Pour plates (approximately 20 mL per plate) into a laminar flow hood. Cover the plates and allow them to dry at room temperature in the laminar flow hood for 4 hours, until the surface of the culture medium is no longer sticky to the touch and there are no water droplets.

[0035] Testing revealed that the acidic YPD selective medium prepared in this embodiment has a natural pH of 3.8, which meets the acid-tolerant growth requirements of Pichia kudrica.

[0036] Phase 3: Coating and Cultivation 1. Serial dilution: The heat-shocked bacterial suspension was serially diluted 10-fold, and 10-fold dilutions were selected. -4 10 -5 10 -6 Three gradients.

[0037] 2. Spreading: Take 100 μL of each gradient dilution and spread it onto a selective plate, with 3 replicates for each gradient.

[0038] 3. High-temperature incubation: Invert the plates and place them in a 40℃ constant temperature incubator for 48 hours. Results are shown below. Figure 1 The colony growth is shown in the acidic YPD medium cultured at 40°C.

[0039] 4. Process monitoring: Observe and record the colony morphology every 12 hours. Typical target colonies are milky white, smooth and moist, with neat edges, and are round with a diameter of 2-3 mm.

[0040] Phase 4: Purification and Validation Picking a single colony: Use a sterile inoculation needle to pick a single colony with a typical morphology and streak it onto a regular YPD slant.

[0041] Purification culture: Incubate at 30℃ for 24 hours to obtain pure strain.

[0042] Molecular identification: Genomic DNA was extracted, the ITS sequence was amplified and sequenced, and the obtained gene sequence was uploaded to the NCBI database for BLAST homology comparison. The 26S rDNA sequence of this strain is similar to that of *Pichia gondii* (Kudelazvichis). Pichiakudriavzevii The homology of the strain was 99.9%, and the purified strain was identified as *Kudelazwichiia*.

[0043] Performance testing: 1. Test subject: The Kudria zweipichia yeast obtained in Example 1; Control group: Standard strain of common Saccharomyces cerevisiae (SC-Ref).

[0044] 2. Test Item 1: Esterification Capacity Assessment (Ethyl Acetate and Isoamyl Alcohol) 2.1 Test Principles and Methods Fermentation model: The fermentation liquid of simulated liquor (corn flour liquefaction liquid, initial sugar content 12°Bx, pH 4.5) was used.

[0045] Cultivation conditions: 5% inoculum, static fermentation at 30℃ for 7 days (simulating the early to middle stages of baijiu fermentation).

[0046] Detection method: After fermentation, the wine samples were centrifuged and filtered, and the contents of ethyl acetate and isoamyl alcohol were quantitatively analyzed by headspace gas chromatography-mass spectrometry (HS-GC-MS).

[0047] Repeatability: Three parallel replicates were set up for each sample.

[0048] 2.2 Test Result Data Group Average ethyl acetate content (mg / L) Average isoamyl alcohol content (mg / L) Total esters / higher alcohols ratio Sensory evaluation description Example 1 185.4±5.2 62.8±3.1 2.95 Rich in fruit aroma, with prominent ester aroma, and no obvious off-flavors. control group 45.2±2.1 95.6±4.5 0.47 The ester aroma is faint, the alcohol flavor is strong, and it has a typical yeasty taste. 2.3 Results Analysis and Derivation High-yield characteristics of ethyl acetate: The ethyl acetate yield in Example 1 reached 185.4 mg / L, which is 4.1 times that of the control group of ordinary brewer's yeast.

[0049] This demonstrates the effectiveness of the screening strategy of this invention. Under the stress of "40℃ + 0.3% acetic acid", only strains with extremely high activities of acetate kinase (ACS) and alcohol acetyltransferase (ATF1) survived and grew dominantly. This "stress activation" mechanism directionally enriched genotypes that highly expressed ester synthase systems.

[0050] Moderate formation of isoamyl alcohol: The isoamyl alcohol content is 62.8 mg / L, which is within the ideal range for baijiu flavor (50-80 mg / L).

[0051] Excessive isoamyl alcohol content can lead to a fusel oily taste and bitterness in the liquor, while insufficient isoamyl alcohol content results in a lack of fullness. In Example 1, while maintaining a high ester production capacity, the amino acid metabolic pathway (Ehrlich pathway) was regulated in a balanced manner, and no excessive accumulation of isoamyl alcohol was observed. This indicates that the strain has excellent metabolic flux distribution capabilities and is suitable for high-quality baijiu brewing.

[0052] Flavor balance: In Example 1, the ratio of total esters to higher alcohols was as high as 2.95, significantly higher than that of the control strain. This indicates that the liquor produced by this strain has a "prominent ester aroma and a harmonious and mellow taste," consistent with the flavor characteristics of high-quality baijiu.

[0053] Comparative Example 1: Acidic YPD Selective Medium under 30℃ Culture Conditions This comparative example used the same acidic YPD selective medium as Example 1, but was cultured at 30°C. Everything else was the same as in Example 1. Colony growth is shown in [see attached image]. Figure 2 As shown.

[0054] Comparative Example 2: Conventional YPD selective medium under 40℃ culture conditions This comparative example used standard YPD selective medium, and all other conditions were the same as in Example 1; colony growth is shown in [Figure 1]. Figure 3 As shown. The formulation and preparation steps of the conventional YPD medium are the same as those of the acidic YPD selective medium described above, except that acetic acid is not added. Its pH is 6.5.

[0055] The colony growth of Comparative Example 1 and Comparative Examples 1 and 2 is shown in the table below.

[0056] Group Contamination rate of miscellaneous bacteria Target strain isolation rate strain purity Results illustration Example 1 0% 100% 99.9% Figure 1 Comparative Example 1 30% 70% 85% Figure 2 Comparative Example 2 95% 5% 60% Figure 3 The above comparison results show that using acidic culture medium or 40°C high-temperature culture alone can only reduce contamination by other microorganisms and improve the isolation rate of the target strain to a certain extent. However, the method of the present invention, which combines acidic YPD selective culture medium with 40°C selective culture in Example 1, can achieve a contamination rate of 0% and a target strain isolation rate of 100%, and the purity of the isolated strain reaches 99.9%, which is significantly better than other culture methods. This fully demonstrates that the method of the present invention can efficiently and specifically isolate Pichia pastoris from medium- and high-temperature Daqu.

[0057] Comparative Example 4: Lack of thermal shock pretreatment Without undergoing 49°C heat shock and 4°C cold shock treatment, the coating was directly diluted and applied, with the remaining steps the same as in Example 1.

[0058] Results: The target colony on the plate was accompanied by a small number of heat-resistant bacterial spots and tiny yeast colonies, with a purity of approximately 60%.

[0059] Performance testing: The esterification capacity is compared with that of Example 1 in the following table. Group Average ethyl acetate content (mg / L) Average isoamyl alcohol content (mg / L) Total esters / higher alcohols ratio Example 1 185.4±5.2 62.8±3.1 2.95 control group 45.2±2.1 95.6±4.5 0.47 Comparative Example 4 142.6±18.5 71.4±6.2 2.00 Data comparison shows that the ethyl acetate yield of Example 1 (185.4 mg / L) was 29.9% higher than that of Comparative Example 4 (142.6 mg / L).

[0060] Heat shock pretreatment is crucial for "purification." It physically eliminates many competitors beforehand, allowing subsequent chemical / temperature screening to more precisely target the true *Pichia goudryana*, thus identifying superior strains with purer metabolic pathways and more stable ester-producing gene expression. The low dispersion of the data demonstrates that this step improves the reproducibility and stability of the screening process.

Claims

1. A method for isolating Pichia kudrica, characterized in that, Includes the following steps: (1) Sample preparation: The medium- and high-temperature Daqu sample was ground and then serially diluted to obtain a bacterial suspension; (2) Selective coating: The bacterial suspension is coated onto a selective culture medium; (3) High temperature constant temperature culture: The selective culture medium after coating is placed in a 38℃-42℃ environment for culture until a single colony grows; The selective culture medium is YPD basal medium supplemented with chloramphenicol and acetic acid; The final concentration of the chloramphenicol is 150-250 mg / L, used to inhibit bacterial growth; The final concentration of the acetic acid is 0.2%-0.5% (v / v), which is used to create an acidic environment to inhibit the growth of mold and non-acid-resistant bacteria. The aforementioned medium-high temperature Daqu is a Daqu in which the content of Kudria zweibichi yeast in the Daqu fungus accounts for more than 10% in the early stage of fermentation.

2. The method for isolating Pichia kudrica (a type of yeast) according to claim 1, characterized in that, Step (1) Perform differential heat shock pretreatment before sample processing: Place the Aspergillus suspension in a 48℃-50℃ water bath for 12-18 minutes, and then quickly transfer it to a 4℃ ice bath for 3-5 minutes to shock it.

3. The method for isolating Pichia kudrica (a type of yeast) according to claim 1, characterized in that, The method for preparing the selective culture medium includes: After sterilizing the components of YPD basal culture medium, cool it to 50-60℃, then add sterile filtered chloramphenicol solution and acetic acid, mix well and pour into plates; The final concentration of chloramphenicol is 200 mg / L, and the final concentration of acetic acid is 0.3%.

4. The method for isolating Kudria zweichi yeast according to claim 3, characterized in that, The basic components of the YPD culture medium are: yeast extract 10g / L, glucose 20g / L, peptone 20g / L, and agar 15-20g / L.

5. The method for isolating Kudria zweichi yeast according to claim 1, characterized in that, The medium-high temperature Daqu samples mentioned are Daqu fermented from day 2 to day 10.

6. The method for isolating Kudria zweichi yeast according to claim 1, characterized in that, In step (3), the culture time is 48-72 hours; before coating culture, the selective culture medium plate is left to stand and dry at room temperature for 0.5-24 hours after preparation, until there are no free water droplets on the surface.

7. The method for isolating Kudria zweichi yeast according to claim 1, characterized in that, The method also includes step (4): picking out the morphologically typical single colonies that grew in step (3), inoculating them into ordinary YPD medium for purification culture, and obtaining pure strains of Pichia kudrica.

8. The method according to claim 7, characterized in that, The method also includes functional screening of the obtained pure strains, selecting strains with at least one of the following characteristics: high ester production capacity, high ethanol tolerance, or high acid tolerance for use in baijiu brewing.

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