Torulaspora delbrueckii, screening culture method and application
By screening and identifying Delb's spore yeast, the problem of flavor and taste changes during the fermentation of moussalaise was solved, the traditional flavor was retained and the fermentation cycle was shortened, providing theoretical guidance for industrial application.
Patent Information
- Application Number
- CN202510808550.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-17
- Publication Date
- 2025-09-23
AI Technical Summary
The natural fermentation cycle of Moussalais is long and is affected by the environment and miscellaneous bacteria. The existing yeast will change its traditional flavor and taste during use and cannot retain its unique flavor.
Torulella delbone was isolated from the environment of Mussalais wine and identified using nutrient agar, sugar fermentation medium, carbon source assimilation medium and nitrogen source assimilation medium. It was cultured on YPD basal medium and the medium composition was optimized by single factor experiment and response surface methodology.
Preserve the traditional flavor and taste of Moussalais, shorten the fermentation cycle, and provide theoretical guidance for the industrial application of Moussalais brewing.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial strains, and in particular to Delbur's spore-forming yeast, a screening and culturing method and an application thereof. Background Art
[0002] Musalaisi is a traditional beverage from the Awati region of Xinjiang. Its unique ingredients and brewing techniques give it a distinct style and characteristics. By inheriting the brewing techniques of our ancestors, Musalaisi has been preserved and is known as a "living fossil" of ancient Western Region wine. Due to the unique fermentation process, Musalaisi contains a wealth of active substances, which not only have high nutritional value but also possess certain health benefits. As a key component of local economic development, Musalaisi holds great significance in both cultural heritage and economic value.
[0003] In recent years, with the rapid development of Xinjiang, moussalaise has attracted widespread attention from scholars both domestically and internationally. Yang Baoqiu et al., through quantitative analysis of the alcohol tolerance of multiple yeast strains, found that moussalaise yeast exhibits high alcohol tolerance. Zhu Lixia et al., through quantitative descriptive evaluation of Xinjiang moussalaise samples and statistical analysis of their sensory characteristics using partial least squares regression, found that its sensory characteristics are complex, variable, and unstable. They also analyzed the strengths, current development status, and remaining challenges of moussalaise, and provided an objective assessment of its future development. Yeast, as a single-celled eukaryotic microorganism, has the ability to convert organic matter into alcohol and therefore plays a crucial role in the production and quality improvement of moussalaise. Liu Zhen et al., through screening and analysis of yeasts from moussalaise, found that some yeasts can degrade toxic and harmful substances such as 5-hydroxymethylfurfural. Yang Chuangju et al. discovered that moussalaise contains yeasts with sugar-lowering and acid-lowering abilities, potentially improving its quality. Qiao Tongtong et al. discovered yeasts that enhance the caramel and fruity aromas of moussalaise, improving its aroma quality.
[0004] Moussalais is mostly fermented naturally during the fermentation process, but the natural fermentation cycle is long and is greatly affected by the environment and miscellaneous bacteria. Although the existing yeast can shorten the fermentation cycle and inhibit the growth of miscellaneous bacteria to a certain extent during use, it will change the traditional flavor and taste of Moussalais, and thus the unique flavor produced by the natural fermentation of Moussalais cannot be obtained. Summary of the Invention
[0005] In response to the above-mentioned problems, the present invention aims to provide a Delb's spore yeast, which is screened from Moussalais fruit wine and used for Moussalais fermentation. It can overcome the shortcomings of existing yeasts in taste and flavor homogeneity during the fermentation process and can retain the traditional flavor and taste of Moussalais.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] In a first aspect, the present invention provides a Delb's spore yeast, characterized in that the Delb's spore yeast was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on January 6, 2025, with a deposit number of CGMCC NO.7.581.
[0008] In a second aspect, the present invention provides a screening and identification medium, characterized in that it is used for screening, identifying and culturing the above-mentioned Delb's spore yeast; the screening and identification medium comprises:
[0009] Nutrient agar medium, sugar fermentation medium, assimilation carbon source medium, assimilation nitrogen source medium and YPD basal medium;
[0010] The nutrient agar medium is used to obtain microorganisms in the environment of Musalaith;
[0011] The sugar fermentation medium is used to identify whether the screened strain can utilize glucose, sucrose, lactose, and maltose to produce acid and gas;
[0012] The assimilation carbon source culture medium is used to identify whether the screened strain can assimilate glucose, sucrose, maltose, xylose, and soluble starch;
[0013] The assimilation nitrogen source culture medium is used to identify whether the screened strain can assimilate ammonium sulfate and ammonium chloride;
[0014] The YPD basic culture medium is used to culture the identified Delbur's spore yeast.
[0015] Furthermore, the nutrient agar medium has the following components:
[0016] Peptone 10g / L, sodium chloride 5g / L, beef extract powder 3g / L, agar 15g / L.
[0017] Furthermore, the sugar fermentation medium has the following components:
[0018] Peptone 10g / L, beef extract 5g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2g / L, bromocresol purple 0.04g / L, glucose 5g / L, sucrose 5g / L, lactose 5g / L, maltose 5g / L.
[0019] Furthermore, the assimilation carbon source culture medium has the following components:
[0020] Peptone 2g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 0.2g / L.
[0021] Furthermore, the assimilated nitrogen source culture medium has the following components:
[0022] Glucose 20g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L.
[0023] Furthermore, the YPD basal medium has the following components:
[0024] Glucose 10-30g / L, peptone 10-30g / L, yeast extract powder 5-25g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, agar 20g / L.
[0025] In a third aspect, the present invention provides a method for screening and culturing Torulella delb's yeast, characterized in that the Torulella delb's yeast is obtained by screening and culturing using the above-mentioned culture medium, comprising the following steps:
[0026] S1. Screening candidate strains from the environment surrounding Musalais wine using nutrient agar medium;
[0027] S2. Screening the candidate strains obtained in step S1 for strains having properties of Torulella delbrueckii using a sugar fermentation medium, an assimilated carbon source medium, and an assimilated nitrogen source medium;
[0028] S3. The strain screened in S2 was subjected to 18S rDNA gene sequencing and verification analysis to determine that the strain belonged to the genus Delb's spore yeast. The strain was named Delb's spore yeast MA-11 and cultured using YPD basal medium.
[0029] Furthermore, S2 includes the following steps:
[0030] S2-1: Conduct sugar fermentation experiments on the candidate strains screened in S1 using sugar fermentation medium. Based on the experimental results, strains that can ferment glucose and sucrose but cannot ferment lactose and maltose are screened to obtain primary strains;
[0031] S2-2: The primary strains screened in S2-1 are subjected to carbon source assimilation experiments using an assimilation carbon source medium. Based on the experimental results, strains that can assimilate glucose and sucrose but cannot assimilate maltose, xylose, and soluble starch are screened to obtain secondary strains;
[0032] S2-3: The secondary strains screened in S2-2 were subjected to a nitrogen source assimilation experiment using an assimilation nitrogen source medium. Based on the experimental results, a strain capable of assimilating ammonium sulfate and ammonium chloride was screened out, and the strain was confirmed to be of the genus Delb's spore yeast.
[0033] In a fourth aspect, the present invention provides an application of Delbur's spore yeast in the field of fruit wine brewing.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] The present invention screens a strain of Torulella delb's spore yeast from the surrounding environment of Moussalais fruit wine and uses it in Moussalais fermentation. This method overcomes the shortcomings of existing yeasts in terms of taste and flavor homogeneity during fermentation, while preserving the traditional Moussalais flavor and mouthfeel. Furthermore, the contents of glucose, peptone, and yeast extract in the culture medium are optimized through single-factor experiments and response surface methodology, aiming to further increase the bacterial count of Torulella delb's spore yeast, providing theoretical guidance for the industrial application of Moussalais brewing.
[0036] Specifically, the present application screens a strain with the properties of Delbur's spore-forming yeast from the surrounding environment of Musalais fruit wine through nutrient agar medium, sugar fermentation medium, assimilated carbon source medium, and assimilated nitrogen source medium, and then cultures the strain through YPD basal medium to obtain the culture medium formula most suitable for the growth of the strain, and applies the strain in the field of fruit wine brewing, which has certain reference significance for using beneficial microorganisms in its own environment for fermentation during the Musalais brewing process. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 This is the phylogenetic tree of strain MA-11 based on the 18SrDNA sequence of the present invention.
[0038] Figure 2 Graph showing the effect of glucose concentration on colony counts in the present invention.
[0039] Figure 3 Graph showing the effect of peptone concentration on colony counts according to the present invention.
[0040] Figure 4 This is a graph showing the effect of yeast extract powder concentration on colony counts.
[0041] Figure 5 It is the contour line and response surface diagram of the interaction effect between various factors of the present invention on the number of colonies. DETAILED DESCRIPTION
[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.
[0043] The present invention discloses a Torulaspora delbrueckii, whose Latin name is Torulaspora delbrueckii. The Torulaspora delbrueckii was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on January 6, 2025, with a deposit number of CGMCC NO.7.581.
[0044] The Delb's spore-forming yeast can be used in fruit wine brewing, particularly in the field of improved Moussalaise brewing.
[0045] The Delb's spore yeast of the present application is obtained by screening, identifying and culturing through a screening and identification medium;
[0046] Specifically, when screening for the genus Delb's spore yeast, it is necessary to conduct a comprehensive sugar fermentation experiment, a carbon source assimilation experiment, and a nitrogen source assimilation experiment, rather than relying on a single experiment. This is because different experiments reflect different dimensions of yeast physiological metabolism, and the information from a single experiment is insufficient to fully characterize the characteristics of the strain. Therefore, the screening and identification culture medium of this application includes:
[0047] Nutrient agar medium, sugar fermentation medium, assimilation carbon source medium, assimilation nitrogen source medium and YPD basal medium;
[0048] Among them, nutrient agar medium is used to obtain microorganisms in the environment of Musalaith;
[0049] The ingredients are: peptone 10g / L, sodium chloride 5g / L, beef extract powder 3g / L, and agar 15g / L.
[0050] Sugar fermentation medium is used to identify whether the screened strains can utilize glucose, sucrose, lactose, and maltose to produce acid and gas;
[0051] If it can ferment glucose and sucrose but cannot ferment lactose and maltose, it preliminarily meets the sugar fermentation performance of Torulella delbrueckii.
[0052] The ingredients are: peptone 10g / L, beef extract 5g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2g / L, bromocresol purple 0.04g / L, glucose 5g / L, sucrose 5g / L, lactose 5g / L, and maltose 5g / L.
[0053] It should be noted that each liter of each ingredient is based on 1 L of distilled water.
[0054] Assimilation carbon source culture medium is used to identify whether the screened strains can assimilate glucose, sucrose, maltose, xylose, and soluble starch;
[0055] If it can assimilate glucose and sucrose but cannot assimilate maltose, xylose and soluble starch, it can be said that it meets the basic characteristics of Delb's spore yeast in assimilating carbon sources.
[0056] The ingredients are: peptone 2g / L, sodium chloride 5g / L, and dipotassium hydrogen phosphate 0.2g / L, wherein each liter of each ingredient is calculated based on 1L of distilled water.
[0057] Assimilation nitrogen source culture medium is used to identify whether the screened strains can assimilate ammonium sulfate and ammonium chloride;
[0058] If it can assimilate ammonium sulfate and ammonium chloride, then the basic characteristics of Delb's spore yeast for assimilating nitrogen sources are met;
[0059] The ingredients are: 20 g / L glucose, 1 g / L dipotassium hydrogen phosphate, and 0.5 g / L magnesium sulfate, wherein each liter of each ingredient is calculated based on 1 L of distilled water.
[0060] YPD basal medium was used to culture the identified Delb's spore yeast.
[0061] The ingredients are: glucose 10-30g / L, peptone 10-30g / L, yeast extract powder 5-25g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, and agar 20g / L.
[0062] Each liter of each ingredient is based on 1 L of distilled water.
[0063] In addition, the Delbur's spore yeast of the present application is obtained by screening and culturing using the above-mentioned culture medium, comprising the following steps:
[0064] S1. Screening strains from the environment (air) surrounding Musalais wine using nutrient agar medium;
[0065] The specific operation is to place the nutrient agar culture medium open in the environment surrounding the Musalais fruit wine (at a height of 1m, using a five-point sampling method). After 5 minutes, cover the culture dish with a lid and seal it with sealing film, then place it in a constant temperature incubator at 28°C for 1-3 days, observe the growth of the strain, and preliminarily screen out strains with milky white or cream-colored colonies, smooth and shiny surfaces, neat edges, moist, soft textures, and easy-to-pick-up textures; and strains with opaque or translucent colonies rather than dry or powdery forms. The ascospores of the preliminarily screened strains are observed under a microscope using malachite green staining. If multilateral budding and typical ascospores are observed, the strain is identified as a candidate strain.
[0066] S2. Screening the candidate strains obtained in step S1 for strains having properties of Torulella delbrueckii using a sugar fermentation medium, an assimilated carbon source medium, and an assimilated nitrogen source medium;
[0067] Specifically, S2-1: performing a sugar fermentation experiment on the candidate strains screened in S1 using a sugar fermentation medium, and screening out strains that can ferment glucose and sucrose but cannot ferment lactose and maltose based on the experimental results to obtain primary strains;
[0068] S2-2: The primary strains screened in S2-1 are subjected to carbon source assimilation experiments using an assimilation carbon source medium. Based on the experimental results, strains that can assimilate glucose and sucrose but cannot assimilate maltose, xylose, and soluble starch are screened to obtain secondary strains;
[0069] S2-3: The secondary strains screened in S2-2 were subjected to a nitrogen source assimilation experiment using an assimilation nitrogen source medium. Based on the experimental results, a strain capable of assimilating ammonium sulfate and ammonium chloride was screened out, and the strain was confirmed to be of the genus Delb's spore yeast.
[0070] S3. The strain screened in S2 was subjected to 18S rDNA gene sequencing and verification analysis to determine that the strain belonged to the genus Delb's spore yeast. The strain was named Delb's spore yeast MA-11 and cultured using YPD basal medium.
[0071] It should be noted that the preparation methods of the nutrient agar medium, sugar fermentation medium, assimilated carbon source medium, assimilated nitrogen source medium and YPD basal medium in this application are all the same. Here, the preparation method of the nutrient agar medium is used as an example for description:
[0072] Step 1: Weigh 10 g of peptone, 5 g of sodium chloride, 3 g / L of beef extract powder, and 15 g / L of agar, and dissolve them in 1 L of distilled water;
[0073] Step 2: Stir and dissolve, then sterilize at 121°C for 20 minutes;
[0074] Step 3: After sterilization, place it in the clean bench and cool it down for later use.
[0075] Example 1
[0076] In this embodiment, the specific content of each component in the YPD basal medium is:
[0077] YPD basal medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, 1 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, and 20 g / L agar.
[0078] Example 2
[0079] The process steps of this embodiment are the same as those of Example 1, with the only difference being that the amount of glucose added to the YPD basal medium in this embodiment is 25 g / L.
[0080] Example 3
[0081] The process steps of this embodiment are the same as those of Example 1, with the only difference being that the amount of glucose added to the YPD basal medium in this embodiment is 30 g / L.
[0082] Example 4
[0083] The process steps of this embodiment are the same as those of Example 1, except that the amount of peptone added to the YPD basal medium in this embodiment is 15 g / L.
[0084] Example 5
[0085] The process steps of this comparative example are the same as those of Example 1, except that the amount of peptone added to the YPD basal medium in this comparative example is 25 g / L.
[0086] Example 6
[0087] The process steps of this embodiment are the same as those of Example 1, with the only difference being that the amount of yeast extract powder added to the YPD basal medium in this embodiment is 10 g / L.
[0088] Example 7
[0089] The process steps of this embodiment are the same as those of Example 1, with the only difference being that the amount of yeast extract powder added to the YPD basal medium in this embodiment is 20 g / L.
[0090] Comparative Example 1
[0091] The process steps of this comparative example are the same as those of Example 1, except that the amount of glucose added to the YPD basal medium in this comparative example is 10 g / L.
[0092] Comparative Example 2
[0093] The process steps of this comparative example are the same as those of Example 1, except that the amount of glucose added to the YPD basal medium in this comparative example is 15 g / L.
[0094] Comparative Example 3
[0095] The process steps of this comparative example are the same as those of Example 1, except that the amount of peptone added to the YPD basal medium in this comparative example is 10 g / L.
[0096] Comparative Example 4
[0097] The process steps of this comparative example are the same as those of Example 1, except that the amount of peptone added to the YPD basal medium in this comparative example is 30 g / L.
[0098] Comparative Example 5
[0099] The process steps of this comparative example are the same as those of Example 1, except that the amount of yeast extract powder added to the YPD basal medium in this comparative example is 5 g / L.
[0100] Comparative Example 6
[0101] The process steps of this comparative example are the same as those of Example 1, except that the amount of yeast extract powder added to the YPD basal medium in this comparative example is 25 g / L.
[0102] 1 Materials and Methods
[0103] 1.1 Materials and Instruments
[0104] Delb's spore yeast MA-11 was obtained from Awati County, Xinjiang (40°34′58″N, 80°25′33″E).
[0105] Glucose, peptone, and yeast extract powder were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; magnesium sulfate and potassium dihydrogen phosphate were purchased from Tianjin Kemeiou Chemical Reagent Co., Ltd.; agar was purchased from Sinopharm Chemical Reagent Co., Ltd. A high-precision analytical balance (ME) was purchased from Mettler-Toledo; a vertical high-pressure steam sterilizer (LDZF-50L) was purchased from Shanghai Shen'an Medical Instrument Factory; and a precision constant-temperature incubator (BPH-9402) was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.
[0106] culture medium
[0107] Nutrient agar medium: peptone 10 g / L, sodium chloride 5 g / L, beef extract powder 3 g / L, agar 15 g / L;
[0108] YPD basal medium: glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, agar 20 g / L;
[0109] Sugar fermentation medium: peptone 10 g / L, beef extract 5 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 2 g / L, bromocresol purple 0.04 g / L, glucose 5 g / L, sucrose 5 g / L, lactose 5 g / L, maltose 5 g / L;
[0110] Assimilative carbon source medium: peptone 2g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 0.2g / L;
[0111] Assimilative nitrogen source culture medium: glucose 20 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L.
[0112] 1.2 Experimental methods
[0113] 1.2.1 Strain isolation and purification
[0114] Take 25 ml of each sample of Delb's spore yeast MA-11, mix them in 225 ml of sterile water, and then dilute them in a 10-fold gradient to 10 -6 100 μL of the dilution solution from each sample was evenly applied to nutrient agar. After incubation in a 28°C biochemical incubator for 48 h, single colonies were selected and repeatedly streaked for isolation and purification. After purification on nutrient agar plates, the strains were transferred to nutrient agar slants and stored at 4°C.
[0115] 1.2.2 Physiological and biochemical tests
[0116] (1) Sugar fermentation test
[0117] The fermentation carbon sources required were identified as glucose, sucrose, lactose and maltose.
[0118] The above sugars were added to the basic culture medium containing the Dulbecco's fermentation tube respectively. After activation, the strain was inoculated into the culture medium and cultured at 28°C for 2-4 weeks before observation. The presence of bubbles in the Dulbecco's tube was considered positive, and the absence of bubbles was considered negative.
[0119] (2) Carbon source assimilation test
[0120] The assimilated substances were identified as glucose, sucrose, maltose, xylose and starch.
[0121] Add the above sugars to the assimilative carbon source culture medium and culture at 28°C for 2-4 weeks. Observe the turbidity. Turbidity is recorded as positive, otherwise negative. Three parallel tests were performed, with the medium without sugar added as the control.
[0122] (3) Assimilation nitrogen source test
[0123] The assimilated substances were identified as ammonium sulfate and ammonium chloride.
[0124] The above nitrogen sources were added to the assimilative nitrogen source culture medium respectively, and cultured at 28℃ for 1 week. Three groups were tested in parallel, and the culture medium without nitrogen source was used as the control. The turbidity was observed, and turbidity was recorded as positive, otherwise it was recorded as negative.
[0125] 1.2.3 Identification of strain 18S rDNA
[0126] The selected strains were activated and sent to Shanghai Paisonno Biotechnology Co., Ltd. for 18S rDNA gene sequencing. Fungal genomic DNA was extracted using a DNA kit and amplified using primer ITS1 (TCCGTAGGTGAACCTGCGG) using this DNA as a template. Sequencing results were compared with the NCBI database, and a phylogenetic tree was constructed.
[0127] 1.2.4 Single-factor experimental design
[0128] (1) Glucose single factor test
[0129] The culture conditions and other components of the YPD basal medium remained unchanged. Glucose was added to the YPD basal medium at a rate of 10, 15, 20, 25, and 30 g / L, and the effects of different additions on the colony count were observed.
[0130] (2) Peptone single factor test
[0131] The culture conditions and other components of YPD medium remained unchanged. Peptone was added to YPD basal medium at a rate of 10, 15, 20, 25, and 30 g / L, and the effects of different additions on the colony count were observed.
[0132] (3) Yeast extract powder single factor test
[0133] The culture conditions and other components of YPD basal medium remained unchanged. Yeast extract powder was added at a rate of 5, 10, 15, 20, and 25 g / L, respectively, to observe the effect of different addition amounts of yeast extract powder on the colony count.
[0134] 1.2.5 Response surface experiment optimization
[0135] According to the results of the single-factor optimization experiment of strain culture, three factors A (glucose), B (peptone), and C (yeast extract powder) were selected, and the Box-Behnken experimental design was used for the response surface experiment. The Box-Behnken experimental factors and levels are shown in Table 1.
[0136] Table 1 Box-Behnken design factors and levels
[0137]
[0138] 2 Results and Analysis
[0139] 2.1 Strain identification
[0140] 2.1.1 Biochemical Identification of the Strain: Strain MA-11, which was found as a single colony in the culture medium, was selected for biochemical analysis and 18S rDNA gene sequencing. The results of MA-11 sugar fermentation, carbon source assimilation, and nitrogen source assimilation tests are shown in Table 2.
[0141] Table 2 Results of physiological and biochemical tests in yeast
[0142]
[0143]
[0144] Note: “+” indicates a positive result; “-” indicates a negative result.
[0145] As shown in Table 1, strain MA-11 cannot ferment lactose and maltose, but can ferment glucose and sucrose; cannot assimilate xylose and soluble starch, but can assimilate glucose, sucrose, and maltose; and can assimilate ammonium sulfate and ammonium chloride. Based on the "Handbook of Fungal Identification" and the "Handbook of Characteristics and Identification of Yeasts," strain MA-11 can be preliminarily identified as Torulaspora.
[0146] 2.1.2 Molecular biological identification of strains The sequencing results of the 18S rDNA gene of the MA-11 strain were compared with the NCBI database by BLAST homology. The ITS sequences of yeasts from NCBI were selected for comparison analysis. The phylogenetic tree was constructed using MEGA 6.0 software. Figure 1 It was found that strain MA-11 was a member of the genus Torulaspora. Combined with the results of biochemical tests,
[0147] 2.2 Optimization of culture medium components
[0148] 2.2.1 Glucose single-factor test
[0149] From Examples 1-3, Comparative Examples 1 and 2, and the experiment on the effect of glucose concentration on the number of colonies, it is shown that different concentrations of glucose will affect the number of colonies. By setting a series of different glucose concentrations on the number of colonies, the effect of Figure 2 As shown in the figure, when the concentration of glucose is 20 g / L and other conditions remain unchanged, the number of colonies reaches the highest ×10 5 CFU / mL, so the glucose concentration of YPD basal medium was selected as 20g / L.
[0150] 2.2.2 Peptone single factor test
[0151] From the experiment of Example 1, Example 4 and 5, Comparative Examples 3 and 4, and the effect of peptone concentration on the number of colonies, it is shown that different concentrations of peptone will affect the number of colonies. The effect of a series of different peptone concentrations on the number of colonies is shown in FIG. Figure 3 When the concentration of peptone was 20 g / L and other conditions remained unchanged, the colony count reached a maximum of 9.9 × 10 5 CFU / mL, so the peptone concentration of YPD basal medium was selected as 20g / L.
[0152] 2.2.3 Single-factor experiment with yeast extract powder
[0153] From the experiment of Example 1, Example 6 and 7, Comparative Examples 5 and 6, and the effect of yeast extract powder concentration on the number of colonies, it is shown that different concentrations of yeast extract powder will affect the number of colonies. The effect of a series of different yeast extract powder concentrations on the number of colonies is shown in FIG. Figure 4When the concentration of yeast extract powder was 15 g / L and other conditions remained unchanged, the colony count reached a maximum of 7.75×10 5 CFU / mL, so the concentration of yeast extract powder in YPD basic medium was selected as 15g / L.
[0154] 2.2.4 Response surface experiment optimization
[0155] According to the results of the single-factor experiment, Design-Expert13.0 software was used to conduct a three-factor three-level central combination experiment on the concentrations of A (glucose), B (peptone), and C (yeast extract powder) according to the central combination experimental design principle of the Box-Behnken experiment, and variance analysis was performed. The central combination experimental design and result analysis are shown in Tables 3 and 4.
[0156] Table 3 Response surface test and response value
[0157]
[0158]
[0159] Table 4 Results and analysis of variance of response surface test for colony count
[0160]
[0161]
[0162] The Box-Behnken experimental design and the data in the result table 4 were fitted and analyzed, and the binary polynomial regression equation of A (glucose), B (peptone), and C (yeast extract powder) on Y (colony count) was obtained as follows:
[0163] Y=11.46-0.1625A-0.6000B-0.3875C-0.4750AB-0.4500AC+0.7750BC-2.71A 2 -1.58B 2 -2.35C 2
[0164] As shown in Table 3, the P value of the model is <0.0001, which is extremely significant, indicating that the model is effective. The lack of fit term P = 0.6851 (>0.05) is not significant, indicating that the functional relationship between the factors under investigation and the response value of the model is extremely significant, and there is almost no lack of fit. 2 ) and adjusted coefficient of determination (R 2adj ) were 0.9884 and 0.9735, respectively, indicating that the model has a good degree of fit and a small experimental error, and is suitable for the optimization and prediction of culture conditions. The P value shows that the linear terms B and C have a significant effect on the number of colonies (<0.05), while the effect of A on the number of colonies is not significant (>0.05); the interaction terms AB, AC, and BC have a significant effect on the number of colonies (<0.05); the quadratic term A 2 、B 2 、C 2 The effects on the number of colonies were extremely significant (<0.0001). From the F value, we can see that the effects of the three factors on the number of colonies are from large to small: B (peptone) > C (yeast extract powder) > A (glucose).
[0165] Using Design-Expert 13.0 software, we can draw the contour lines and response surface diagrams of the interaction between factors A (glucose), B (peptone), and C (yeast extract powder) on the number of colonies. Figure 5 The surface plot can directly reflect the interaction effect of various factors on the colony count. The steeper the slope of the surface, the greater the influence of the factor on the colony count; the closer the shape of the contour map is to an ellipse, the greater the influence of the factor on the colony count. Through the binary polynomial regression equation and further analysis, when the YPD basal medium contains 20.303 g / L glucose, 18.849 g / L peptone, and 14.367 g / L yeast extract powder, and other conditions remain unchanged, the colony count is 11.558×10 5 CFU / mL.
[0166] 2.2.5 Model Validation
[0167] In order to verify the effectiveness of the model and to ensure the actual operation is controllable, the glucose concentration in the YPD basal medium was set to 20 g / L, peptone 19 g / L, and yeast extract powder 14 g / L. Other conditions remained unchanged and the culture was carried out. The colony count was 11.45 × 10 5 The CFU / mL was basically consistent with the predicted value, indicating that the model can better reflect the trend of the relationship between colony count and factor changes.
[0168] In summary, the present invention takes the fermentation strain screened from Musalais as the research object, and screens out strains with the properties of Delb's spore-forming yeast through nutrient agar medium, sugar fermentation medium, assimilated carbon source medium, and assimilated nitrogen source medium, and then cultivates them through YPD basic medium. First, through a single factor experiment, the concentrations of glucose, peptone, and yeast extract in the culture medium were determined to be 20g / L, 20g / L, and 15g / L, respectively. Subsequently, a three-factor three-level experiment was performed using the Box-Behnken design, and the optimal culture medium formula for the strain was obtained as follows: glucose 20g / L, peptone 19g / L, yeast extract 14g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, and agar 20g / L. Under these conditions, the number of colonies reached 11.45×10 5 CFU / mL. This indicates that glucose is the primary carbon and energy source for yeast. Appropriate glucose concentrations provide sufficient energy for yeast metabolism, growth, and reproduction. If the glucose concentration is too low, yeast growth may be slow due to lack of energy, limiting reproduction and ultimately resulting in a low colony count. Excessive glucose concentrations, on the other hand, can lead to excessive osmotic pressure, inhibiting yeast growth. Peptone provides yeast with the nitrogen source necessary for the synthesis of cellular proteins, enzymes, and other biomacromolecules. Yeast extract powder is rich in nutrients such as B vitamins, nucleotides, and amino acids, which play a crucial role in promoting yeast growth and metabolism. Through a synergistic effect, the energy provided by glucose promotes the absorption and utilization of nutrients in peptone and yeast extract powder by yeast. However, certain components in peptone and yeast extract powder may affect the yeast's glucose uptake and metabolic pathways, enabling yeast to more efficiently utilize glucose for growth and reproduction, resulting in a certain change in the total colony count of Torulella delbrueckii MA-11. The results of the present invention have certain reference significance for utilizing beneficial microorganisms in the environment of moussalaise for fermentation during the brewing process of moussalaise.
[0169] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.
Claims
1. A Delb's spore yeast, characterized in that The Delb's spore yeast was deposited in the General Microbiology Center of the China Culture Collection Administration on January 6, 2025, with the deposit number CGMCC NO.7.
581.
2. A screening and identification culture medium, characterized in that: Used for screening, identifying and culturing the Delb's spore yeast according to claim 1; the screening and identification culture medium comprises: Nutrient agar medium, sugar fermentation medium, assimilation carbon source medium, assimilation nitrogen source medium and YPD basal medium; The nutrient agar medium is used to obtain microorganisms in the environment of Musalaith; The sugar fermentation medium is used to identify whether the screened strain can utilize glucose, sucrose, lactose, and maltose to produce acid and gas; The assimilation carbon source culture medium is used to identify whether the screened strain can assimilate glucose, sucrose, maltose, xylose, and soluble starch; The assimilation nitrogen source culture medium is used to identify whether the screened strain can assimilate ammonium sulfate and ammonium chloride; The YPD basic culture medium is used to culture the Delbur's spore yeast identified by the above culture media.
3. A screening and identification culture medium according to claim 2, characterized in that, Nutrient agar medium, the composition is as follows: Peptone 10g / L, sodium chloride 5g / L, beef extract powder 3g / L, agar 15g / L.
4. A screening and identification culture medium according to claim 2, characterized in that, The sugar fermentation medium has the following components: Peptone 10g / L, beef extract 5g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2g / L, bromocresol purple 0.04g / L, glucose 5g / L, sucrose 5g / L, lactose 5g / L, maltose 5g / L.
5. A screening and identification culture medium according to claim 2, characterized in that, The assimilation carbon source culture medium has the following components: Peptone 2g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 0.2g / L.
6. A screening and identification culture medium according to claim 2, characterized in that, The assimilation nitrogen source culture medium has the following components: Glucose 20g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L.
7. A screening and identification culture medium according to claim 2, characterized in that, The YPD basal medium has the following components: Glucose 10-30g / L, peptone 10-30g / L, yeast extract powder 5-25g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, agar 20g / L.
8. A method for screening and culturing Torulella delb's yeast, characterized in that: The Delb's spore yeast is obtained by screening and culturing using the culture medium according to any one of claims 2 to 7, comprising the following steps: S1. Screening candidate strains from the environment surrounding Musalais wine using nutrient agar medium; S2. Screening the candidate strains obtained in step S1 for strains having properties of Torulella delbrueckii using a sugar fermentation medium, an assimilated carbon source medium, and an assimilated nitrogen source medium; S3. The strain screened in S2 was subjected to 18S rDNA gene sequencing and verification analysis to determine that the strain belonged to the genus Delb's spore yeast. The strain was named Delb's spore yeast MA-11 and cultured using YPD basal medium.
9. The screening and culturing method of Torulella delb's yeast according to claim 8, characterized in that: The S2 comprises the following steps: S2-1: Conduct sugar fermentation experiments on the candidate strains screened in S1 using sugar fermentation medium. Based on the experimental results, strains that can ferment glucose and sucrose but cannot ferment lactose and maltose are screened to obtain primary strains; S2-2: The primary strains screened in S2-1 are subjected to carbon source assimilation experiments using an assimilation carbon source medium. Based on the experimental results, strains that can assimilate glucose and sucrose but cannot assimilate maltose, xylose, and soluble starch are screened to obtain secondary strains; S2-3: The secondary strains screened in S2-2 were subjected to a nitrogen source assimilation experiment using an assimilation nitrogen source medium. Based on the experimental results, a strain capable of assimilating ammonium sulfate and ammonium chloride was screened out, and the strain was confirmed to be of the genus Delb's spore yeast.
10. Use of the Torulella delbone yeast according to claim 1 in the field of fruit wine brewing.