Kazakstan yeast with high protein yield and application of Kazakstan yeast

Abstract

The invention relates to the technical field of microorganisms, in particular to Kazakstein yeast with high protein yield and application of the Kazakstein yeast. The invention provides Kazachstania humii CMRC 12Y, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Kazachstania humii CMRC 12Y is CGMCC No.34656. The Kazachstania humii CMRC 12Y can be used for preparing the Kazachstania humii CMRC 12Y. The bacterial strain is separated from traditional fermented food, has the characteristics of good safety, fast growth, high mycoprotein content and the like, and can realize high biomass growth and high protein expression, and the mycoprotein produced by the bacterial strain has good sensory flavor, can be used for preparing microbial protein related products, and has important application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a Kazakhstan yeast with high protein yield and application thereof. BACKGROUND

[0002] At present, protein acquisition mainly relies on planting and breeding, which requires a large amount of land and water resources, causes serious environmental pollution, and is difficult to meet the huge pressure of population growth and consumption upgrading on protein demand growth. Under this situation, it is an inevitable trend to tap alternative protein sources and achieve efficient protein production to realize sustainable and diversified protein supply system. Microbial cell protein (i.e. single cell protein) is one of the ideal alternative protein sources.

[0003] Yeast has a long history of application in food, has the advantages of high protein content (generally 30%-55%), large cell volume, and easy separation and recovery.

[0004] Kazakhstan yeast (Kazachstania microarthrodii) is a yeast strain with unique characteristics and wide application. At present, the strain is mainly applied in food fermentation, which is beneficial to improve the sensory flavor quality of fermented food, but its research in microbial protein production has not been reported. Chinese patent application CN202010878100.X discloses a yeast for producing flavor by solid-state fermentation and its application, and the isolated Kazakhstan yeast (Kazachstania microarthrodii) CGMCC19813 can improve the yield of flavor substances in solid-state fermented liquor. Chinese patent application CN202211131858.2 discloses Kazakhstan yeast XJ-65 and its application in pepper fermentation, and the strain as a fermentation agent can significantly improve the content of flavor substances in pepper products and improve the quality of pepper products. Chinese patent application CN202411811668.4 discloses Kazakhstan yeast JM1503 and its application in composite fermentation agent, and the isolated Kazakhstan yeast (Kazachstania microarthrodii) JM1503 has strong fermentation capacity and can improve the taste, luster and color of steamed buns. Kazachstania humilis Kazachstania humilis Kazachstania humilis SUMMARY

[0005] The present application provides a Kazakhstan yeast with high protein yield and application thereof.

[0006] In order to obtain a yeast strain with high protein yield and food safety, the present application screens a Kazakhstan yeast (Kazachstania microarthrodii) with excellent growth performance and high cell protein content from traditional natural fermented sour soup, which is named as CMRC 12Y. The strain can be used for microbial protein production and provides an excellent strain for microbial protein production. Kazachstania humilis

[0007] Specifically, the present application provides the technical solutions described below.​​​​

[0008] In a first aspect, the present application provides a Saccharomyces eugamiferus with dwarf phenotype, Kazachstania humilis ) CMRC 12Y, which was deposited with the China General Microbiological Culture Collection Center (CGMCC, Beijing, China) on May 23, 2025, and was named as Kazachstania humilis , and the accession number is CGMCC No. 34656.

[0009] The Saccharomyces eugamiferus CMRC 12Y has the characteristics of fast growth, high cell protein content, and acid tolerance, and has good flavor, and is suitable for use as a protein production strain.

[0010] In a second aspect, the present application provides a microbial preparation comprising the Saccharomyces eugamiferus CMRC 12Y.

[0011] In the present application, the microbial preparation can be a solid preparation or a liquid preparation.

[0012] In addition to comprising the Saccharomyces eugamiferus CMRC 12Y, the microbial preparation can further comprise other microorganisms (such as bacteria or fungi for preparing microbial protein), and / or can further comprise excipients allowed in the field of microbial preparations.

[0013] Preferably, in the microbial preparation, the Saccharomyces eugamiferus CMRC 12Y exists in the form of viable bacteria.

[0014] In a third aspect, the present application provides a preparation method of the above-mentioned microbial preparation, which comprises the step of culturing the Saccharomyces eugamiferus CMRC 12Y.

[0015] Preferably, the temperature of the culture is 28-32℃.

[0016] Preferably, the culture medium used in the culture is YPD culture medium.

[0017] In a fourth aspect, the present application provides the use of the Saccharomyces eugamiferus CMRC 12Y or the microbial preparation in the preparation of microbial protein.

[0018] Preferably, the microbial protein is microbial cell protein.

[0019] In a fifth aspect, the present application provides the use of the Saccharomyces eugamiferus CMRC 12Y or the microbial preparation in the preparation of single-cell protein.

[0020] In a sixth aspect, the present application provides use of the Saccharomyces cariocanus CMRC 12Y or the microbial preparation in the preparation of a yeast culture.

[0021] In a seventh aspect, the present application provides use of the Saccharomyces cariocanus CMRC 12Y or the microbial preparation in the preparation of a food or feed.

[0022] The use according to the fourth to seventh aspects above comprises culturing the Saccharomyces cariocanus CMRC 12Y or the microbial preparation, and collecting the bacterial cells.

[0023] Preferably, the temperature of the culture is 28-32℃.

[0024] Preferably, the culture comprises seed culture and fermentation culture.

[0025] The medium used in the seed culture comprises the following components: glucose 15-25 g / L, peptone 15-25 g / L, and yeast powder 3-7 g / L.

[0026] The medium used in the fermentation culture comprises the following components: glucose 60-70 g / L, corn syrup dry powder 5-15 g / L, urea 1-3 g / L, potassium dihydrogen phosphate 8-12 g / L, and magnesium sulfate 3-5 g / L.

[0027] Preferably, during the fermentation culture, glucose solution and ammonium sulfate solution are fed.

[0028] Preferably, the rotation speed of the fermentation culture is 500-600 rpm, and the aeration amount is 4-6 LPM.

[0029] In an eighth aspect, the present application provides a method for preparing microbial protein, which comprises culturing the Saccharomyces cariocanus CMRC 12Y or the microbial preparation, and collecting the bacterial cells.

[0030] Preferably, the temperature of the culture is 28-32℃.

[0031] Preferably, the culture comprises seed culture and fermentation culture.

[0032] The medium used in the seed culture comprises the following components: glucose 15-25 g / L, peptone 15-25 g / L, and yeast powder 3-7 g / L.

[0033] The medium used in the fermentation culture comprises the following components: glucose 60-70 g / L, corn syrup dry powder 5-15 g / L, urea 1-3 g / L, potassium dihydrogen phosphate 8-12 g / L, and magnesium sulfate 3-5 g / L.

[0034] Preferably, in the fermentation culture process, glucose solution and ammonium sulfate solution are fed.

[0035] Preferably, the rotation speed of the fermentation culture is 500-600 rpm, and the aeration amount is 4-6 LPM.

[0036] The present application has at least the following beneficial effects: the present application provides a dwarf Kazakhstan yeast CMRC 12Y, which has the characteristics of good safety, fast growth, high cell protein content, acid resistance, etc., can realize high biomass growth and high protein expression, has the advantages of green, environmental protection, no pollution, high expression rate, etc., and the produced protein product has good sensory flavor, can be used for the preparation of microbial protein related products, and has important application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0038] Figure 1 Growth curves of CMRC 12Y strain in Example 1 of the present application at different pH values. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application more clear, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0040] The control strain CGMCC No. 18725 involved in the following examples was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 23, 2019, and was named Saccharomyces cerevisiae Saccharomyces cerevisiae , with the preservation number of CGMCC No. 18725. The strain has been disclosed in Chinese patent CN202011257450.0.

[0041] In the following examples, the composition of YPD liquid medium is (1L): yeast extract 10.0g, peptone 20.0g, glucose 20.0g, pH 6.5.

[0042] The composition of the PDA solid medium is (1 L): potato infusion powder 5.0 g, glucose 20.0 g, agar 20.0 g, chloramphenicol 0.1 g.

[0043] Example 1 Obtaining of the yeast strain CMRC 12Y From the naturally fermented white vinegar soup in Guizhou, 25 g of the sample was aseptically weighed, 225 mL of sterile normal saline was added, and after homogenization, different concentrations of dilutions were made. The bacterial suspensions of different dilutions were evenly coated on the PDA solid medium, and after 48 h of culture at 28°C, the colonies were randomly picked from the culture plate with the highest dilution and transferred to the YPD liquid medium to obtain 25 yeast strains.

[0044] 1. Determination of the growth performance of the strain After three purifications, the candidate strains were inoculated in the YPD liquid medium under sterile conditions and placed in a constant temperature incubator at 30°C for 24 hours. During this period, the absorbance value (OD 600 ) at a wavelength of 600 nm was continuously detected. The 10 strains with the highest OD 600 within 24 hours were selected. Among them, the strain CMRC 12Y had the shortest generation time and lag phase, and the most excellent growth performance (Table 1).

[0045] Table 1 Detection of the growth performance of the yeast strain

[0046] 2. Determination of the protein content of the bacterial cells The candidate strains were inoculated in 300 mL of YPD medium at an inoculation amount of 2%, and cultured at 30°C and 150 rpm for 24 h. The bacterial cells were collected by centrifugation after the strain culture was placed at 60°C for 20 min. The bacterial cells were washed twice with sterile water and then vacuum freeze-dried to reduce the water content to less than 5%. The freeze-dried sample was ground thoroughly and then the Kjeldahl method was used to determine the protein content of the bacterial cells. 0.1 g of the freeze-dried sample was weighed into a digestion tube with an accuracy of 0.1 mg. After adding 2 pieces of Kjelt catalyst Cu3.5 (equivalent to 7 g of K2SO4 and 0.8 g of CuSO4·5H2O) into the digestion tube, 10 mL of concentrated sulfuric acid was added, and the sample was soaked. The digestion tube was placed in position, the water pump was turned on, and the sample was digested at 420°C for 60 min and cooled for 15 min. The cooled digestion solution was diluted with 50 mL of distilled water, 25-30 mL of 4% boric acid receiving solution was added to the receiving bottle, and 50 mL of 40% NaOH was added to the digestion solution, which was titrated with 0.5 N hydrochloric acid standard titrant.

[0047] The calculation formula of the protein content in the sample is:

[0048] Protein = Nitrogen x 6.25 T = sample titration of hydrochloric acid consumption (mL) B = blank experiment of hydrochloric acid consumption (mL) N = number of moles or equivalent number of hydrochloric acid, accurate to four decimal places.

[0049] The protein content of the CMRC 12Y strain was determined to be 60%.

[0050] 3. Acid resistance test The CMRC 12Y strain was inoculated at 10 6 CFU / mL into different pH (3.0, 5.0, 7.0) YPD liquid medium, and placed in a constant temperature incubator at 30°C for 40 hours, during which the absorbance (OD 600 ) at wavelength 600 nm was continuously detected. The results showed that (Fig. 1) Figure 1 ) with the decrease of pH value, the growth of the strain was not significantly affected, and it had strong acid resistance.

[0051] 4. Species identification The ITS and 26S rDNA sequences of the CMRC 12Y strain were sequenced, wherein the ITS sequence is shown as SEQ ID NO. 1, and the 26S rDNA sequence is shown as SEQ ID NO. 2. The homology of the strain with Kazachstania exiguua (Kazachstania exigua) is >99%. Kazachstania humilis

[0052] According to the method provided in the operation manual of the VITEK 2 type automatic microorganism identification instrument of BIOMERIEUX company, the colony morphology and physiological and biochemical properties of the strain were identified, and the results are shown in Table 2.

[0053] Table 2 Colony morphology and physiological and biochemical identification of the strain CMRC 12Y

[0054] Through morphological, physiological and biochemical properties and molecular biology identification, the CMRC 12Y strain is Kazachstania exigua, and the strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Postcode 100101) on May 23, 2025, and named Kazachstania exigua, and the preservation number is CGMCC No. 34656. Kazachstania humilis Kazachstania humilis

[0055] ​​​Example 2 Determination of biomass of Saccharomyces cariocanus CMRC 12Y in shake flask After Saccharomyces cariocanus CMRC 12Y was activated overnight, it was inoculated into YPD medium at an inoculation amount of 1-2% (V / V), and then cultured in a shake flask at 30°C for 24 h. The biomass and cell protein content were determined.

[0056] After the bacterial suspension was centrifuged at 4°C for 10 min, the supernatant was discarded, the bacterial cells were washed with sterile water, and the bacterial cells were collected after being centrifuged at 4°C for another 10 min. After being pre-frozen at -20°C for 2 h, the bacterial cells were placed in a vacuum freeze dryer, and vacuum drying was performed (cold trap temperature -45°C, vacuum degree 10-20 Pa) for 22-24 h. When the water content of the bacterial cells was reduced to 2.5-3%, the drying was stopped. The mass of the sample (dry weight) was determined, which was the biomass of the yeast cells. After the freeze-dried sample was fully ground, the Kjeldahl method was used to determine the cell protein content. The specific method is described in Example 1. The results showed that the biomass of CMRC 12Y was 4.4 g / L after being cultured in a shake flask for 24 h, and the cell protein content was 61%.

[0057] Example 3 Determination of biomass of Saccharomyces cariocanus CMRC 12Y in a fermenter Saccharomyces cariocanus CMRC 12Y was inoculated into a seed culture medium at an inoculation amount of 1-2% (V / V), and then cultured at 30°C for 24 h. After that, it was transferred into a fermenter at an inoculation amount of 6-10% (V / V). The initial liquid volume in the fermenter was 3 L. The formula of the seed culture medium was as follows: glucose 20 g / L, peptone 20 g / L, and yeast powder 5 g / L. The formula of the fermentation medium was as follows: food-grade glucose 65 g / L, corn syrup dry powder 10 g / L, urea 1.8 g / L, potassium dihydrogen phosphate 10 g / L, and magnesium sulfate 4 g / L. After 10 h of fermentation, a mixed solution of 50% (w / v) glucose solution and 60 g / L ammonium sulfate solution was used for feeding, and the feeding rate is shown in Table 3. The fermentation temperature was 30°C, the pH was controlled at 5.0 using 25% ammonia water, the rotation speed was 500-600 rpm, and the aeration volume was 4-6 lpm until the end of fermentation. The bacterial cells collected by centrifugation according to the method in Example 2 above were the yeast protein precipitate. After freeze-drying according to the method in Example 2 above, the yeast protein powder was obtained. The mass of the yeast protein powder (dry weight) was the biomass of the yeast. The results showed that the biomass of CMRC 12Y was 50.3 g / L after being cultured in a fermenter for 30 h.

[0058] Table 3 Feeding rate of CMRC 12Y in a fermenter

[0059] The protein content in the sample was detected according to the protein content detection method in Example 1 above, i.e. the protein content in the bacterial cell. The results showed that the protein content in the bacterial cell of CMRC 12Y was 61.7%.

[0060] Example 4 Sensory flavor evaluation of the protein produced by the yeast CMRC 12Y Sensory flavor evaluation: The yeast protein precipitate and the yeast protein powder prepared according to the method in Example 3 were subjected to sensory flavor evaluation. In terms of smell, 5 points were given if there was no unpleasant smell (e.g. earthy smell) and there was aroma, and the points were lower if there was unpleasant smell or the aroma was lighter; in terms of taste, 5 points were given if the taste was smooth and there was no unpleasant taste, and the points were lower if the taste was rough and there were sand-like feeling or the unpleasant taste was heavier (e.g. alkaline taste); in terms of overall acceptance, 5 points were given if it was acceptable, and the points were lower if it was less acceptable. The results were evaluated mainly by olfactory sensory evaluation of people of different genders and different ages. The number of people participating in the sensory evaluation in each group was 10, the male to female ratio was 1:1, and the age was between 25 and 45. The results are shown in Table 4. The smell of the yeast protein precipitate, the taste of the protein powder and the overall acceptance of CMRC 12Y were all higher than those of the control Saccharomyces cerevisiae strain. Therefore, the protein produced by CMRC 12Y is palatable and easy to be accepted by people, and it has important application prospects as a raw material for food.

[0061] Table 4 Sensory flavor evaluation scores of different yeast proteins

[0062] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Dwarf Kazakhstan yeast ( Kazachstania humilis CMRC 12Y, characterized in that, It is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 34656.

2. A microbial preparation, characterized in that, The microbial preparation comprises the yeast Saccharomyces cariocanus (Saccharomyces exiguus) according to claim 1 Kazachstania humilis ) CMRC 12Y.

3. The method for preparing the microbial preparation according to claim 2, characterized in that, The method comprises the step of culturing the dwarf Kazachstania yeast of claim 1 Kazachstania humilis ) CMRC 12Y.

4. The method of claim 3, wherein, The temperature of the culture is 28-32℃.

5. The use of the microorganism preparation of the microorganism of the microorganism of claim 1 (Kazachstania exiguua) CMRC 12Y or claim 2 for the production of microbial proteins. Kazachstania humilis ) CMRC 12Y or claim 2 for the production of microbial proteins.

6. The use of the microorganism preparation of the microorganism of the microorganism of claim 1 (Kazachstania exigua) CMRC 12Y or claim 2 for the production of single-cell protein. Kazachstania humilis ) CMRC 12Y or claim 2 for the production of single-cell protein.

7. The reduced Kazachstania yeast of claim 1, Kazachstania humilis ) Use of CMRC 12Y or the microbial preparation of claim 2 for the preparation of a yeast culture.

8. The reduced Kazachstania yeast of claim 1, Kazachstania humilis ) Use of CMRC 12Y or the microbial preparation of claim 2 for the preparation of food or feed.

9. A method for producing a microbial protein, characterized by, The method comprises cultivation of the dwarf Kazakhstan yeast (Saccharomyces Kazachstania humilis ) CMRC 12Y or the microbial preparation of claim 2, collecting the cells.

10. The method of claim 9, wherein, The temperature of the culture is 28-32℃.

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