Pichia pastoris CMRC 13Y with high protein yield and application of pichia pastoris CMRC 13Y

Abstract

The invention relates to the technical field of microorganisms, and provides pichia pastoris CMRC 13Y with high protein yield and application of the pichia pastoris CMRC 13Y. The pichia pastoris CMRC 13Y is separated from naturally-fermented dried milk cakes, the classification name of the pichia pastoris CMRC 13Y is Pichia kudriavzevii, and the preservation number of the pichia pastoris CMRC 13Y is CGMCC No.34655. The pichia pastoris CMRC 13Y has high acid resistance, and the growth of the pichia pastoris CMRC 13Y is not obviously influenced along with the reduction of the pH value within the pH value range of 3.0-7.0; a yeast protein product prepared from the strain CMRC 13Y is subjected to sensory flavor determination, has no earthy smell in the aspect of smell, has fragrance, is fine and smooth in mouth feel, and has no unpleasant taste; the strain can achieve high-biomass growth and high-protein expression, has the advantages of being green, environmentally friendly, high in protein yield and the like, can be used for preparing microbial protein related products and is wide in application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Pichia pastoris strain CMRC13Y with high protein yield and its application. BACKGROUND

[0002] Protein is one of the most important nutrients for human and animal life activities. The current acquisition of food and feed protein mainly relies on traditional agricultural farming and planting, which requires a large amount of land and water resources, and causes serious environmental pollution. Under this situation, it is an inevitable trend to explore alternative protein and achieve efficient production of alternative protein to ensure the sustainable supply of protein.

[0003] Microbial cell protein (i.e. single cell protein) is one of the ideal sources of alternative protein. Among various microorganisms, yeast has a long history of application in food and animal feed, and has a relatively high protein content in cell dry weight, which is suitable for producing microbial cell protein. In this study, a strain of Pichia kudriavzevii (CMRC13Y) with excellent growth performance and high cell protein content was screened from traditional natural fermented dairy products, which can provide an excellent strain for protein production. At present, Pichia kudriavzevii is mainly applied in food fermentation such as wine, fermentation of corn deep processing by-products, production of perfume ingredients, animal intestinal health, preparation of cosmetic moisturizing raw materials, acid soil improvement, etc. Its application in microbial cell protein production has not been reported. Pichia kudriavzevii

[0004] ​CN202311351680.7 discloses a strain of acid-tolerant Pichia kudriavzevii GAAS-JG-1 and its application in preparing high-acidity fruit fermented wine. The strain can increase the total ester content of high-acidity fruit fermented wine. CN202011584482.1 discloses a strain of Pichia kudriavzevii and its application. The isolated Pichia kudriavzevii Y50 can shorten the production cycle of pickled chili peppers and improve product safety and flavor quality. CN202411727950.4 discloses a strain of acid-tolerant Pichia kudriavzevii and its application in fermenting corn deep processing by-products. The strain can reduce the content of reducing sugar in corn soaking water, significantly increase the content of crude protein in fermented spouted corn skin, and improve the spouting process of corn syrup. CN202310255105.0 discloses Pichia kudriavzevii and its use. The strain can achieve a 2-octanone yield of 46.25 mg / L, expanding the natural source of 2-octanone perfume. CN202310115826.1 discloses Pichia kudriavzevii with intestinal health protection function and its application. The strain can improve the growth performance of weaned piglets, reduce the diarrhea rate, and protect intestinal health. CN202410806125.7 discloses Pichia kudriavzevii and its application. The strain can obtain a bacteria solution with moisturizing activity after fermentation culture, which is used for preparing moisturizing materials. CN201710767138.8 discloses the application of Pichia kudriavzevii in soil and a method thereof. After inoculation into acidic soil and natural propagation culture for 30-90 days, the strain can increase the soil pH value and improve the soil structure. SUMMARY

[0005] The purpose of the present application is to provide a Pichia kudriavzevii strain CMRC 13Y with high protein yield and its application.

[0006] To achieve the purpose of the present application, in the first aspect, the present application provides a Pichia kudriavzevii strain CMRC 13Y with high protein yield isolated from naturally fermented beancurd, which is classified and named as Pichia kudriavzevii (Pichia kudriavzevii) Pichia Pichia kudriavzevii ), which has been preserved in the General Microbiological Center of China Microbial Strain Preservation Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences, Postcode 100101, on May 23, 2025, with the preservation number CGMCC No. 34655 and the preservation date.

[0007] In the second aspect, the present application provides a microbial agent containing the Pichia kudriavzevii strain CMRC 13Y.

[0008] In the third aspect, the present application provides the application of the Pichia kudriavzevii strain CMRC 13Y in microbial cell protein production.

[0009] In a fourth aspect, the present application provides a method for producing microbial cell protein, wherein the Pichia pastoris CMRC 13Y is fermented, and the cell protein is collected from the fermentation product.

[0010] Further, the Pichia pastoris is inoculated into YPD liquid medium for fermentation culture.

[0011] Further, the fermentation culture is performed at a temperature of 28-32℃.

[0012] In a specific embodiment of the present application, the seed liquid of the Pichia pastoris CMRC 13Y is inoculated into the medium at an inoculation amount of 1% v / v, and is shaken at a temperature of 28-32℃ and a speed of 100-150 rpm for 24-30 h (shaken at a temperature of 30℃ and a speed of 150 rpm for 24 h).

[0013] Under the above culture conditions, the harvested yeast biomass and protein content are high, thereby improving the efficiency of yeast cell protein production.

[0014] In the present application, the cell content of the seed liquid is 10 6 -10 8 CFU / mL.

[0015] Further, the fermentation is performed at a pH value of 3.0-7.0 (such as 3.0, 5.0 or 7.0).

[0016] The Pichia kudriavzevii CMRC 13Y of the present application has the characteristics of good safety, fast growth, high cell protein content, etc.; at the same time, it has strong acid resistance, and the growth of the strain is not significantly affected as the pH value decreases within the range of 3.0-7.0; the yeast protein product prepared from the strain CMRC 13Y is subjected to sensory flavor determination, and has no fishy smell and has aroma in terms of smell, and has delicate and smooth taste and no unpleasant taste in terms of taste; the strain can realize high-biomass growth and high-protein expression, has the advantages of green, environmental protection, high protein yield, etc., and can be used for the preparation of microbial protein related products, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Fig. 2 shows the growth curve of the CMRC 13Y strain in YPD liquid medium at different pH values in a preferred embodiment of the present application. DETAILED DESCRIPTION

[0018] The present application aims to provide the Pichia kudriavzevii CMRC 13Y, which can be applied to microbial protein production.

[0019] The application provides a Pichia kudriavzevii CMRC 13Y, which is obtained from naturally fermented beancurd, has fast growth, high cell protein content and good flavor, and is suitable for being used as a microbial cell protein production strain. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 23, 2025, and is classified and named as Pichia kudriavzevii (P. kudriavzevii) CMRC 13Y, with a preservation number of CGMCC No. 34655. Pichia kudriavzevii

[0020] The application provides an application of the Pichia kudriavzevii in microbial protein production.

[0021] Further, the fermentation culture condition is 30 DEG C.

[0022] The following examples are used to illustrate the application, but are not used to limit the scope of the application. If not specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.

[0023] The composition of the YPD liquid culture medium used in the following examples is as follows (1L): yeast extract 10.0 g, peptone 20.0 g, glucose 20.0 g.

[0024] The composition of the PDA solid culture medium is as follows (1L): potato infusion powder 5.0 g, glucose 20.0 g, agar 20.0 g, chloramphenicol 0.1 g.

[0025] The control strain CGMCC No. 18725 involved in the following examples has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 23, 2019, and is classified and named as Saccharomyces cerevisiae, and the strain has been disclosed in the patent CN202011257450.0.

[0026] Example 1: Obtaining of Pichia kudriavzevii CMRC 13Y 25 g of a sample is aseptically weighed from the naturally fermented beancurd in Huoluoguoer City, 225 mL of sterile normal saline is added, and the mixture is uniformly homogenized and then diluted at different concentrations. The bacterial suspensions at different dilutions are uniformly coated on PDA solid culture medium, and after 28 DEG C culture for 48 h, colonies are randomly picked from the culture plate at the highest dilution and transferred to YPD liquid culture medium, to obtain 25 strains of yeast.

[0027] 1. Strain growth performance determination ​After 3 times of purification, the candidate strains were inoculated into YPD liquid medium under sterile conditions at an inoculation amount of 1%, and were placed in a microbial growth curve instrument for 24 hours of culture at 30°C, during which the absorbance value (OD) at a wavelength of 600 nm was detected every half hour. 600 The OD 600 of the highest strain within 24 hours was selected, i.e., CMRC 13Y. Although CMRC 13Y had a longer lag phase compared with the control strain Saccharomyces cerevisiae CGMCC No. 18725, the OD 600 of the strain was higher than that of the control strain after 24 hours of growth, indicating that the strain had higher biomass production potential (Table 1).

[0028] Table 1 Growth performance detection of different yeast strains

[0029] 2. Determination of protein content of the bacterial cells The candidate strains were inoculated into 300 mL of YPD medium at an inoculation amount of 1%, and were cultured at 30°C and 150 rpm for 24 hours of shaking culture. The bacterial cell culture was allowed to stand at 60°C for 20 minutes, and then was centrifuged to collect the bacterial cells. The bacterial cells were washed twice with sterile water, and then were subjected to vacuum freeze-drying to reduce the water content to less than 5%. The freeze-dried sample was fully ground, and then was subjected to determination of the protein content by the Kjeldahl method. 0.1 g was weighed into a digestion tube with an accuracy of 0.1 mg. After 2 pieces of Cu3.5 catalyst were added into the digestion tube (equivalent to 7 g of K2SO4 and 0.8 g of CuSO4·5H2O), 10 mL of concentrated sulfuric acid was added, and the sample was immersed. The digestion tube was placed in position, the water pump was opened, and the sample was digested at 420°C for 60 minutes and was cooled for 15 minutes. The cooled digestion solution was diluted with 50 mL of distilled water, 25-30 mL of 4% boric acid receiving solution was added into a receiving bottle, and 50 mL of 40% NaOH was added into the digestion solution, which was titrated with 0.1 N hydrochloric acid standard titration solution.

[0030] The calculation formula of the protein content in the sample is as follows:

[0031] Protein % = Nitrogen % × 6.25 T = amount of hydrochloric acid consumed in titration of the sample (ml); B = amount of hydrochloric acid consumed in blank experiment (ml); N = number of moles or equivalent number of hydrochloric acid (accurate to four decimal places); It was determined that the protein content of CMRC 13Y was 60.3%.

[0032] 3. Acid resistance test The CMRC 13Y strain was inoculated into YPD liquid medium at a concentration of 10 6 CFU / mL at different pH values (3.0, 5.0, and 6.0), and was cultured at 30°C for 48 hours in a microbial growth curve instrument. The absorbance (OD 600 ) at a wavelength of 600 nm was detected every half hour during the culture. Figure 1 As shown in Table 1, the growth of the strain was not significantly affected by the decrease in pH value, indicating that the strain has strong acid resistance.

[0033] 4. Species identification The 26S rDNA sequence (SEQ ID NO: 1) and ITS sequence (SEQ ID NO: 2) of the strain were sequenced. The 26S rDNA sequence of the strain has a homology of 99.82% with Pichia kudriavzevii KC481678.1, and the ITS sequence of the strain has a homology of 99.37% with Pichia kudriavzevii KU987877.1. Therefore, the homology of the strain with Pichia kudriavzevii is >99%. Pichia Pichia kudriavzevii

[0034] The colony morphology and physiological and biochemical properties of the strain were identified according to the method provided in the operation manual of the VITEK 2 full-automatic microbial identification instrument of BIOMERIEUX. The results are shown in Table 2.

[0035] Table 2. Colony morphology and physiological and biochemical identification of the strain CMRC 13Y

[0036] According to morphological, physiological and biochemical properties, and molecular biology identification, the CMRC 13Y strain is Pichia kudriavzevii. Pichia Pichia kudriavzevii The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Huayuancun, Beijing, China, and the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China) on May 23, 2025, and is named Pichia kudriavzevii. Pichia kudriavzevii The preservation number is CGMCC No. 34655.

[0037] Example 2. Determination of the biomass of Pichia kudriavzevii CMRC 13Y in a shake flask Pichia kudriavzevii CMRC 13Y was inoculated into YPD medium at an inoculation amount of 1-2% (V / V), and was cultured at 30°C for 24 hours in a shake flask. The biomass and protein content of the strain were determined.

[0038] ​After centrifugation of the bacterial suspension at 4°C for 10 min, the supernatant was discarded, and the bacterial cells were washed with sterile water. After centrifugation at 4°C for another 10 min, the bacterial cells were collected, pre-frozen at -20°C for 2 h, and then dried in a vacuum freeze dryer (cold trap temperature -45°C, vacuum degree 0.1-0.2 mbar) 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. After 24 h of cultivation of CMRC 13Y in a shake flask, the biomass was 5.1 g / L, and the protein content of the bacterial cells was 60%.

[0039] Example 3 Determination of the biomass of Pichia pastoris CMRC 13Y in a fermenter Pichia pastoris CMRC 13Y was inoculated into a seed culture medium at an inoculation amount of 1-2% (V / V). After 24 h of cultivation at 30°C, the obtained seed solution (bacterial content about 10 8 CFU / mL) 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 seed culture medium contained 20 g / L glucose, 20 g / L proteose peptone, and 5 g / L yeast powder. The fermentation medium contained 50 g / L food-grade glucose, 8 g / L corn syrup dry powder, 1.8 g / L urea, 10 g / L potassium dihydrogen phosphate, 4 g / L magnesium sulfate, 10 mg / L zinc sulfate, 12 mg / L iron sulfate, and 2 mg / L manganese sulfate. 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. During the 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 was as follows: from the 7th hour to the 10th hour after the start of fermentation, the feeding was performed at a constant rate of 15 mL / h; from the 11th hour to the 18th hour after the start of fermentation, the feeding was performed at a constant rate of 30 mL / h; from the 19th hour to the 22nd hour after the start of fermentation, the feeding was performed at a constant rate of 46 mL / h; from the 23rd hour to the 26th hour after the start of fermentation, the feeding was performed at a constant rate of 55 mL / h; and from the 27th hour to the 30th hour after the start of fermentation, the feeding was performed at a constant rate of 41 mL / h.

[0040] According to the above-mentioned method for determining the mass of the sample (dry weight) after freeze-drying in Example 2, the mass was the biomass of the yeast. After 30 h of cultivation of CMRC 13Y in a fermenter, the biomass was 50.8 g / L. According to the above-mentioned method for determining the protein content, the protein content of the sample was the protein content of the bacterial cells. The protein content of CMRC 13Y was 59.5%.

[0041] Example 4 Sensory flavor evaluation of the protein produced by Pichia pastoris CMRC 13Y Sensory flavor evaluation: The prepared yeast protein product was subjected to sensory flavor determination. In terms of smell, no unpleasant smell (e.g. earthy smell) and with aroma of 5 points, the lighter the unpleasant smell or aroma, the lower the score; in terms of taste, smooth and delicate taste without unpleasant taste of 5 points, the heavier the unpleasant taste (e.g. alkaline taste) or the coarser the taste, the lower the score; in terms of overall acceptability, acceptable of 5 points, the lower the acceptance, the lower the score. The results were mainly evaluated by different genders and different age groups through olfactory sensory evaluation (Table 3).

[0042] Table 3 Sensory flavor evaluation of different yeast proteins

[0043] Although the present application has been described in detail with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A high-protein Pichia pastoris strain CMRC 13Y, characterized in that, It is Kudria zweibichi yeast ( Pichia kudriavzevii (The accession number is CGMCC No. 34655).

2. A microbial agent, characterized in that, Contains the Pichia pastoris CMRC 13Y as described in claim 1.

3. The application of Pichia pastoris CMRC 13Y as described in claim 1 in the production of microbial cell protein.

4. A method for producing microbial cell protein, characterized in that, Fermenting the Pichia pastoris CMRC 13Y as described in claim 1, and collecting cell protein from the fermentation product.

5. The method according to claim 4, characterized in that, The Pichia pastoris CMRC 13Y was inoculated into YPD liquid medium for fermentation culture.

6. The method according to claim 5, characterized in that, The fermentation culture temperature is 28-32℃.

7. The method according to claim 6, characterized in that, The seed culture of Pichia pastoris CMRC13Y was inoculated into the culture medium at an inoculation rate of 1% v / v, and cultured with shaking at 28-32℃ and 100-150 rpm for 24-30 h.

8. The method according to claim 7, characterized in that, The bacterial content of the seed liquid is 10. 6 -10 8 CFU / mL.

9. The method according to any one of claims 4-8, characterized in that, Fermentation is carried out under conditions of pH 3.0-7.

0.

10. The method according to claim 9, characterized in that, Fermentation is carried out under conditions of pH 3.0, 5.0, or 7.0.

Images