Candida utilis BYC-YP-118 and application thereof

Abstract

The invention discloses candida utilis BYC-YP-118 and application thereof, the candida utilis BYC-YP-118 is preserved in China Center for Type Culture Collection, the preservation date is June 23, 2025, and the preservation number is CCTCC NO: M20251460. The content of the mycoprotein is more than or equal to 60% and can reach 67.32% to the maximum, the mycoprotein is rich in valine, lysine, threonine, tryptophan, phenylalanine, methionine, isoleucine, leucine and other essential amino acids for a human body, the nutritional value of the yeast protein is improved, the yeast protein can be used as a high-quality feed additive to effectively replace imported soybean meal and fish meal, the feed cost is reduced, and the economic benefit is increased. Sustainable development of animal husbandry is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial application, and particularly relates to a Candida utilis BYC-YP-118 and application thereof. BACKGROUND

[0002] With the development of animal husbandry, the necessary protein feed for livestock is increasingly scarce, and a large amount of protein feed such as soybean meal and fish meal needs to be imported from abroad every year, resulting in a substantial increase in feed costs and affecting food security. Single-cell protein (SCP) is a high-quality protein substitute, which is the first product of the fermentation process and can be used as a feed additive. SCP has the advantages of short production cycle, high production efficiency, production not restricted by seasons and climate, and small fermentation production area. Yeast is a commonly used strain for producing single-cell protein, and the protein content in yeast dry weight is about 40% to 60%. The protein contains various essential amino acids and is a high-quality protein additive. Candida utilis is a safe organism (GRAS) certified by the US FDA, which has a fast growth rate, high single-cell protein content, and is rich in various nutrients such as nucleotides, enzymes, B vitamins, and oligosaccharides. It is also an extremely important microorganism in industry and can be used to produce various biological products.

[0003] In the prior art, the application of Candida utilis in yeast protein production faces many challenges. For example, CN117264787A discloses a method for producing Candida utilis protein, although the cell density is high, the protein content is only about 30%, which is relatively low and cannot meet the demand for high-efficiency protein feed. This limits the potential of Candida utilis as a protein substitute. CN118620753A shows a route for producing yeast protein by modifying Saccharomyces cerevisiae. Although this method provides a reference path for genetic modification, it is targeted at Saccharomyces cerevisiae rather than Candida utilis, and the modification process is complex and has limited applicability. Similarly, the literature by Yu Jing et al. shows a mutagenesis method for Candida utilis, which provides a starting strain and technical reference for screening high-yield strains, but does not solve the problems of low protein content and fermentation scale-up. CN115677818A and CN116925938A respectively show a yeast protein purification production process. Although these processes can extract protein, the production process is long and involves multiple steps of separation and purification, resulting in low recovery rate, increased production cost and operation difficulty.

[0004] In summary, the existing Candida utilis protein production process has the problems of complex production process and low protein content, which limits the application of Candida utilis in yeast protein. SUMMARY

[0005] The present application aims to overcome the defects of the prior art and provide a Candida utilis BYC-YP-118.

[0006] Another object of the present application is to provide a preparation method of the Candida utilis BYC-YP-118.

[0007] The technical solution of the present application is as follows:

[0008] A Candida utilis BYC-YP-118, which is preserved in the China Center for Type Culture Collection on June 23, 2025, and has a preservation number of CCTCC NO: M20251460.

[0009] The Candida utilis BYC-YP-118 is used for preparing a yeast protein.

[0010] In a preferred embodiment of the present application, the yeast protein is in a dry powder form and contains valine, lysine, threonine, tryptophan, phenylalanine, methionine, isoleucine and leucine.

[0011] A fermentation production method of a yeast protein, which uses the Candida utilis BYC-YP-118.

[0012] In a preferred embodiment of the present application, the method comprises the following steps:

[0013] (1) The Candida utilis BYC-YP-118 stored at -80℃ is streaked on a YPD solid culture medium and cultured at 30℃ for 24-32h;

[0014] (2) A colony with a diameter of about 3-5mm on the plate is picked and inoculated in 50mL of YPD liquid culture medium, which is cultured at 30℃ and 160rpm for 48h to obtain a first-stage seed liquid;

[0015] (3) The first-stage seed liquid is inoculated in 200mL of fermentation medium CM7 at an inoculation amount of 2-3%, which is cultured at 30℃ and 160rpm for 16h to obtain a second-stage seed liquid;

[0016] (4) The second-stage seed liquid is transferred to a 5L fermenter containing 2L of fermentation medium CMX9 or CMX11 at an inoculation amount of 10%, and 600g / L of glucose and 20% of ammonia water solution or 500g / L of glucose and 125g / L of urea solution are added, which is cultured at a temperature of 28℃, a rotation speed of 500rpm, a ventilation amount of 10vvm and a pH of 4.5-5 for 24h to obtain a third-stage seed liquid;

[0017] (5) the third level seed liquid is transferred into 50L fermenter with 20L fermentation medium CMX11 and 600g / L glucose and 20% ammonia water solution is added, and the fermentation is carried out at 28℃, 500rpm, 10vvm and pH 4.5-5 for 48h;

[0018] (6) the bacteria are collected by a tubular centrifuge and are treated by spray drying to obtain yeast protein.

[0019] Further preferably, the fermentation medium CM7 comprises glucose 100g / L, corn syrup dry powder 15g / L, industrial grade ammonium sulfate 25g / L, industrial grade diammonium hydrogen phosphate 5g / L, magnesium sulfate 1g / L, zinc sulfate 0.1g / L, vitamin B1 0.06g / L, vitamin B6 0.02g / L, vitamin B7 0.003g / L.

[0020] Further preferably, the fermentation medium CMX9 comprises industrial glucose 65g / L, industrial corn powder 10g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 8g / L, zinc sulfate heptahydrate 20mg / L, ferrous sulfate 12mg / L, manganese sulfate 2mg / L, copper sulfate 2mg / L, calcium chloride 0.555g / L.

[0021] Further preferably, the fermentation medium CMX11 comprises industrial glucose 65g / L, industrial corn powder 10g / L, potassium dihydrogen phosphate 10g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 8g / L, zinc sulfate heptahydrate 20mg / L, ferrous sulfate 12mg / L, manganese sulfate 2mg / L, copper sulfate 2mg / L, calcium chloride 0.555g / L.

[0022] The present application has the following advantages:

[0023] 1. The content of the bacterial protein of the present application is ≥60%, and the highest can reach 67.32%, which is rich in valine, lysine, threonine, tryptophan, phenylalanine, methionine, isoleucine and leucine and other essential amino acids for human body, improves the nutritional value of the yeast protein, can be used as high-quality feed additive, effectively replaces imported soybean meal and fish meal, reduces the feed cost, and promotes the sustainable development of the livestock and poultry industry.

[0024] 2. The present application provides a simple fermentation process of yeast protein: after plate and seed culture, inoculation is carried out in the fermentation medium, and the culture is carried out at 28-32℃, and large-scale fermentation is carried out through two-stage amplification (such as 5L and 50L tank) to obtain a large amount of bacteria.

[0025] 3、The fermentation period of the present application is short (within 48h), the wet weight of the bacterial body reaches 441-450g / L, the dry weight is 90-100g / L, the yield is high, the process is not affected by season and climate, the occupation is small, the operation is simple, the recovery rate is high, and the present application has good industrial amplification production potential.

[0026] 4、The application of the strain and process of the present application can improve the production efficiency of single-cell protein, reduce environmental pollution, and support the development of food safety and green breeding. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is the colony morphology of the Candida humicola strain obtained by mutagenesis and screening in Example 1 of the present application.

[0028] Figure 2 The figure is the wet weight and residual sugar and ethanol data of the Candida humicola fermentation culture (5L) in Example 2 of the present application.

[0029] Figure 3 The figure is the wet weight and residual sugar and ethanol data of the Candida humicola fermentation culture (5L) in Example 3 of the present application (48h).

[0030] Figure 4 The figure is the component detection result of the yeast dry powder obtained in Example 4 of the present application. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further described and explained in the following specific embodiments in combination with the drawings.

[0032] The formulations of the culture media in the following examples are as follows:

[0033] The YPD solid culture medium includes 20g / L of proteose peptone, 10g / L of yeast extract, 10g / L of glucose, and 15g / L of agar powder.

[0034] The YPD liquid culture medium is the same as the YPD solid culture medium except that it does not contain agar powder.

[0035] The fermentation medium CM7 includes 100g / L of glucose, 15g / L of corn syrup dry powder, 25g / L of industrial grade ammonium sulfate, 5g / L of industrial grade diammonium hydrogen phosphate, 1g / L of magnesium sulfate, 0.1g / L of zinc sulfate, 0.06g / L of vitamin B1, 0.02g / L of vitamin B6, and 0.003g / L of vitamin B7.

[0036] The fermentation medium CMX9 includes 65g / L of industrial glucose, 10g / L of industrial corn powder, 10g / L of potassium dihydrogen phosphate, 8g / L of magnesium sulfate heptahydrate, 20mg / L of zinc sulfate heptahydrate, 12mg / L of ferrous sulfate, 2mg / L of manganese sulfate, 2mg / L of copper sulfate, and 0.555g / L of calcium chloride.

[0037] The fermentation medium CMX11 includes industrial glucose 65 g / L, industrial corn flour 10 g / L, potassium dihydrogen phosphate 10 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 8 g / L, zinc sulfate heptahydrate 20 mg / L, ferrous sulfate 12 mg / L, manganese sulfate 2 mg / L, copper sulfate 2 mg / L, calcium chloride 0.555 g / L.

[0038] Each of the above media is sterilized at 115°C high temperature and high pressure and then stored.

[0039] Example 1

[0040] (1) Starting strain screening: Dilute the leachate of the rotten fruits and vegetables, streak on YPD solid medium, and culture at 30°C for 48 h. Select the colonies that are round, slightly white and opaque, and smooth and moist on the surface, inoculate in YPD liquid medium, and culture at 30°C for 48 h. Select the strains with wine smell, and name them as BYC-YP-1.

[0041] (2) ARTP mutagenesis:

[0042] a. Preparation of bacterial suspension: inoculate the BYC-YP-1 strain in YPD liquid medium and culture to the logarithmic phase. After washing with sterile water for 3-4 times, dilute to OD600 of 1.0, and take 1 mL of bacterial suspension into an EP tube.

[0043] b. Sample preparation: in a clean bench, take 10 μL of diluted bacterial solution and evenly coat it on a sterile metal slide for mutagenesis, and then place it in a sterile culture dish.

[0044] c. Mutagenesis treatment: place the treated slide in the ARTP mutagenesis instrument, set the parameters to make the lethality rate 90%. The irradiation dose is 160 Gy, the power is 160 W, the ventilation volume is 10 SLM, and the mutagenesis time is 30 s. At the same time, set up a blank control group with mutagenesis time of 0 s.

[0045] (3) Plate coating: dilute the bacterial solution after mutagenesis (10 -6 -10 -8 ), take 40-60 μL of bacterial solution and drop it on YPD solid medium, evenly coat it, and culture at 30°C for 36-48 h.

[0046] (4) Preliminary screening: compare the colony diameters with the control group, select the strains with diameter difference within 5% for rescreening, and select a total of 8 strains.

[0047] (5) Rescreening: pick single colony and inoculate into 5 mL YPD liquid medium, incubate at 30℃, 160 rpm for 16-24 h; then inoculate the bacterial liquid into 50 mL YPD liquid medium, continue to incubate under the same conditions for 16-24 h; then take 5 mL bacterial liquid and inoculate into 200 mL fermentation medium CM7, incubate for 24-48 h. Then centrifugal collect the bacterial cells, dry at 103±2℃ for 16 h, and detect the protein content according to the standard of GB / T 6432-2018. Among them, the strain with the highest protein content is 67.32%, named BYC-YP-118 (colony photo as shown in Figure 1 Table 1 No. 8 of the following Table 1).

[0048] Table 1

[0049] Number Colony size mm Protein content % Starting strain 5.11 54 1 5.44 65.34 2 5.45 57.22 3 5.47 66.72 4 5.47 65.81 5 5.43 65.45 6 5.45 65.41 7 5.5 59.52 8 5.5 67.32

[0050] Example 2

[0051] (1) The Candida utilis strain BYC-YP-118 stored at -80℃ was streaked on YPD solid medium and incubated at 30℃ for 24 h.

[0052] (2) A colony with a diameter of about 4 mm on the plate was picked and inoculated into 50 mL YPD liquid medium, incubated at 30℃, 160 rpm for 48 h to obtain a primary seed liquid.

[0053] (3) The primary seed liquid was inoculated into 200 mL fermentation medium CM7 at an inoculation amount of 2-3%, and incubated at 30℃, 160 rpm for 16 h to obtain a secondary seed liquid.

[0054] (4) The secondary seed liquid was inoculated into a 5L fermenter (containing 2L fermentation medium CMX9) at an inoculation amount of 10%, and 500g / L glucose and 125g / L urea solution were added. Incubate at 30℃, 500 rpm, 10vvm, pH 4.5-5 for 48 h, the wet weight of the bacterial cells is 441g / L, and the dry weight is 90.2g / L (see Figure 2 ). Then centrifugal collect the bacterial cells at 10000 rpm, dry at 103±2℃, and the protein content of the dry bacterial cells is 60.13%.

[0055] Example 3

[0056] (1) The Candida utilis strain BYC-YP-118 stored at -80℃ was streaked on YPD solid medium and incubated at 30℃ for 24-32 h.

[0057] (2) Take a colony with a diameter of about 4 mm from the plate and inoculate it into 50 mL of YPD liquid medium. Incubate at 30°C and 160 rpm for 48 h to obtain a primary seed solution.

[0058] (3) Inoculate the primary seed solution into 200 mL of fermentation medium CM7 at an inoculation amount of 2-3%. Incubate at 30°C and 160 rpm for 16 h to obtain a secondary seed solution.

[0059] (3) Inoculate the secondary seed solution into a 5L fermenter (containing 2L of fermentation medium CMX11) at an inoculation amount of 10%, and add 600g / L of glucose and 20% ammonia water solution. Incubate at 28°C, 500 rpm, 10vvm, and pH 4.5-5 for 48 h. The wet weight of the bacterial cells reaches 450g / L, and the dry weight reaches 100g / L (see Figure 3 ). Then, centrifuge the bacterial cells at 10000 rpm, and dry them at 103±2°C. The protein content of the dry bacterial cells is 61.15%.

[0060] Example 4

[0061] (1) Streak the Candida utilis strain BYC-YP-118 stored at -80°C on YPD solid medium, and incubate at 30°C for 24-32 h.

[0062] (2) Take a colony with a diameter of about 4 mm from the plate and inoculate it into 50 mL of YPD liquid medium. Incubate at 30°C and 160 rpm for 48 h to obtain a primary seed solution.

[0063] (3) Inoculate the primary seed solution into 200 mL of fermentation medium CM7 at an inoculation amount of 2-3%. Incubate at 30°C and 160 rpm for 16 h to obtain a secondary seed solution.

[0064] (4) Inoculate the secondary seed solution into a 5L fermenter (containing 2L of fermentation medium CMX11) at an inoculation amount of 10%, and add 600g / L of glucose and 20% ammonia water solution. Incubate at 28°C, 500 rpm, 10vvm, and pH 4.5-5 for 24 h to obtain a tertiary seed solution.

[0065] (5) Inoculate the tertiary seed solution into a 50L fermenter (containing 20L of fermentation medium CMX11) at an inoculation amount of 10%, and add 600g / L of glucose and 20% ammonia water solution. Incubate at 28°C, 500 rpm, 10vvm, and pH 4.5-5 for 48 h. Then, collect the bacterial cells by a tubular centrifuge, and spray-dry them to obtain dry yeast powder with a protein content of 60.2% (see Figure 4The application is illustrated in the accompanying drawings, of which:

[0066] The above description is only the preferred embodiment of the application, and cannot limit the scope of the application. Any equivalent changes and modifications according to the patent scope and content of the application should still be within the scope of the application.

Claims

1. A type of Candida utilis BYC-YP-118, characterized in that: It is deposited at the China Center for Type Culture Collection, with the deposit date being June 23, 2025, and the accession number being CCTCC NO: M20251460.

2. The application of the Candida utilis BYC-YP-118 as described in claim 1 in the preparation of yeast protein.

3. The application as described in claim 2, characterized in that: The yeast protein is in the form of a dry powder and contains valine, lysine, threonine, tryptophan, phenylalanine, methionine, isoleucine, and leucine.

4. A method for fermenting yeast protein, characterized in that: The *Candida utilis* BYC-YP-118 as described in claim 1 was used.

5. The fermentation production method as described in claim 4, characterized in that: Includes the following steps: (1) Streaking of Candida utilis BYC-YP-118 stored at -80℃ onto YPD solid medium and culturing at 30℃ for 24-32h; (2) Pick colonies with a diameter of about 3-5 mm on the plate, inoculate them into 50 mL of YPD liquid medium, and incubate them at 30℃ and 160 rpm for 48 h to obtain the first-stage seed liquid; (3) Inoculate the primary seed culture at an inoculation rate of 2-3% into 200mL of fermentation medium CM7 and culture at 30℃ and 160rpm for 16h to obtain the secondary seed culture; (4) The secondary seed culture was transferred to a 5L fermenter at an inoculation rate of 10%. The fermenter contained 2L of fermentation medium CMX9 or CMX11, and 600g / L glucose and 20% ammonia solution or 500g / L glucose and 125g / L urea solution were added. The culture was carried out for 24h at a temperature of 28℃, a rotation speed of 500rpm, an aeration rate of 10vvm, and a pH of 4.5-5 to obtain the tertiary seed culture. (5) The third-level seed liquid was transferred to a 50L fermenter at an inoculation rate of 10%. The fermenter contained 20L of fermentation medium CMX11 and added 600g / L glucose and 20% ammonia solution. The fermenter was cultured for 48h at a temperature of 28℃, a rotation speed of 500rpm, an aeration rate of 10vvm, and a pH of 4.5-5. (6) Collect the cells using a tubular centrifuge and then spray dry them to obtain yeast protein.

6. The fermentation production method as described in claim 5, characterized in that: The fermentation medium CM7 comprises 100 g / L glucose, 15 g / L corn steep liquor powder, 25 g / L industrial grade ammonium sulfate, 5 g / L industrial grade diammonium hydrogen phosphate, 1 g / L magnesium sulfate, 0.1 g / L zinc sulfate, 0.06 g / L vitamin B1, 0.05 g / L vitamin B6, and 0.003 g / L vitamin B7.

7. The fermentation production method as described in claim 5, characterized in that: The fermentation medium CMX9 comprises 65 g / L industrial glucose, 10 g / L industrial corn flour, 10 g / L potassium dihydrogen phosphate, 8 g / L magnesium sulfate heptahydrate, 20 mg / L zinc sulfate heptahydrate, 12 mg / L ferrous sulfate, 2 mg / L manganese sulfate, 2 mg / L copper sulfate, and 0.555 g / L calcium chloride.

8. The fermentation production method as described in claim 5, characterized in that: The fermentation medium CMX11 comprises 65 g / L industrial glucose, 10 g / L industrial corn flour, 10 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, 8 g / L magnesium sulfate heptahydrate, 20 mg / L zinc sulfate heptahydrate, 12 mg / L ferrous sulfate, 2 mg / L manganese sulfate, 2 mg / L copper sulfate, and 0.555 g / L calcium chloride.

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