Method for producing yeast single-cell protein by taking acetate and urea as raw materials

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for producing yeast single-cell protein by taking acetate and urea as raw materials, which comprises the following steps: carrying out activation culture on a yeast strain, and then carrying out amplification culture to obtain a seed solution; carrying out first centrifugation on the seed solution, collecting seed solution precipitates, carrying out first washing and resuspension, then inoculating the resuspended seed solution into a fermentation culture medium, and carrying out fermentation culture to obtain a fermentation solution; and after carrying out second centrifugation on the fermentation liquor, collecting fermentation liquor precipitate, and carrying out second washing and drying to obtain the yeast single-cell protein, wherein a carbon source in the fermentation culture medium is acetate, and a nitrogen source in the fermentation culture medium is urea. According to the method disclosed by the invention, acetate and urea are respectively used as a unique carbon source and a unique nitrogen source, so that application of acetate as the unique carbon source in yeast single-cell protein production is realized, and a new technical thought is provided for single-cell protein production.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to a method for producing yeast single-cell protein using acetate and urea as raw materials. Background Art

[0002] Single-cell protein is a protein produced by microbial cells, mainly including yeast protein, algal protein, bacterial protein, fungal protein, etc., and is a new type of protein feed resource that has attracted much attention. Compared with traditional protein resources, microbial cell protein has the advantages of high protein content, rich nutritional components, high production efficiency, no occupation of arable land, and wide sources of production raw materials. However, the industrial production of single-cell protein faces problems such as high cost, low raw material conversion efficiency, insufficient protein synthesis ability, and lack of new strains. As essential nutrients for microbial growth and reproduction, carbon sources and nitrogen sources can provide carbon and nitrogen skeletons for cell growth and product synthesis in cell life activities. In the production process of single-cell protein, the cost of carbon sources and nitrogen sources exceeds 60% of the total cost of single-cell protein production. Therefore, exploring and developing processes for producing single-cell protein using low-cost and sustainable carbon and nitrogen sources is crucial for reducing production costs. Acetate is a new type of carbon source with great potential. Currently, it can be synthesized through methods such as cellulose hydrolysis, anaerobic digestion, syngas fermentation, electrochemistry, and chemical catalysis, and is simple to obtain and low in cost. Urea is one of the compounds with the highest nitrogen content, and its industrial synthesis process is mature, making it a simple and easily available microbial nitrogen source. Currently, many microorganisms have been proven to have the ability to assimilate natural acetate and urea, and these microorganisms can use acetate and urea as carbon and nitrogen sources to synthesize microbial cell protein.

[0003] As a single-cell protein production strain with excellent performance, yeast has the advantages of fast reproduction, reasonable amino acid ratio of the obtained protein, high digestibility of the obtained protein, short production cycle, industrialization feasibility, controllable product quality, and no biosafety risk, and is one of the most ideal new alternative proteins. Currently, several technologies for producing single-cell protein using yeast with acetate and other carbon-containing compounds as combined carbon sources have emerged. For example, the carbon source of the fermentation medium in the prior art CN117025430A includes a composition of acetate and ketone compounds, and the carbon sources of the primary screening medium and the secondary screening medium in the prior art CN117050892A include acetate and glucose as a combined carbon source, or a mixed carbon source composed of acetate and any one or several of formate, bicarbonate, and carbonate and glucose as a combined carbon source. However, there is no relevant report on using acetate as the sole carbon source for yeast to produce single-cell protein.

[0004] Therefore, a new production method is needed to fill the technical gap in using acetate as the sole carbon source for single-cell protein production. Summary of the Invention

[0005] The problem to be solved by this application is to provide a method for producing yeast single-cell protein using acetate and urea as raw materials, using acetate and urea as the sole carbon source and the sole nitrogen source respectively, realizing the application of acetate as the sole carbon source in the production of yeast single-cell protein, and providing a new technical idea for the production of single-cell protein.

[0006] To solve the above technical problems, this application adopts the following technical solutions: On the one hand, this application provides a method for producing yeast single-cell protein using acetate and urea as raw materials, including the following steps: After activating and culturing the yeast strain, then performing an enlarged culture to obtain a seed liquid; performing a first centrifugation on the seed liquid, collecting the precipitate of the seed liquid for the first washing and resuspension, and then inoculating the resuspended bacterial liquid into a fermentation medium for fermentation culture to obtain a fermentation broth; and after performing a second centrifugation on the fermentation broth, collecting the fermentation broth precipitate for the second washing and drying to obtain the yeast single-cell protein; Among them, the carbon source in the fermentation medium is acetate, and the nitrogen source in the fermentation medium is urea.

[0007] In the above technical solution, by first activating and culturing the yeast strain and then performing an enlarged culture, rapid reproduction of yeast can be achieved within a short time, ensuring that there are sufficient numbers of yeast in the seed liquid. Centrifugation and resuspension can remove the nutrients in the seed liquid, avoiding affecting the utilization of acetate by the yeast strain in subsequent steps.

[0008] Further, the yeast strain includes at least one of Pichia kudriavzevii ( Pichia kudriavzevii ), Pichia stipitis ( Scheffersomyces stipitis ), Rhodotorula glutinis ( Rhodotorula glutinis ), Rhodotorula mucilaginosa ( Rhodotorula mucilaginosa ), Candida utilis ( Candida utilis ), Kluyveromyces marxianus ( Kluyveromyces marxianus ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha ( Hansenula polymorpha ), Rhodosporidium toruloides ( Rhodosporidium toruloides ), Pichia pastoris ( Pichia pastoris ), Hansenula anomala ( Hansenula anomala ), Cryptococcus albidus ( Naganishia albida ), Pichia galeiformis ( Pichia manshurica ), Melanopsichia magna ( Filobasidium magnum ), Candida cylindracea (Candida cylindracea ), and Pichia jadinii ( Pichia jadinii ).

[0009] In the above technical solution, by limiting the type of yeast strain, the stable production of single-cell protein can be ensured during the production process.

[0010] Furthermore, the acetate includes at least one of sodium acetate, potassium acetate, and ammonium acetate.

[0011] In the above technical solution, by limiting the type of acetate, the stable utilization of acetate by yeast during the production process can be ensured, and the stable production of single-cell protein can be guaranteed.

[0012] Furthermore, the OD value of the resuspended bacterial liquid is 10 - 30, and the inoculation amount of the resuspended bacterial liquid on the fermentation medium is 1% - 5% v / v.

[0013] In the above technical solution, by limiting the concentration of the resuspended seed liquid and the inoculation amount on the fermentation medium, the intensified competition among yeast can be avoided, and at the same time, the accumulation of excessive metabolic wastes can be prevented.

[0014] Furthermore, the activation culture is carried out in a YPD solid medium, the temperature of the activation culture is 25°C - 30°C, and the time of the activation culture is 48 - 72 h.

[0015] In the above technical solution, by limiting the temperature and time of the activation culture, the yeast strain can be fully activated and have sufficient activity in subsequent expansion and fermentation cultures.

[0016] Furthermore, the expansion culture is carried out in a liquid YPD liquid medium with a shaker at a speed of 150 - 200 rpm, the temperature of the expansion culture is 25°C - 30°C, and the time of the expansion culture is 24 - 48 h.

[0017] In the above technical solution, by limiting the temperature and time of the expansion culture, the yeast strain can grow and reproduce sufficiently, ensuring an adequate amount of microorganisms in the subsequent fermentation process.

[0018] Furthermore, the fermentation culture is carried out with a shaker at a speed of 150 - 200 rpm, the temperature of the fermentation culture is 25°C - 30°C, and the time of the fermentation culture is 72 - 120 h.

[0019] In the above technical solution, by limiting the temperature and time of the fermentation culture, the vital activities of the yeast strain can be ensured to be more stable, producing a better fermentation effect and reducing the possibility of contamination by other strains.

[0020] Furthermore, the preparation method of the fermentation medium includes: Filter and sterilize the urea to obtain sterile urea; and mix the acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, trace elements, and vitamins, adjust the pH to 6-7, perform high-pressure steam sterilization, and then add the sterile urea to obtain the fermentation medium.

[0021] In the above technical solution, sterilizing each component of the fermentation medium can avoid fungal contamination. Filtering and sterilizing urea can prevent decomposition during high-pressure steam sterilization.

[0022] Further, the final concentration of urea in the fermentation medium is 1-5 g / L.

[0023] Further, the final concentration of acetate in the fermentation medium is 5-40 g / L.

[0024] Further, the final concentration of potassium dihydrogen phosphate in the fermentation medium is 5-7 g / L.

[0025] Further, the final concentration of dipotassium hydrogen phosphate in the fermentation medium is 2-3.5 g / L.

[0026] Further, the final concentration of magnesium sulfate in the fermentation medium is 0.5-1 g / L.

[0027] In the above technical solution, limiting the final concentrations of urea, acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and magnesium sulfate can ensure the synergy among the components, thereby reducing the inhibitory effect of acetate on the vital activities of yeast strains.

[0028] Further, the trace elements include EDTA, zinc sulfate heptahydrate, cobalt chloride hexahydrate, manganese chloride tetrahydrate, copper sulfate pentahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate, sodium molybdate dihydrate, boric acid, and potassium iodide.

[0029] Exemplarily, the trace elements include, based on the final concentration in the fermentation medium: EDTA 15 mg / L Zinc sulfate heptahydrate 4.5 mg / L Cobalt chloride hexahydrate 0.3 mg / L Manganese chloride tetrahydrate 1 mg / L Copper sulfate pentahydrate 0.3 mg / L Calcium chloride dihydrate 4.5 mg / L Ferrous sulfate heptahydrate 3 mg / L Sodium molybdate dihydrate 0.4 mg / L Boric acid 1 mg / L Potassium iodide 0.1 mg / L.

[0030] In the above technical solution, defining the types of various trace elements and the final concentrations of these trace elements in the fermentation medium can improve the fermentation efficiency of yeast strains and at the same time reduce the inhibitory effect of acetate on the life activities of yeast strains.

[0031] Further, the vitamins include biotin, calcium pantothenate, hydrochloric acid, inositol, vitamin B1, vitamin B6, and p-aminobenzoic acid.

[0032] Exemplarily, the vitamins include, based on the final concentration in the fermentation medium: Biotin 0.05 mg / L Calcium pantothenate 1 mg / L Hydrochloric acid 1 mg / L Inositol 25 mg / L Vitamin B1 1 mg / L Vitamin B6 1 mg / L p-Aminobenzoic acid 0.2 mg / L.

[0033] In the above technical solution, defining the types of vitamins and the final concentrations of these vitamins in the fermentation medium can improve the fermentation efficiency of yeast strains and at the same time reduce the inhibitory effect of acetate on the life activities of yeast strains.

[0034] On the other hand, the present application provides an application of the above method in the production of single-cell protein, and the application includes the production of protein feed.

[0035] The present application has the following beneficial effects: 1. The method of the present application successfully uses acetate as the sole carbon source to achieve the efficient production of single-cell protein by yeast, and the entire production cycle is only 6-8 days, greatly improving the efficiency and yield of yeast using acetate to produce single-cell protein.

[0036] 2. The method of the present application is simple to operate, the raw materials are cheap and easy to obtain, it can be applied to a variety of yeast strains, and the final single-cell protein content is relatively high, which is a very industrially valuable method for producing single-cell protein. Specific Embodiments

[0037] Next, the technical solutions in some embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments provided by the present application, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present application.

[0038] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is to be construed in an open, inclusive sense, i.e., "including, but not limited to".

[0039] When describing some embodiments, the expressions "at least one of A, B, and C" and "at least one of A, B, or C" may be used, and both have the same meaning, and both include the following combinations of A, B, and C: only A, only B, only C, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0040] Example 1 Culture Medium YPD solid culture medium, the formula of which is: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, and agar 15 - 20 g / L; pH is 5.5 - 6.5.

[0041] YPD liquid culture medium, the formula of which is: yeast extract 10 g / L, peptone 20 g / L, and glucose 20 g / L; pH is 5.5 - 6.5.

[0042] Acetate - urea fermentation culture medium, the formula of which includes: acetate (at least one of sodium acetate, potassium acetate, ammonium acetate) 5 - 40 g / L, urea 1 - 5 g / L, potassium dihydrogen phosphate 5 - 7 g / L, dipotassium hydrogen phosphate 2 - 3.5 g / L, magnesium sulfate 0.5 - 1 g / L; trace elements: EDTA 15 mg / L, zinc sulfate heptahydrate 4.5 mg / L, cobalt chloride hexahydrate 0.3 mg / L, manganese chloride tetrahydrate 1 mg / L, copper sulfate pentahydrate 0.3 mg / L, calcium chloride dihydrate 4.5 mg / L, ferrous sulfate heptahydrate 3 mg / L, sodium molybdate dihydrate 0.4 mg / L, boric acid 1 mg / L, potassium iodide 0.1 mg / L; vitamins: biotin 0.05 mg / L, calcium pantothenate 1 mg / L, nicotinic acid 1 mg / L, inositol 25 mg / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, p - aminobenzoic acid 0.2 mg / L; pH is 6 - 7; the preparation steps include: filtering and sterilizing the urea to obtain the sterile urea, then mixing the acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, the trace elements, and the vitamins, adjusting the pH to 6 - 7 with potassium hydroxide solution, performing high - pressure steam sterilization, and then adding the sterile urea to obtain the fermentation culture medium.

[0043] Example 2 Preparation of Seed Liquids of Different Yeast Strains After taking out the yeast strains stored in glycerol tubes, streak them onto the YPD solid medium in Example 1 respectively, and activate and culture them in an incubator at 25°C to 30°C for 48 to 72 h. Then transfer the activated yeast strains to the YPD liquid medium in Example 1, and perform enlarged culture in a shaker at 25°C to 30°C and 150 to 200 rpm for 24 to 48 h.

[0044] Exemplarily, for the yeast strains such as Pichia kudriavzevii, Pichia stipitis, Rhodotorula glutinis, Rhodotorula mucilaginosa, Candida utilis, Kluyveromyces marxianus, Yarrowia lipolytica, Hansenula polymorpha, Rhodosporidium toruloides, Pichia pastoris, Hansenula anomala, Cryptococcus albidus, Pichia galeiformis, Ustilago magnistipitis, Candida cylindracea, Pichia jadinii, etc. stored in glycerol tubes in the present application, after taking them out, streak them onto the YPD solid medium in Example 1 respectively, activate and culture them in an incubator at 30°C for 48 h, then transfer the activated yeast strains to the YPD liquid medium in Example 1, and perform enlarged culture in a shaker at 30°C and 180 rpm for 24 h to obtain the seed solutions of different yeast strains.

[0045] Example 3 Preparation of yeast single cell protein from seed solution After centrifuging the seed solution at a speed of 4000 rpm for 5 min, remove the supernatant. After washing the precipitate three times with sterile water, resuspend it with sterile water to form a bacterial solution with an OD value of 10 to 30, and then inoculate it into the fermentation medium at an inoculation amount of 1% to 5% (v / v), and perform fermentation culture on a shaker at 25°C to 30°C and a speed of 150 to 200 rpm for 72 to 120 h. After fermentation is completed, centrifuge the fermentation broth at a speed of 4000 rpm for 5 min, collect the precipitate, wash it three times with sterile water to thoroughly remove the residual urea, and finally freeze-dry it at -45°C for 24 h to obtain the yeast single cell protein. Exemplarily, after centrifuging the seed solutions obtained in Example 2 at a speed of 4000 rpm for 5 min respectively, remove the supernatant. After washing the precipitate three times with sterile water, resuspend it with sterile water to form a bacterial solution with an OD value of 10, and then inoculate it into the acetate-urea fermentation medium in Example 1 at an inoculation amount of 1% (v / v), and perform fermentation culture on a shaker at 30°C and a speed of 180 rpm for 72 h. After fermentation is completed, centrifuge the fermentation broth at a speed of 4000 rpm for 5 min, collect the precipitate, wash it three times with sterile water to thoroughly remove the residual urea, and finally freeze-dry it at -45°C for 24 h to obtain the yeast single cell protein.

[0046] Example 4 Determination of crude protein content of yeast single cell protein The crude protein content is determined by the Kjeldahl method. The specific method is as follows: (1)Sample treatment: The yeast single-cell protein sample obtained by fermenting the seed liquid using different yeast strains in Example 3 is heated and digested together with concentrated sulfuric acid and a catalyst (such as potassium sulfate, copper sulfate, etc.) to convert the organic nitrogen in the sample into inorganic nitrogen (ammonium sulfate), obtaining a digestion solution. (2)Distillation and absorption: Transfer the digestion solution into the reaction chamber of a Kjeldahl nitrogen analyzer, add an excessive amount of concentrated sodium hydroxide solution to convert ammonium ions into ammonia gas, and then drive the ammonia gas into a receiving flask containing an excessive amount of boric acid solution through distillation. After the boric acid accepts the ammonia gas, a tetraborate ammonium solution is formed. (3)Titration and calculation: Titrate the tetraborate ammonium solution with a standard hydrochloric acid until the pH value of the tetraborate ammonium solution returns to its original level. The number of moles of the standard hydrochloric acid consumed in the titration is the number of moles of ammonia. The protein content of different samples can be calculated according to the following formula, as shown in Table 1: In the formula, V1—the volume of the standard hydrochloric acid solution required for the blank control (mL); V2—the volume of the standard hydrochloric acid solution consumed in titrating the sample (mL); c—the concentration of the standard hydrochloric acid (mol / L); m—the mass of the sample (g); V’—the volume of the digestion solution used for distillation (mL); V—the total volume of the sample digestion solution (mL); 0.014—the number of grams per milliequivalent of ammonia; 6.25—the average coefficient for converting ammonia into crude protein.

[0047] Table 1: Protein content of yeast single cells synthesized by different yeast solutions using an acetate-urea fermentation medium Number Yeast strain Protein content (%) Y1 Pichia kudriavzevii 35.54 Y2 Scheffersomyces stipitis 52.84 Y3 Rhodotorula glutinis 31.23 Y4 Rhodotorula mucilaginosa 37.26 Y7 Candida utilis 48.30 Y8 Kluyveromyces marxianus 39.96 Y9 Yarrowia lipolytica 40.10 Y10 Hansenula polymorpha 39.24 Y11 Rhodosporidium toruloides 40.46 Y12 Pichia pastoris 51.18 Y13 Hansenula anomala 35.11 Y14 Naganishia albida 32.23 Y15 Pichia manshurica 35.00 Y16 Filobasidium magnum 42.98 Y17 Pichia kudriavzevii 40.87 Y18 Candida cylindracea 44.63 Y19 Pichia jadinii 54.97 It can be seen from the data in the table that by the method of this application, the protein content of yeast single cells synthesized by different yeast strains using an acetate-urea fermentation medium is all above 30%. Among them, the protein content of Pichia stipitis, Pichia pastoris, and Pichia jadinii is above 50%.

[0048] The above is only the preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be changed within the scope of the concept described herein through the above teachings or the technology or knowledge in the relevant field. Any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for producing yeast single cell protein using acetate and urea as raw materials, characterized in that, It includes the following steps: After activating and culturing the yeast strain, it is further expanded cultured to obtain a seed solution; Perform a first centrifugation on the seed solution, collect the precipitate of the seed solution for the first washing and resuspension, and then inoculate the resuspended bacterial solution into the fermentation medium for fermentation culture to obtain a fermentation broth; After performing a second centrifugation on the fermentation broth, collect the precipitate of the fermentation broth for the second washing and drying to obtain the yeast single-cell protein; Among them, the carbon source in the fermentation medium is acetate, and the nitrogen source in the fermentation medium is urea.

2. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 1, characterized in that The yeast strains include Pichia kudriavzevii ( Pichia kudriavzevii ), Pichia stipitis ( Scheffersomyces stipitis ), Rhodotorula glutinis ( Rhodotorula glutinis ), Rhodotorula mucilaginosa ( Rhodotorula mucilaginosa ), Candida utilis ( Candida utilis ), Kluyveromyces marxianus ( Kluyveromyces marxianus ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha ( Hansenula polymorpha ), Rhodosporidium toruloides ( Rhodosporidium toruloides ), Pichia pastoris ( Pichia pastoris ), Hansenula anomala ( Hansenula anomala ), Cryptococcus albidus ( Naganishia albida ), Pichia galeiformis ( Pichia manshurica ), Melanopsichia magna ( Filobasidium magnum ), Candida cylindracea (Candida cylindracea ), and Pichia jadinii ( Pichia jadinii ), and at least one of them.

3. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 1, characterized in that, The acetate includes at least one of sodium acetate, potassium acetate, and ammonium acetate.

4. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 1, characterized in that, The OD value of the resuspended bacterial solution is 10 - 30, and the inoculation amount of the resuspended bacterial solution on the fermentation medium is 1% - 5% v / v.

5. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 1, characterized in that, The activation culture is carried out in a YPD solid medium, the temperature of the activation culture is 25°C - 30°C, and the time of the activation culture is 48 - 72 h; And / or, the expansion culture is carried out in a liquid YPD liquid medium with a shaker at a speed of 150 - 200 rpm, the temperature of the expansion culture is 25°C - 30°C, and the time of the expansion culture is 24 - 48 h; And / or, the fermentation culture is carried out with a shaker at a speed of 150 - 200 rpm, the temperature of the fermentation culture is 25°C - 30°C, and the time of the fermentation culture is 72 - 120 h.

6. The method for producing yeast single-cell protein using acetate and urea as raw materials according to claim 1, wherein, The preparation method of the fermentation medium includes: Filter and sterilize the urea to obtain sterile urea; and Mix the acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, trace elements, and vitamins, adjust the pH to 6 - 7, perform high-pressure steam sterilization, and then add the sterile urea to obtain the fermentation medium.

7. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 6, characterized in that, The final concentration of urea in the fermentation medium is 1 - 5 g / L; And / or, the final concentration of acetate in the fermentation medium is 5 - 40 g / L; And / or, the final concentration of potassium dihydrogen phosphate in the fermentation medium is 5 - 7 g / L; And / or, the final concentration of dipotassium hydrogen phosphate in the fermentation medium is 2 - 3.5 g / L; And / or, the final concentration of the magnesium sulfate in the fermentation medium is 0.5 - 1 g / L.

8. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 6, characterized in that, The trace elements include EDTA, zinc sulfate heptahydrate, cobalt chloride hexahydrate, manganese chloride tetrahydrate, copper sulfate pentahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate, sodium molybdate dihydrate, boric acid, and potassium iodide.

9. The method for producing yeast single cell protein using acetate and urea as raw materials according to claim 6, characterized in that, The vitamins include biotin, calcium pantothenate, hydrochloric acid, inositol, vitamin B1, vitamin B6, and p-aminobenzoic acid.

10. Use of the method for producing yeast single cell protein from acetate and urea as claimed in any one of claims 1 to 9 in the production of single cell protein, characterized in that, The application includes the production of protein feed.