Geotrichum candidum Do-Ya-2-12 and method for producing single-cell protein through fermentation thereof

Abstract

The invention relates to the technical field of biological fermentation. The invention provides a strain of Geotrichum candidum Do-Ya-2-12, and the preservation number of the strain of Geotrichum candidum Do-Ya-2-12 is CGMCC (China General Microbiological Culture Collection Center) No. 33104. The strain of the geotrichum candidum Do-Ya-2-12 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the strain is CGMCC No.33104, the preservation time of the strain is December 17, 2024, the preservation center is called CGMCC for short, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain provided by the invention can replace a conventionally used carbon source and nitrogen source with a corn soaking solution or corn steep liquor, is environment-friendly, improves the conversion rate of lactic acid by microorganisms such as yeast, reduces the production cost, simplifies the production process, and realizes efficient output of single-cell protein.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation. Background Art

[0002] Single cell protein (SCP) is produced by microbial fermentation, and these microorganisms include bacteria, yeasts, and algae, etc. Compared with traditional plant and animal protein feeds, SCP has significant advantages: First, high production efficiency: Microorganisms grow rapidly and can reproduce in large quantities in a short time; Second, environmentally friendly: The production process has low requirements for land and water resources and can reduce greenhouse gas emissions; Third: Strong sustainability: Using renewable resources as raw materials, such as by-products like corn steep liquor; Fourth: Wide adaptability: It can be cultured under various conditions and is not restricted by seasons or climate. Yeast cells are rich in nutrients and contain almost all essential amino acids, especially lysine, threonine, leucine, and phenylalanine, etc., with relatively high contents.

[0003] In the process of producing single cell protein (SCP), the cost of the substrate accounts for a quite large part of the total cost. Therefore, choosing cheap and sustainable raw materials is crucial for reducing the costs of enterprises. These raw materials can include industrial and agricultural wastes such as biogas slurry, brewery spent grains, molasses, apple pomace, and pig manure. These biodegradable agro-industrial wastes are considered as environmentally friendly substrates for fermenting SCP production. A suitable substrate should have the characteristics of high levels of nutrients, non-toxicity, and renewability.

[0004] Corn steep liquor (CSL) is a by-product in the process of corn starch production. However, corn steep liquor has problems such as high environmental protection pressure, high COD, and high concentration costs, and the unit price of the corn steep liquor formed after being concentrated 5 times is low. Nevertheless, corn steep liquor is rich in vitamins, minerals, amino acids, and proteins and is an important source of nitrogen. These components make corn steep liquor an ideal substrate for microbial growth. However, corn steep liquor contains a high concentration of lactic acid, and many yeasts are difficult to directly utilize lactic acid at this concentration. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Geotrichum candidum Do-Ya-2-12, with a preservation number of CGMCC No. 33104. The Geotrichum candidum Do-Ya-2-12 has been preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation number of this strain is: CGMCC No. 33104. The preservation time of this strain is: December 17, 2024. The center is abbreviated as CGMCC, and the address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides a method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein, which includes the following steps: ferment Geotrichum candidum Do-Ya-2-12 in a seed medium at a temperature of 30°C, a culture time of 24 h, and a rotation speed of 200 rpm to obtain a Geotrichum candidum Do-Ya-2-12 seed liquid; transfer the Geotrichum candidum Do-Ya-2-12 seed liquid into 2 L of the optimal fermentation medium, and ferment at 30°C and a pH value of 4. After the lactic acid is consumed, add 1.5 L of the feeding medium until the lactic acid is consumed again, and then the fermentation ends.

[0007] Furthermore, the composition of the optimal fermentation medium is: per liter of corn steep liquor contains 0.023 g of zinc sulfate, 0.031 g of magnesium sulfate, 0.003 g of copper sulfate, and 0.39 g of potassium sulfate.

[0008] Furthermore, the inoculation amount of the Geotrichum candidum Do-Ya-2-12 seed liquid is 15% of the volume of the optimal fermentation medium.

[0009] Furthermore, the preparation method of the Geotrichum candidum Do-Ya-2-12 seed medium is to mix corn steep liquor and YPD medium in a one-to-one volume ratio.

[0010] Furthermore, the YPD medium includes: 20 g / L of glucose, 20 g / L of peptone, and 10 g / L of yeast extract.

[0011] Furthermore, the feeding medium is: corn steep liquor diluted 2 times; the corn steep liquor is corn steep liquor concentrated 5 times.

[0012] The strain provided by the present invention can replace the conventionally used carbon source and nitrogen source with corn steep liquor or corn steep water, which is environmentally friendly, improves the conversion rate of lactic acid by microorganisms such as yeast, reduces the production cost, simplifies the production process, and realizes the efficient production of single-cell protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a morphological diagram of the strain Do-Ya-2-12 on YPD agar medium.

[0014] Figure 2 It is a jagged normal distribution diagram.

[0015] Figure 3 It is a corresponding relationship diagram between the residuals and the predicted values of the equation.

[0016] Figure 4 It is a corresponding relationship diagram between the predicted values and the actual test values.

[0017] Figure 5 It is a 3D model diagram of the interaction between zinc sulfate and magnesium sulfate.

[0018] Figure 6 It is a contour diagram of zinc sulfate and magnesium sulfate.

[0019] Figure 7 It is a 3D model diagram of the interaction between zinc sulfate and copper sulfate.

[0020] Figure 8 It is a contour diagram of zinc sulfate and copper sulfate.

[0021] Figure 9 It is a 3D model diagram of the interaction between zinc sulfate and potassium sulfate.

[0022] Figure 10 It is a contour diagram of zinc sulfate and potassium sulfate.

[0023] Figure 11 It is a 3D model diagram of the interaction between magnesium sulfate and copper sulfate.

[0024] Figure 12 It is a contour diagram of magnesium sulfate and copper sulfate.

[0025] Figure 13 It is a 3D model diagram of the interaction between magnesium sulfate and potassium sulfate.

[0026] Figure 14 It is a contour diagram of magnesium sulfate and potassium sulfate.

[0027] Figure 15 It is a 3D model diagram of the interaction between copper sulfate and potassium sulfate.

[0028] Figure 16 It is a contour diagram of copper sulfate and potassium sulfate.

[0029] Figure 17 It is a graph showing the changes in dissolved oxygen, pH value, sulfuric acid added, and feeding in the fed-batch fermentation process of a fermenter.

[0030] Figure 18 It is a graph showing the changes in lactic acid content, cell dry weight, protein content, and crude protein yield in the fed-batch fermentation process of a fermenter. Specific Embodiments

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it according to the description in the specification.

[0032] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0033] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0034] In the description of the present invention, the orientation or positional relationship indicated by terms such as "lateral", "longitudinal", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0035] Example 1

[0036] Determination of the Composition of Corn Steeping Liquid

[0037] The corn steeping liquid was obtained from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd. and stored in a -80°C refrigerator. The COD, total nitrogen, ammonia nitrogen, and total phosphorus of the corn steeping liquid were measured by a water quality analyzer; the content of trace elements was sent for testing, and the results are shown in Table 1.

[0038] Table 1 Numerical Values of the Composition Content of Corn Steeping Liquid

[0039]

[0040] Example 2

[0041] Isolation, Purification, and Identification of Geotrichum candidum

[0042] Sample Collection: In August 2023, samples were collected from the Daqu of Xijiu fermentation on the 0th day in Xijiu Town, Xishui County, Guizhou Province. The obtained samples were stored in 50 mL centrifuge tubes and brought back to the laboratory at low temperature.

[0043] Sample separation: Using the method of dilution coating, take 5 mL of the sample and place it into a sterile 50 mL centrifuge tube. Each centrifuge tube contains 45 mL of sterile distilled water, and the final dilution factor is (1:10). Then perform gradient dilution. Transfer the diluted solution aseptically onto YPD and PDA agar media and spread it evenly.

[0044] Incubate the plates (5 plates for each dilution concentration) at 17 °C, 25 °C, and 30 °C for 3 - 5 days. Pick single colonies similar in morphology to the Geotrichum candidum strain and transfer them to a new YPD plate using a sterile inoculation loop to ensure obtaining pure cultures. Purify the cultures again to ensure that there is no growth of contaminants in the colonies.

[0045] Colony morphology: On the YPD plate as Figure 1 , the colony diameter is 40 - 50 mm, white, rough, wrinkled, and filamentous on the front. The strain is named D0 - Ya - 2 - 12.

[0046] Molecular identification method: Use the alkaline lysis method to extract fungal DNA. Using the universal primers NL1 / NL4 as primers, perform PCR amplification. The D1 / D2 base sequence of strain D0 - Ya - 2 - 12 is shown as SEQ ID NO.1 in the sequence listing.

[0047] Through observing colony characteristics by solid plate culture, observing cell morphology under a microscope, and identifying by 16S rDNA and ITS sequencing, it is determined that D0 - Ya - 2 - 12 is Geotrichum candidum. This strain has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit number of strain Do - Ya - 2 - 12 is CGMCC No. 33104, and the deposit date of strain Do - Ya - 2 - 12 is December 17, 2024. The abbreviation of this center is CGMCC, and the address of this center is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0048] Example 3

[0049] Optimization of culture temperature

[0050] Inoculate the strain into a 250 mL conical flask containing 100 mL of corn steep liquor, with an inoculum amount of 0.05 g, and culture at 200 rpm for 48 h. There are three replicates for each strain. Study the effect of different culture temperatures in the fermentation medium on protein production during fermentation. The culture temperatures are 25 °C, 30 °C, and 35 °C respectively. Leave an appropriate amount of supernatant in the fermented broth after culturing to measure the remaining lactic acid content, dry the cell precipitate at 105 °C to measure its dry weight; take a small amount of the dried substance and measure its crude protein content. Multiply the cell dry weight by the crude protein content to obtain the crude protein yield. The results are shown in Table 2.

[0051] Table 2 Effects of Different Culture Temperatures on Protein Production by Fermentation

[0052]

[0053] As can be seen from the data in Table 2, when the culture temperature is 30°C, the more lactic acid is consumed, the higher the crude protein yield. Therefore, 30°C is selected as the culture temperature for the follow-up.

[0054] Example 4

[0055] Optimization of Initial pH

[0056] Based on the fermentation method of Example 3, the effects of the initial pH of fermentation culture on protein production by fermentation were studied, and the pH values were the natural pH value, 4.5, 5.5, and 6.5 respectively. The natural pH value means that no adjustment of the pH value is made to the liquid culture medium used for fermentation, and the natural pH is 4. The results are shown in Table 3.

[0057] Table 3 Effects of Initial pH Value of Fermentation Culture on Protein Production by Fermentation

[0058]

[0059] As can be seen from the data in Table 3, the yield of crude protein produced by fermentation is the highest at the natural pH value. However, when the pH value is adjusted to 6.5, an abnormally high lactic acid consumption is detected, because an acid-base reaction occurs and lactic acid is consumed. Therefore, the natural pH value is selected for the follow-up.

[0060] Example 5

[0061] Short-Term Domestication of Strains

[0062] Since the corn steep liquor is a high-lactic acid environment with a relatively high acidity, considering that the strains in the early stage are not adapted to the high-lactic acid environment and thus cannot utilize lactic acid quickly, the effects of short-term domestication of the strains on fermentation were studied. A small amount of bacterial cells were scraped and inoculated into each test tube containing 5 mL of liquid domestication medium for 12 h, and five different domestication media were set, namely CSL, YPD, CSL:YPD = 1:1 (volume ratio), CSL:YPD = 1:2 (volume ratio), CSL:YPD = 1:3 (volume ratio). The cultures in the above test tubes were respectively inoculated into conical flasks at an inoculation amount of 10% (v / v) and cultured at 200 rpm at 30°C for 24 h. Then, they were inoculated into the corn steep liquor medium at an OD = 2 and an inoculation amount of 10% and cultured for 48 h. The following indicators were measured subsequently, and the results are shown in Table 4.

[0063] The CSL medium is the corn steep liquor.

[0064] The YPD medium is: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

[0065] Table 4 Effects of short-term domestication on protein production by fermentation

[0066]

[0067] As can be seen from the data in Table 4, after short-term domestication, the final protein yield increased. When the ratio of YPD to CSL was 1:1, the protein yield was the highest.

[0068] Example 6

[0069] Optimization of inoculum size

[0070] Based on the fermentation method described in Example 5, the effects of different inoculum sizes (5%, 10%, 15% and 20%) on protein production by fermentation were studied. The results are shown in Table 5.

[0071] Table 5 Effects of inoculum size on protein production by fermentation

[0072]

[0073] As can be seen from the data in Table 5, at an inoculum size of 15%, the maximum cell dry weight and the highest crude protein yield in fermentation were obtained.

[0074] Example 7

[0075] Addition of metal elements

[0076] Based on the fermentation method described in Example 5, the effects of adding metal elements (magnesium sulfate, zinc sulfate, copper sulfate, potassium sulfate) on protein production by fermentation were studied. The results are shown in Table 6

[0077] Table 6 Effects of four metal elements with different contents on protein production by fermentation

[0078]

[0079] As can be seen from the data in Table 6, when 0.2 g / L of zinc sulfate, 0.2 g / L of magnesium sulfate, 0.003 g / L of copper sulfate and 0.4 g / L of potassium sulfate were added, the highest protein yield was obtained.

[0080] Example 8

[0081] Response surface experiment on addition of metal elements

[0082] According to the results of the single-factor experiment on the addition of metal elements, a four-factor and three-level experiment was designed, as shown in Table 7. Using the Box-Behnken design method (BBD) of Design-Expert 13 software, a response surface experiment was designed with the protein yield as the response value. Among them, the experimental results were analyzed using Design-Expert 13 software to confirm the optimal fermentation elements and predict the optimal values.

[0083] Table 7 Response surface test coefficients and levels

[0084]

[0085] Based on the results of single-factor experiments, the concentration ranges of each metal element were determined. Taking their concentrations as independent variables, which were represented by A, B, C, and D respectively, and using the crude protein yield (y) of Geotrichum candidum as the response value, a Box-Behnken experimental design was carried out, and the results are shown in Table 8.

[0086] Table 8 Response surface test design and results

[0087]

[0088] Using Design-Expert 10.0 software, Table 8 was fitted into a multiple regression analysis to obtain the regression equation:

[0089]

[0090] The results of the variance analysis of the response surface regression model are shown in Table 9, and the error statistical analysis of the regression equation is shown in Table 10. From Tables 9 and 10, it can be seen that the model P < 0.0001, R 2 = 0.9904, indicating that the model is significant. The deviation between the actual test value and the theoretical value is very small, and the obtained regression model can well predict the response value. At the same time, the lack-of-fit term is 0.1238 > 0.05, showing insignificance, indicating that the fitting degree of the model and the experiment is good. The P value is greater than 0.5, which is an insignificant level, indicating that the missing fitting values in the experiment do not affect the construction of the model. Considering the P values of the 4 factors, the P values of zinc sulfate and potassium sulfate are less than 0.01, which is an extremely significant level, indicating that they have an obvious impact on the test results; while the P values of magnesium sulfate and copper sulfate are greater than 0.05, which is an insignificant level.

[0091] Table 9 Variance analysis of the response surface regression model

[0092]

[0093] Table 10 Error statistical analysis of the regression equation

[0094]

[0095] The results of the normal distribution law of the residuals, the corresponding relationship between the residuals and the predicted values of the equation, and the corresponding relationship between the predicted values and the actual test values are as Figure 2 、 3 shown. It can be seen from the figure that the fitting model using the response surface method has good adaptability. Figures 4 - 15 The response surface diagram reflecting the interaction between the crude protein yield for any two variables. The steeper the surface diagram, the more obvious the interaction between the variables.Figure 8 , 9 It can be seen that the interaction term (AD) has the most significant effect on the crude protein yield.

[0096] Through the screening and prediction of the response surface by Design Expert 13 software, the optimal metal element composition for protein production is obtained as follows: zinc sulfate 0.024 g / L, magnesium sulfate 0.313 g / L, copper sulfate 0.003 g / L, potassium sulfate 0.367 g / L, and the predicted protein yield is 7.613 g / L. The dry cell weight measured in three parallel experiments is 16.78 g / L, the protein content is 45.17%, and the crude protein yield is 7.578 g / L.

[0097] Example 9

[0098] Enlarged culture in a fermenter

[0099] According to Examples 1 to 8, the optimal fermentation conditions are as follows:

[0100] The optimal seed medium composition is: a one-to-one volume mixture of corn steep liquor (CSL) and YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract);

[0101] The optimal fermentation medium composition is: adding 0.023 g of zinc sulfate, 0.031 g of magnesium sulfate, 0.003 g of copper sulfate, and 0.39 g of potassium sulfate to each liter of corn steep liquor; the temperature is 30°C, the pH is natural, the inoculation amount is 15%, and the fermentation time is 24 h;

[0102] The feeding medium composition is: corn steep liquor diluted twice, and the corn steep liquor is obtained by concentrating corn steep liquor 5 times.

[0103] Using the Geotrichum candidum strain D0-Ya-2-12 isolated by the present invention, inoculated into the seed medium (CSL and YPD with a volume ratio of one to one), and using a 5 L fermenter, fermented at a temperature of 30°C, a culture time of 24 h, and a rotation speed of 200 rpm to obtain a seed liquid.

[0104] Transfer the seed liquid of Geotrichum candidum strain D0-Ya-2-12 (inoculated at a volume fraction of 15%) into a 5 L stirred fermenter containing 2 L of the optimal fermentation medium, and ferment at 30°C. When the lactic acid is consumed, add 1.5 L of the feeding medium until the lactic acid is consumed again, and the fermentation ends. During the fermentation process, adjust the pH value to about 4 using 20% sulfuric acid and 40% sodium hydroxide by volume.

[0105] Set the initial rotational speed and initial aeration volume to automatic, and control the dissolved oxygen at 25%. Start slowly feeding the culture medium when the lactic acid drops below 0.3 g / L. Increase the feeding rate when the dissolved oxygen is greater than 30%.

[0106] Take about 20 mL of samples every 1 - 2 hours to detect pH, CDW, lactic acid content, ammonia nitrogen, total nitrogen, SCP (washed 3 times with sterile water), and observe the change of dissolved oxygen.

[0107] When the lactic acid is consumed completely, the fermentation is completed. Centrifuge or filter to collect the thalli, wash the thalli 3 times with sterile water, and place the washed thalli in an electrothermal constant temperature drying oven at 105 °C to dry to a constant weight, then the single - cell protein is obtained.

[0108] During the fermentation process, the changes of each parameter are as Figure 3 shown in Table 11.

[0109] Table 11 Numerical table of dissolved oxygen, pH value, sulfuric acid, and feeding changes during the fermentation process

[0110]

[0111] The biomass of the single - cell protein mycelium obtained by fermentation is 140.71 g (46.90 g / L), the crude protein content is 52.86%, and the crude protein yield is 74.37 g (24.79 g / L).

[0112] The analysis of the thallus composition obtained by fermenting Geotrichum candidum D0 - Ya - 2 - 12 under the optimal fermentation conditions of Example 9 is shown in Table 12. %DM represents the percentage of dry matter.

[0113] Table 12 Analysis of thallus composition components

[0114]

[0115] The total amino acid content of the obtained SCP is 35.66%, which is significantly higher than 30.98% of soybean meal (Table 13). In particular, the types of non - essential amino acids in SCP are more abundant than those in soybean meal. Aspartic acid, asparagine, alanine, serine, proline, and glutamic acid are not found in soybean meal. Glutamic acid is the most abundant amino acid, accounting for 5.30% of the cell dry weight. Glutamic acid is the main cell nitrogen donor, effectively supplementing the cell nitrogen currency and providing resources for the cell to re - invest in nitrogen synthetic metabolism, such as aspartic acid synthesis, to support macromolecule synthesis. In addition, it also contains arginine (1.95%) which is indispensable for fish growth. These unique characteristics may contribute to improving the reproductive performance and disease resistance of animals and fish. Generally speaking, the SCP produced from corn steep liquor is more nutritious and can be regarded as a potential protein feed for replacing soybean meal.

[0116] Table 1 Amino acid composition of SCP products

[0117]

[0118] In summary, the nutritional value of the mycoprotein fermented from Geotrichum candidum D0-Ya-2-12 is very high, and compared with expensive plant proteins and animal proteins, the mycoprotein also has the advantages of low development cost and high nutritional value.

[0119] The SCP products are rich in protein content and have a very high nutritional value. They can provide some essential amino acids for animal growth and can also improve the immunity of animals.

[0120] The equipment quantities and treatment scales described here are used to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be apparent to those skilled in the art.

[0121] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the examples shown and described here.

Claims

1. Geotrichum candidum Do-Ya-2-12, with the preservation number of CGMCC No. 33104.

2. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 1, characterized in that It includes the following steps: Ferment Geotrichum candidum Do-Ya-2-12 in a seed medium at a temperature of 30°C, a culture time of 24 h, and a rotation speed of 200 rpm to obtain a Geotrichum candidum Do-Ya-2-12 seed solution. Transfer the Geotrichum candidum Do-Ya-2-12 seed solution into 2 L of the optimal fermentation medium and ferment at 30°C and a pH value of 4. After the lactic acid is consumed, add 1.5 L of the feeding medium until the lactic acid is consumed again, and the fermentation ends.

3. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 2, characterized in that, The composition of the optimal fermentation medium is as follows: per liter of corn steep liquor contains 0.023 g of zinc sulfate, 0.031 g of magnesium sulfate, 0.003 g of copper sulfate, and 0.39 g of potassium sulfate.

4. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 2, characterized in that, The inoculation amount of the Geotrichum candidum Do-Ya-2-12 seed solution is 15% of the volume of the optimal fermentation medium.

5. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 2, characterized in that, The preparation method of the Geotrichum candidum Do-Ya-2-12 seed medium: Mix corn steep liquor and YPD medium in a volume ratio of 1:

1.

6. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 5, characterized in that, The YPD medium includes: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

7. The method for fermenting Geotrichum candidum Do-Ya-2-12 to produce single-cell protein according to claim 2, characterized in that, The feeding medium is corn steep liquor diluted 2 times; the corn steep liquor is corn steep liquor concentrated 5 times.

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