A method for producing pyruvic acid by fermentation using pyruvic acid fermentation waste microbial bodies as seed culture medium nitrogen source

Abstract

The application discloses a method for producing pyruvic acid by using pyruvic acid fermentation waste microbial bodies as seed culture medium nitrogen source, and belongs to the technical field of biological fermentation. The application uses the pyruvic acid fermentation waste microbial bodies as seed culture medium nitrogen source, improves the seed activity, and further effectively improves the production intensity of pyruvic acid. The application also optimizes the drying process of the waste microbial body dry powder, so that the dry weight of the microbial bodies obtained by the fermentation strain growing in the microbial powder prepared by the spray drying process is increased by 61.54% and 53.66% respectively compared with the hot air drying and freeze drying methods, and the pyruvic acid yield is increased by 32.73% and 25.14% respectively. The application uses the waste microbial body dry powder to replace soybean peptone, can reduce the production cost of the biological method for preparing pyruvic acid, keeps the yield at a level equivalent to the original yield, and is favorable for the industrial production of pyruvic acid.

Description

Technical Field

[0001] This invention relates to a method for producing pyruvate by using waste microorganisms from pyruvate fermentation as a seed culture medium and nitrogen source, belonging to the field of bio-fermentation technology. Background Technology

[0002] Pyruvate is an important industrial raw material with wide applications in various fields. In the pharmaceutical industry, pyruvate is converted into L-tyrosine, L-leucine, L-DOPA, vitamin B6, and vitamin B1 using enzymatic methods. 12 In agriculture, pyruvate is used to prepare grain protectants and hydrogenated atropine. In the food industry, pyruvate is an important ingredient in functional beverages. In addition, pyruvate is widely used in the chemical industry to synthesize important chemical products such as ethyl pyruvate and ceramides.

[0003] In industrial production, pyruvate is mainly produced through microbial fermentation, with *Gnaphalium glabripennis* being the preferred strain. In our previous research, *Gnaphalium glabripennis* showed better results than yeast extract in pyruvate production using soybean peptone as a seed nitrogen source, and soybean peptone is currently widely used in industrial pyruvate fermentation. However, soybean peptone is expensive, with domestically produced soybean peptone priced at 20,000-30,000 RMB / ton and imported soybean peptone at 70,000-100,000 RMB / ton. Furthermore, the waste microbial cells from pyruvate fermentation are a byproduct of pyruvate production; direct discharge would cause environmental pollution. Therefore, reducing fermentation costs and effectively utilizing waste microbial cells are problems that need to be addressed to achieve green and efficient production. Summary of the Invention

[0004] The purpose of this invention is to improve the utilization value of waste microorganisms from pyruvate fermentation and to solve the problem of high cost of existing pyruvate production culture media, so that the improved fermentation method can maintain a pyruvate production level that is basically equivalent to the original fermentation method while reducing costs.

[0005] This invention provides the application of pyruvate fermentation waste cells in increasing pyruvate production.

[0006] In one embodiment, the pyruvate fermentation waste cells are prepared by the following method: collecting pyruvate fermentation broth, centrifuging to collect solids, resuspending the solids, washing, and drying.

[0007] In one embodiment, the drying includes, but is not limited to, one or more of the following: hot air drying, freeze drying, spray drying, inactivating hot air drying, inactivating freeze drying, and inactivating spray drying.

[0008] In one embodiment, the application uses the pyruvate fermentation waste cells as a nitrogen source for the seed culture medium to prepare the seed liquid of fermenting microorganisms.

[0009] In one embodiment, the seed culture medium contains: 25-35 g / L glucose, 5-35 g / L waste bacterial protein powder, 0.4-0.6 g / L MgSO4·7H2O, and 0.8-1.2 g / L KH2PO4.

[0010] The present invention also provides a method for producing pyruvic acid, comprising the following steps:

[0011] Step 1) Cultivate the fermenting microorganisms in a seed culture medium containing pyruvate fermentation waste cells to obtain seed liquid;

[0012] Step 2) Transfer the seed culture prepared in Step 1) to the fermentation medium and ferment at 28-35℃ for at least 50 hours.

[0013] In one embodiment, the seed culture medium contains 5-35 g / L of waste bacterial protein powder.

[0014] In one embodiment, the seed culture medium comprises the following components: 30 g / L glucose, 5–35 g / L waste bacterial protein powder, 0.5 g / L MgSO4·7H2O, and 1 g / L KH2PO4.

[0015] In one embodiment, the culture in step 1) is carried out at 30°C and 220 rpm for 16 to 18 hours.

[0016] In one embodiment, the fermenting microorganism is *Candida glabrata*, including but not limited to *Candida glabrata* CCTCC M 202019, *Candida glabrata* TgU-(pY26-PYC2-PCK1), *Candida glabrata* TgU-(pY26-Shrew1p-MPC1), and *Candida glabrata* 4H2 (published in the paper "Fluorescence-activated dropletsorting for enhanced pyruvic acid accumulation by Candida glabrata").

[0017] In one embodiment, the seed liquid inoculation amount may specifically be 5-10% (v / v).

[0018] In one embodiment, the seed liquid can be prepared by inoculating an activated *Gnaphalium affine* strain into a seed liquid culture medium and culturing it at 30°C and 220 rpm to obtain the seed liquid.

[0019] In one embodiment, the fermentation medium is further supplemented with a vitamin solution; each liter of the vitamin solution contains: 0.004 g biotin, 0.75 mg thiamine, 0.04 g pyridoxine, and 0.8 g nicotinic acid; the amount of vitamin solution added is greater than or equal to 12.5 mL.

[0020] In one embodiment, the fermentation medium contains: 120 g / L glucose, 0.8 g / L MgSO4·7H2O, 2 g / L KH2PO4, 3 g / L CH3COONa, 10 mL urea solution, 10 mL trace element solution, and 10–20 mL vitamin solution.

[0021] In one embodiment, the concentration of the urea solution is 386 g / L.

[0022] In one embodiment, each L of the trace element solution contains: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, and 0.5g ZnCl2.

[0023] In one embodiment, each liter of the vitamin solution contains: 0.004g biotin, 0.75mg thiamine, 0.04g pyridoxine, and 0.8g niacin.

[0024] In one embodiment, the fermentation can be carried out in a fermenter, and the fermentation conditions can be: temperature 30°C, aeration rate 1.5 vvm, rotation speed 500 rpm, and fermentation time 72 h.

[0025] In one embodiment, the method centrifuges the fermentation product obtained in step 2) to obtain pyruvate, and then dries the solid bacterial cells to use as the nitrogen source for the seed culture medium for the next batch of pyruvate production.

[0026] The present invention also provides a process for continuous production of pyruvic acid, comprising the following steps:

[0027] (1) The fermenting microorganisms are cultured in a seed culture medium for a period of time to obtain seed liquid, and then the seed liquid is transferred to a fermentation culture medium for fermentation for a period of time and the fermentation liquid is collected.

[0028] (2) Centrifuge the fermentation broth from step (1), collect the solids, resuspend the solids, wash and dry them;

[0029] (3) Use the bacterial powder obtained by drying in step (2) as the nitrogen source for preparing the culture medium for the next round of seed culture medium, and continue the operations of steps (1) to (3).

[0030] This invention also provides the application of pyruvate fermentation waste cells in improving the activity of *Saccharomyces glaucoma* cells. The pyruvate fermentation waste cells are obtained by centrifuging the pyruvate fermentation broth of *Saccharomyces glaucoma*, collecting the solids, resuspending the solids, washing them, and then drying them.

[0031] In one embodiment, the enhancement of *Saccharomyces glabrata* cell viability includes, but is not limited to, increasing the amount of pyruvate grown in the culture medium and / or cell weight.

[0032] Beneficial effects:

[0033] (1) This invention provides a method for using pyruvate fermentation waste bacterial powder for seed culture medium nitrogen source fermentation to produce pyruvate. By optimizing the seed culture medium composition and the amount of vitamins added in the fermentation culture medium, the microbial activity is improved, thereby effectively increasing the production intensity of pyruvate.

[0034] (2) By optimizing the preparation method of pyruvate fermentation waste bacterial cell dry powder, this invention further improves the growth vitality and pyruvate production capacity of fermentation strains in seed culture medium with bacterial cell dry powder as nitrogen source. The spray drying method can increase the bacterial cell dry weight by 61.54% and 53.66% respectively compared with hot air drying and freeze drying methods, and increase the pyruvate yield by 32.73% and 25.14% respectively.

[0035] (3) The present invention uses waste bacterial powder to replace soybean peptone, which can reduce the production cost of pyruvate preparation by biological method. Compared with using soybean peptone as nitrogen source, the production cost of producing pyruvate is reduced by RMB 280 to 1900 per ton, which is beneficial to the industrial production of pyruvate. Attached Figure Description

[0036] Figure 1 The effect of different nitrogen sources in seed culture media on pyruvate accumulation.

[0037] Figure 2 The effect of waste bacterial protein prepared by different drying methods on pyruvate accumulation.

[0038] Figure 3 The effect of different concentrations of waste bacterial protein powder on pyruvate accumulation.

[0039] Figure 4 The effect of different vitamin supplementation amounts on pyruvate accumulation.

[0040] Figure 5 Comparison of fermentation results of soybean peptone and waste bacterial powder in tanks; where, circle symbol: glucose; triangle symbol: DCW; square symbol: pyruvate. Detailed Implementation

[0041] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0042] Pyruvate content was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Amin HPX-87H column (Bio-Rad, Hercules, CA, USA); UV detection wavelength 210 nm; column oven temperature 40 °C; injection volume 10 μL; flow rate 0.5 mL / min; mobile phase 5 mmol / L dilute H₂SO₄; and pyruvate elution time 9.758 min.

[0043] Fermentation medium: glucose 120 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, CH3COONa 3 g / L. Urea solution 10 mL, trace element solution 10 mL, vitamin solution 10 mL; the urea solution, trace element solution, and vitamin solution were all prepared as stock solutions, sterilized by filtration, and then added before inoculation. The specific formula is as follows:

[0044] Urea solution: 386g, diluted with water to 1L.

[0045] Trace element solution: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, 0.5g ZnCl2, dissolved in 2mol / L HCl and brought to a final volume of 1L.

[0046] Vitamin solution: Biotin 0.004g, Thiamine 0.75mg, Pyridoxine 0.04g, Nicotinic acid 0.8g, dissolved in 2mol / L HCl, and brought to a final volume of 1L.

[0047] Pyruvate fermentation broth: A cell culture medium of *Candida glabrata* fermented in any suitable medium for growth and pyruvate production until the cell wet weight concentration reaches 12 g / L or higher, and used as a pyruvate fermentation broth for preparing waste cell powder. In some embodiments of the present invention, the fermentation broth obtained by fermenting *Candida glabrata* 4H2 in a medium containing glucose, MgSO4, KH2PO4, CH3COONa, urea, trace elements, and vitamins is used as a raw material for preparing waste pyruvate cell powder.

[0048] Example 1: Method for preparing waste pyruvate bacteria

[0049] The pyruvate waste bacterial cell dry powder was prepared by spray drying, and the specific steps are as follows:

[0050] The pyruvate fermentation broth of *Saccharomyces cerevisiae* 4H2 (wet weight concentration of 12 g / L) was centrifuged to remove the supernatant, and then water was added to resuspend and wash the cells. The resuspended cells were then subjected to ultrasonic treatment for 30 minutes in an ultrasonic cleaner. Next, the cells were exposed to hot air in a dryer (inlet temperature 130℃, outlet temperature 80℃), and the moisture was instantly evaporated to obtain dry cell powder.

[0051] Example 2: Effects of different nitrogen sources on pyruvate accumulation in seed culture medium

[0052] 1. Strain activation: The strain Candida glabrata4H2 (published in the paper "Fluorescence-activated droplet sorting for enhanced pyruvic acid accumulation by Candida glabrata") was streaked on YPD solid medium and incubated statically at 30℃ for 48 hours to grow single colonies. Single colonies were picked and streaked on solid seed medium and incubated statically at 30℃ for 20 hours for use in shake flasks to prepare seed culture.

[0053] 2. Shake flask culture: Pick one loopful of the activated bacterial cells from step 1 and inoculate them into a 250mL Erlenmeyer flask containing 25mL of liquid seed culture medium. Incubate at 30℃ and 220rpm for 16h to obtain the seed culture.

[0054] 3. Fermentation culture: The seed culture obtained in step 2 was transferred to a 250 mL Erlenmeyer flask containing 25 mL of fermentation medium at an inoculation rate of 10% (v / v) for fermentation. The fermentation conditions were as follows: temperature 30℃, stirring speed 220 rpm, 40 g / L CaCO3 was added as pH buffer during the culture process, and the fermentation time was 60 h.

[0055] The seed culture medium used in step 2 is as follows:

[0056] Seed culture medium I (using the waste bacterial protein powder prepared in Example 1 as the nitrogen source): glucose 30 g / L, waste bacterial protein powder 10 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L.

[0057] Seed culture medium II (using soybean peptone as nitrogen source): glucose 30 g / L, soybean peptone 10 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L.

[0058] Fermentation medium: glucose 120 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, CH3COONa 3 g / L. Urea solution 10 mL, trace element solution 10 mL, vitamin solution added at 10–15 mL (15 mL / L for fermentation medium transferred from seed medium I, and 10 mL / L for fermentation medium transferred from seed medium II);

[0059] The urea solution, trace element solution, and vitamin solution are all prepared as stock solutions, sterilized by filtration, mixed, and added before inoculation. The specific formulas are as follows:

[0060] Urea solution: 386g, diluted with water to 1L.

[0061] Trace element solution: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, 0.5g ZnCl2, dissolved in 2mol / L HCl and brought to a final volume of 1L.

[0062] Vitamin solution: Biotin 0.004g, Thiamine 0.75mg, Pyridoxine 0.04g, Nicotinic acid 0.8g, dissolved in 2mol / L HCl, and brought to a final volume of 1L.

[0063] Fermentation results as follows Figure 1 As shown, the cell concentrations obtained from fermentation using waste bacterial protein powder and soybean peptone as seed liquid nitrogen sources were 6.7 g / L and 7.6 g / L, respectively, and the pyruvate yields were 23.2 g / L and 23.7 g / L, respectively, with little difference. During fermentation, the cell concentrations of both cells met the requirements for normal growth and acid production. Soybean peptone is more expensive, which would increase production costs. The cost of 1 L of seed culture medium II is approximately 5.5 yuan, an increase of approximately 3.9 yuan compared to seed culture medium I. Considering all factors, bacterial protein powder was chosen as the nitrogen source.

[0064] Example 3: Preparation of bacterial protein dry powder

[0065] The pyruvate fermentation broth was selected from *Saccharomyces cerevisiae* 4H2 pyruvate fermentation broth with a cell wet weight concentration of 12 g / L. Cell protein powder was prepared according to the following methods:

[0066] Hot air drying: The pyruvate fermentation broth was centrifuged to remove the supernatant, and then water was added to resuspend and wash the cells. The resuspended cells were then subjected to ultrasonic treatment for 30 minutes in an ultrasonic cleaner. Next, the cells were placed in a constant temperature (65℃) hot air dryer for 24 hours, with the cells stirred every 8 hours during this period.

[0067] Freeze-drying: The pyruvate fermentation broth was centrifuged to remove the supernatant, and then water was added to resuspend and wash the cells. The resuspended cells were ultrasonically treated for 30 minutes in an ultrasonic cleaner. Next, they were pre-frozen in an ultra-low temperature freezer at -80°C for 4 hours, and then transferred to a freeze dryer for 20 hours.

[0068] Spray drying: The pyruvate fermentation broth was centrifuged to remove the supernatant, and then water was added to resuspend and wash the cells. The resuspended cells were subjected to ultrasonic treatment in an ultrasonic cleaner for 30 minutes. Next, the cells were exposed to hot air in a dryer, where the moisture evaporated instantly to obtain dry cell powder; the inlet air temperature of the drying tower was 130℃, and the exhaust air temperature was 80℃.

[0069] Inactivation and hot air drying: The pyruvate fermentation broth was boiled under normal pressure to inactivate the cells. After cooling to room temperature, the supernatant was removed by centrifugation, and water was added to resuspend and wash the cells. The resuspended cells were then subjected to ultrasonic treatment for 30 minutes in an ultrasonic cleaner. Next, the cells were placed in a constant temperature (65℃) hot air dryer for 24 hours, with the cells stirred every 8 hours during the drying process.

[0070] Inactivation and freeze-drying: The pyruvate fermentation broth was boiled under normal pressure to inactivate the cells. After cooling to room temperature, the supernatant was removed by centrifugation, and water was added to resuspend and wash the cells. The resuspended cells were then subjected to ultrasonic treatment for 30 minutes in an ultrasonic cleaner. Next, the cells were pre-frozen in an ultra-low temperature freezer at -80°C for 4 hours, and then transferred to a freeze dryer for 20 hours.

[0071] Inactivation spray drying: The pyruvate fermentation broth was boiled under normal pressure to inactivate the cells. After cooling to room temperature, the supernatant was removed by centrifugation, and water was added to resuspend and wash the cells. The resuspended cells were then subjected to ultrasonic treatment for 30 minutes in an ultrasonic cleaner. Next, the cells were exposed to hot air in a dryer, where the moisture evaporated instantly to obtain dry cell powder. The inlet air temperature of the drying tower was 130℃, and the exhaust air temperature was 80℃.

[0072] Further comparison of the effectiveness of bacterial protein powder obtained by different drying methods as a nitrogen source ( Figure 2 When hot air drying and freeze drying were used, the cell concentrations at the end of fermentation were 3.9 g / L and 4.1 g / L, respectively, and the pyruvate yields were 16.5 g / L and 17.5 g / L, respectively. When spray-dried cell protein powder was used, the cell concentration and pyruvate yield at the end of fermentation were 6.3 g / L and 21.9 g / L, respectively, and the fermentation effect was significantly better than that of cell protein powder obtained by other drying methods.

[0073] Example 4: Optimization of bacterial protein powder concentration

[0074] 1. Strain activation: The strain Candida glabrata4H2 was streaked on YPD solid medium and incubated at 30°C for 48 hours to grow single colonies. Single colonies were picked and streaked on solid seed medium and incubated at 30°C for 20 hours to prepare seed culture in shake flasks.

[0075] 2. Shake flask culture: Pick one loopful of the activated bacterial cells from step 1 and inoculate them into a 250mL Erlenmeyer flask containing 25mL of liquid seed culture medium. Incubate at 30℃ and 220rpm for 16h to obtain the seed culture.

[0076] 3. Fermentation culture: The seed culture prepared in step 2 was transferred to a 250 mL Erlenmeyer flask containing 25 mL of fermentation medium at an inoculation rate of 10% (v / v) for fermentation. The fermentation conditions were as follows: temperature 30℃, stirring speed 220 rpm, 40 g / L CaCO3 was added as pH buffer during the culture process, and the fermentation time was 60 h.

[0077] The seed culture medium used in step 2 is as follows:

[0078] Seed culture medium: glucose 30 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, and waste bacterial protein powder concentrations of 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L, respectively.

[0079] The results of pyruvate fermentation are as follows Figure 3 As shown, the pyruvate concentration increases with the increase of the concentration of spray-dried waste microbial protein powder. When the mass concentration of spray-dried waste microbial protein powder is 30 g / L, the pyruvate yield can reach a maximum of 46.0 g / L. When the mass concentration of spray-dried microbial protein powder is 35 g / L, the pyruvate yield is 40.5 g / L, which is a decrease.

[0080] Example 5: Optimization of Vitamin Addition in Fermentation Medium

[0081] 1. Strain activation: The strain Candida glabrata4H2 was streaked on YPD solid medium and incubated statically at 30°C for 48 hours to grow single colonies. Single colonies were picked and streaked on solid seed medium and incubated statically at 30°C for 20 hours for shake flask culture.

[0082] 2. Shake flask culture: Pick one loopful of the activated cells from step 1 and inoculate it into a 250mL Erlenmeyer flask containing 25mL of liquid seed culture medium. Incubate at 30℃ and 220rpm for 16h to obtain the seed culture for fermentation inoculation.

[0083] 3. Fermentation tank culture: The seed culture prepared in step 2 was transferred to a 5L fermenter containing 3L of fermentation medium at an inoculation rate of 10% (v / v) for fermentation. The fermentation conditions were as follows: temperature 30℃, stirring speed 500rpm, NaOH was added during the culture process to control the pH of the fermentation system to be maintained at 5.5, and fermentation time 70h.

[0084] Seed culture medium (using waste bacterial protein powder as nitrogen source): glucose 30g / L, waste bacterial protein powder 30g / L, MgSO4·7H2O 0.5g / L, KH2PO4 1g / L.

[0085] Fermentation medium: glucose 120 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, CH3COONa 3 g / L. Urea solution 10 mL, trace element solution 10 mL, and vitamin solution added in volumes of 10 mL, 12.5 mL, 15 mL, 17.5 mL, and 20 mL respectively. The urea solution, trace element solution, and vitamin solution were all prepared as stock solutions, sterilized by filtration, and added before inoculation. The specific formula is as follows:

[0086] Urea solution: 386g, diluted with water to 1L.

[0087] Trace element solution: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, 0.5g ZnCl2, dissolved in 2mol / L HCl and brought to a final volume of 1L.

[0088] Vitamin solution: Biotin 0.004g, Thiamine 0.75mg, Pyridoxine 0.04g, Nicotinic acid 0.8g, dissolved in 2mol / L HCl, and brought to a final volume of 1L.

[0089] Centrifuge the fermentation broth after step 3, collect the supernatant, and measure the pyruvate concentration in the supernatant. The results are as follows: Figure 4 As shown, when the vitamin solution was added in volumes of 10 mL, 12.5 mL, 15 mL, 17.5 mL, and 20 mL, the corresponding pyruvate concentrations were 46.9 g / L, 56.6 g / L, 61.8 g / L, 53.5 g / L, and 58.1 g / L, respectively.

[0090] Example 6: Production of pyruvic acid using waste microbial protein powder in a fermenter

[0091] 1. Strain activation: The experimental strain Candida glabrata4H2 was streaked on YPD solid medium and incubated statically at 30°C for 48 hours to grow single colonies. Single colonies were picked and streaked on solid seed medium and incubated statically at 30°C for 20 hours for inoculation into shake flasks.

[0092] 2. Shake flask culture: Pick one loopful of the activated cells from step 1 and inoculate it into a 250mL Erlenmeyer flask containing 25mL of liquid seed culture medium. Incubate at 30℃ and 220rpm for 16h for fermentation inoculation.

[0093] 3. Fermentation tank culture: The seed culture prepared in step 2 was transferred to a 5L fermenter containing 3L of fermentation medium at an inoculation rate of 10% (v / v) for fermentation. The fermentation conditions were as follows: temperature 30℃, stirring speed 500rpm, NaOH was added during the culture process to control the pH of the fermentation system to be maintained at 5.5, and fermentation time 70h.

[0094] Seed culture medium (using waste bacterial protein powder as nitrogen source): glucose 30g / L, waste bacterial protein powder 30g / L, MgSO4·7H2O 0.5g / L, KH2PO4 1g / L.

[0095] Fermentation medium: glucose 120 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, CH3COONa 3 g / L. Urea solution 10 mL, trace element solution 10 mL, vitamin solution 15 mL; the urea solution, trace element solution, and vitamin solution were all prepared as stock solutions, sterilized by filtration, and then added before inoculation. The specific formula is as follows:

[0096] Urea solution: 386g, diluted with water to 1L.

[0097] Trace element solution: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, 0.5g ZnCl2, dissolved in 2mol / L HCl and brought to a final volume of 1L.

[0098] Vitamin solution: Biotin 0.004g, Thiamine 0.75mg, Pyridoxine 0.04g, Nicotinic acid 0.8g, dissolved in 2mol / L HCl, and brought to a final volume of 1L.

[0099] The fermentation broth from step 3, after fermentation, was centrifuged, and the supernatant was collected. The concentration of pyruvate in the supernatant was measured, and the results are shown in Table 1. The fermentation process curve is shown in... Figure 5As shown, waste protein cell powder was used as the seed culture medium nitrogen source to produce pyruvate in a fermenter. At the end of fermentation, the cell concentration was 21.3 g / L, the pyruvate yield was 61.8 g / L, the sugar-acid conversion rate was 0.55 g / g, and the pyruvate production intensity was 0.88 g / L / h.

[0100] Comparative example: Pyruvic acid production using soybean peptone as a nitrogen source

[0101] 1. Strain activation: The experimental strain Candida glabrata4H2 was streaked on YPD solid medium and incubated statically at 30°C for 48 hours to grow single colonies. Single colonies were picked and streaked on solid seed medium and incubated statically at 30°C for 20 hours for inoculation into shake flasks.

[0102] 2. Shake flask culture: Pick one loopful of the activated cells from step 1 and inoculate it into a 250mL Erlenmeyer flask containing 25mL of liquid seed culture medium. Incubate at 30℃ and 220rpm for 16h for fermentation inoculation.

[0103] 3. Fermentation tank cultivation: After completing step 2, the bacterial culture is transferred to a 5L fermenter containing 3L of fermentation medium at an inoculation rate of 10% (v / v) for fermentation. The fermentation conditions are as follows: temperature 30℃, stirring speed 500rpm, NaOH is added during the cultivation process to control the pH of the fermentation system to be maintained at 5.5, and fermentation time 70h.

[0104] Seed culture medium (using soybean peptone as nitrogen source): glucose 30 g / L, soybean peptone 10 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L.

[0105] Fermentation medium: glucose 120 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, CH3COONa 3 g / L. Urea solution 10 mL, trace element solution 10 mL, vitamin solution 10 mL. The urea solution, trace element solution, and vitamin solution were all prepared as stock solutions, sterilized by filtration, and then added before inoculation. The specific formula is as follows:

[0106] Urea solution: 386g, diluted with water to 1L.

[0107] Trace element solution: 12g MnCl2·4H2O, 2g FeSO4·7H2O, 2g CaCl2·2H2O, 0.05g CuSO4·5H2O, 0.5g ZnCl2, dissolved in 2mol / L HCl and brought to a final volume of 1L.

[0108] Vitamin solution: Biotin 0.004g, Thiamine 0.75mg, Pyridoxine 0.04g, Nicotinic acid 0.8g, dissolved in 2mol / L HCl, and brought to a final volume of 1L.

[0109] The fermentation broth from step 3, after fermentation, was centrifuged, and the supernatant was collected. The concentration of pyruvate in the supernatant was measured, and the results are shown in Table 1. The fermentation process curve is shown in... Figure 5 As shown, pyruvate was produced in a fermenter using soybean peptone as the seed culture medium nitrogen source. At the end of fermentation, the cell concentration was 19.4 g / L, the pyruvate yield was 56.9 g / L, the sugar-acid conversion rate was 0.53 g / g, and the pyruvate production intensity was 0.81 g / L / h.

[0110] Table 1. Pyruvate production in fermenters using nitrogen sources from different seed culture media

[0111]

[0112] Example 6 uses waste bacterial protein powder as a seed culture medium nitrogen source to produce pyruvate. Its yield, sugar-acid conversion rate and production intensity are all better than those of producing pyruvate using soybean peptone as a seed culture medium nitrogen source.

[0113] The inventors also attempted to prepare seed culture using yeast powder and corn steep liquor as nitrogen sources. The results showed that while yeast powder facilitated cell absorption and promoted cell growth, it did not significantly increase pyruvate production. Corn steep liquor's composition is not stable enough; factors such as storage and transportation can alter its composition, affecting pyruvate fermentation.

[0114] Cost reduction estimate for producing pyruvate by fermenting waste microbial protein powder:

[0115] In the first half of 2022, the market price of domestically produced soybean peptone was RMB 20,000-30,000 per ton, while the market price of imported soybean peptone was RMB 70,000-100,000 per ton. Based on the pyruvate yield of 61.8 g / L in Example 6, and assuming an 80% pyruvate extraction yield (referencing patent publication number CN106496022B), and with a market price of RMB 20,000 per ton of domestically produced soybean protein, approximately RMB 405 can be saved per ton of pyruvate produced (requiring 20.2 kg of soybean peptone); with a domestically produced soybean protein price of RMB 100,000 per ton, approximately RMB 2,020 can be saved per ton of pyruvate produced.

[0116] The strain cultured using waste bacterial protein powder as the nitrogen source in the seed culture medium only requires 15 mL of vitamin solution during fermentation. Compared with the 10 mL added when the initial soybean protein was used as the nitrogen source in the seed culture medium (comparative example), the impact of vitamin usage on fermentation cost is almost negligible.

[0117] Regarding the drying process, based on the calculation of spray drying 100kg of waste bacterial protein powder (with an initial solid content of 30%) using a 75kwh spray drying equipment, the energy cost of the spray drying process is about 200 yuan. The waste bacterial protein powder added to the culture medium is 30g / L. After conversion, the energy cost added to the drying process for each ton of pyruvate product is about 120 yuan.

[0118] A comprehensive cost analysis was conducted to determine the impact of replacing soybean peptone with waste microbial protein powder on the overall production process. Using waste microbial protein powder as a seed nitrogen source for pyruvate fermentation reduces production costs by 280 to 1900 yuan per ton of pyruvate produced. Furthermore, only a small portion of the obtained waste microbial protein powder is used as a seed nitrogen source in the pyruvate fermentation process; the majority can be used in bio-fertilizers and animal feed, generating additional economic benefits.

[0119] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of pyruvate fermentation waste cells in the fermentation of *Saccharomyces cerevisiae* to produce pyruvate, characterized in that, The pyruvate fermentation waste cells were used as the nitrogen source for the seed culture medium to cultivate *Gnaphalium affine*. The pyruvate fermentation waste cells were prepared by the following method: collecting the pyruvate fermentation broth of *Gnaphalium affine*, centrifuging to collect the solids, resuspending the solids, washing and drying them; the drying was spray drying, with the inlet air temperature of the drying tower at 130°C and the exhaust air temperature at 80°C.

2. The application according to claim 1, characterized in that, The seed culture medium contains: 25-35 g / L glucose, 5-35 g / L dry powder of the pyruvate fermentation waste cells, 0.4-0.6 g / L MgSO4·7H2O, and 0.8-1.2 g / L KH2PO4.

3. A method for producing pyruvic acid, characterized in that, Includes the following steps: (1) The smooth-skinned yeast was cultured in a seed culture medium containing pyruvate fermentation waste cells to obtain a seed liquid; the pyruvate fermentation waste cells were prepared by the following method: collecting the pyruvate fermentation broth of smooth-skinned yeast, centrifuging to collect the solids, resuspending the solids, washing and drying them; the drying was spray drying, with the inlet air temperature of the drying tower at 130°C and the outlet air temperature at 80°C. (2) Transfer the seed liquid prepared in step (1) to the fermentation medium and ferment at 28~35℃ for at least 50 h.

4. The method according to claim 3, characterized in that, The seed culture medium contains 5-35 g / L of pyruvate fermentation waste cells.

5. The method according to claim 3, characterized in that, The seed culture is prepared by inoculating activated *Saccharomyces cerevisiae* into the seed culture medium and culturing it at 28-30°C and 200-250 rpm. The seed culture medium also contains: glucose, MgSO4, and KH2PO4.

6. The method according to any one of claims 3 to 5, characterized in that, The fermentation medium contains: glucose, MgSO4 4、 KH2PO4, CH3COONa, urea, trace elements and vitamins.

7. A process for continuous production of pyruvic acid, characterized in that, Includes the following steps: (1) The seed culture of *Saccharomyces glabra* was cultured in seed culture medium for a period of time to obtain seed liquid, and then the seed liquid was transferred to fermentation culture medium for a period of time and the fermentation liquid was collected. (2) Centrifuge the fermentation broth from step (1), collect the solids, resuspend the solids, wash and dry them; the drying is spray drying, with the inlet air temperature of the drying tower at 130°C and the outlet air temperature at 80°C. (3) Use the bacterial powder obtained by drying in step (2) as the nitrogen source for the new round of seed culture medium to prepare the culture medium, and continue the operation of steps (1) to (3).

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