Culture method for producing single-cell protein from microorganisms

Abstract

The invention provides a culture method for producing single-cell protein by microorganisms. The single-cell protein is obtained by taking a product obtained by hydrolyzing straws through cellulase as a carbon source, shrimp and crab shells as a nitrogen source and clostridium tyrobutyricum as a microbial provenance through fermentation. According to the method, straw is adopted as an initial raw material of a carbon source, and shrimp and crab shells are adopted as a nitrogen source, so that the production cost is further reduced, and the protein yield is remarkably improved. The output-input ratio of production is increased from the raw material side and the product side, and industrial application is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of microbial single-cell protein, in particular to a culture method for producing single-cell protein by microorganisms at low cost and application thereof. Background Art

[0002] The economic viability of microbial fermentation is highly dependent on culture medium costs, which account for 30%-50% of the total fermentation cost. Carbon sources (e.g., glucose) and nitrogen sources (e.g., yeast extract) contribute 70%-80% of the total culture medium raw material cost. High carbon and nitrogen inputs severely restrict the industrial production of microbial single-cell proteins. Therefore, reducing culture medium costs by substituting inexpensive raw materials is a primary challenge addressed by this method. Furthermore, bacterial cell residues are an inherent byproduct of the fermentation process, and traditional disposal methods primarily result in waste. Utilizing the Clostridium tyrobutyricum cells as a resource can further enhance the added value of the process.

[0003] Microbial single-cell protein production initially used traditional sugar products or commercial carbon sources (glucose, xylose) to construct microbial growth media. This not only increased production costs but also failed to meet the requirements of sustainable production. Building on my previous invention patent (ZL 2022 1 0840433.2), I developed a cost-effective method for producing single-cell protein from Clostridium tyrobutyricum by replacing the commercial carbon source in the original patent with a hydrolyzate of waste straw. Summary of the Invention

[0004] The patent previously applied for by the applicant (application number 202210840433.2) uses waste shrimp and crab shell powder as a nitrogen source to ferment and produce single-cell protein. Although the use of waste shrimp and crab shells reduces production costs, it uses glucose as a carbon source, and the production cost is still relatively high.

[0005] To address the aforementioned challenges of the prior art, the present invention provides a method for cultivating microorganisms to produce single-cell protein. This method uses straw as a carbon source and shrimp and crab shells as a nitrogen source, further reducing production costs while significantly increasing protein yield. This method improves the input-output ratio of production from both the raw material and product sides, facilitating industrial application.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for cultivating single-cell protein produced by microorganisms, which uses a product of cellulase hydrolysis of straw as a carbon source, shrimp and crab shells as a nitrogen source, and Clostridium tyrobutyricum as a microbial source for fermentation to obtain the single-cell protein.

[0007] Preferably, the carbon source is obtained by: The straw is subjected to acid treatment, and then cellulase is added under acidic conditions and hydrolyzed at 50-57° C. for 24-48 hours to obtain the carbon source.

[0008] Preferably, the acid treatment conditions are: adding the straw into the acid solution at a solid-liquid ratio of 5-10% w / v and immersing the straw, and then treating the straw at 108-121° C. for 20-30 min.

[0009] Preferably, the straw is rice straw.

[0010] Preferably, the acid solution is a sulfuric acid solution, and the concentration of the acid solution is 0.02-0.05 M.

[0011] Preferably, the acidic condition is pH=5.

[0012] Preferably, the amount of cellulase added is 0.5-2% w / v.

[0013] Preferably, the acid treatment further comprises the step of filtering out unreacted solids.

[0014] Preferably, the shrimp and crab shells are discarded shrimp and crab shell powder.

[0015] Preferably, the waste shrimp and crab shell powder is made by cleaning, drying and grinding waste shrimp and crab shells in kitchen waste without undergoing acid or alkali treatment.

[0016] Preferably, the carbon source and the nitrogen source satisfy a carbon-nitrogen ratio of 4.56:1.

[0017] Preferably, the product of cellulase hydrolysis of the straw and the shrimp and crab shells are the only two nutritional components of the fermentation.

[0018] The Clostridium tyrobutyricum is Clostridium tyrobutyricum ( Clostridium tyrobutyricum )L319, deposit number GDMCC No: 62289.

[0019] The beneficial effects of the present invention are: 1. The present invention uses straw and shrimp and crab shells as the only two nutrients to produce single-cell protein, further reducing production costs.

[0020] 2. Compared to the prior art (Application No. 202210840433.2), this invention increases protein content from 47.2g / 100g to 52.8g / 100g, a 11.9% increase in protein yield. Furthermore, the proportions of tyrosine, aspartic acid, glutamic acid, and lysine in the product are significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The following is a comparison of the growth of Clostridium tyrobutyricum in different culture media in Example 1 (2). A is the growth curve; B is the maximum specific growth rate.

[0022] Figure 2 The graphs are comparative diagrams of the growth of Clostridium tyrobutyricum in straw, shrimp and crab shell culture medium with different carbon-nitrogen ratios in Example 1 (3). A is the growth curve and B is the maximum specific growth rate.

[0023] Figure 3 This is a comparison chart of the protein content (A) and biomass (B) of Clostridium tyrobutyricum fermented in SSS medium and TGXY medium in Example 1 (4).

[0024] Figure 4 This is a comparison chart of the amino acid composition of bacterial proteins of Clostridium tyrobutyricum in SSS medium and TGXY medium in Example 1 (4). DETAILED DESCRIPTION

[0025] Example 1 (1) 200 g of rice straw (purchased from an online agricultural product processing plant) was placed in a 5 L glass beaker. The rice straw was immersed in a 0.02 M H2SO4 solution at a solid-liquid ratio of 10% (w / v) and treated at 121°C for 30 min. After the liquid cooled to room temperature, the pH was adjusted to 5, the optimal pH for cellulase, using 2 M NaOH solution.

[0026] After adding cellulase at a ratio of 1% (w / v), the beaker was placed at 57°C for hydrolysis for about 48 h.

[0027] The straw hydrolyzate was filtered through a mesh to preliminarily remove the unhydrolyzed rice straw. The rice straw hydrolyzate was then concentrated to 200 mL using a rotary evaporator. The pH was then adjusted to 6 using a 2 M NaOH solution. The solution was then centrifuged at 8,000 rpm for 5 min in a refrigerated centrifuge to further remove small particles of rice straw, mud, and other solid impurities in the rice straw hydrolyzate. The rice straw hydrolyzate was finally obtained and filtered through 0.45 μm before use.

[0028] Take discarded shrimp and crab shells, which come from kitchen waste, clean, dry, grind them, and pass them through a 100-mesh sieve before use.

[0029] (2) Determine the carbon and nitrogen source components of the culture medium: Three experimental groups were set up, each with three replicates, and fermentation was performed using 20 mL of the system in serum bottles.

[0030] The experimental groups are: CE group: 20 mL of rice straw hydrolyzate obtained in step (1) was used as the carbon source (containing approximately 10 g / L glucose and 1.7 g / L xylose), without adding glucose and xylose, and 10 g / L yeast powder and 30 g / L peptone were used as the nitrogen source.

[0031] ABD group: 20 g / L shrimp and crab shell powder was used as nitrogen source, and 10 g / L glucose and 1.5 g / L xylose were used as carbon sources.

[0032] CD group: 20 mL of rice straw hydrolyzate obtained in step (1) was used as the carbon source, without adding glucose and xylose, and 20 g / L shrimp and crab shell powder was used as the nitrogen source.

[0033] The control group was TGXY medium, which contained 30 g / L peptone (nitrogen source), 10 g / L yeast powder (nitrogen source), 10 g / L glucose (carbon source), and 1.5 g / L xylose (carbon source).

[0034] Each culture medium was sterilized at 108°C for 30 minutes and then inoculated with 5% seed from an overnight culture of Clostridium tyrobutyricum L319 (Accession No. GDMCC 62289). The cultures were incubated at 37°C for 50 hours, with growth OD and product yield measured at regular intervals.

[0035] The results are as follows Figure 1 As shown in the figure, whether it is the CE group and CD group using straw hydrolyzate as carbon source, or the TGXY group and ABD group using xylose and glucose as carbon source, they can grow normally and there is no significant difference in the maximum biomass. Figure 1 As shown in Figure 2, the maximum specific growth rate of the CE group was 0.243 h -1 The maximum specific growth rate of the TGXY group was 0.255 h -1 The maximum specific growth rate of the ABD group was 0.241 h -1 The maximum specific growth rate of the CD group was 0.246 h -1 , and there was no significant difference in the maximum specific growth rate among the groups. This shows that in the serum bottle fermentation system, straw hydrolyzate and shrimp and crab shell powder (CE group) have no obvious negative impact on the growth of Clostridium tyrobutyricum and can be used as culture media for Clostridium tyrobutyricum fermentation.

[0036] (3) Ratio of carbon and nitrogen sources in the optimized culture medium: Five experimental groups with different carbon-nitrogen ratios were set up, with three replicates in each group, and fermentation was performed using a 20 mL system in serum bottles.

[0037] The experimental groups are: 30 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate, with a carbon-nitrogen ratio of 3.04:1 (20 mL of straw hydrolyzate, 0.6 g of shrimp and crab shell powder).

[0038] 20 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate, with a carbon-nitrogen ratio of 4.56:1 (20 mL of straw hydrolyzate, 0.4 g of shrimp and crab shell powder).

[0039] 15 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate, with a carbon-nitrogen ratio of 6.08:1 (20 mL of straw hydrolyzate, 0.3 g of shrimp and crab shell powder).

[0040] 10 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate, with a carbon-nitrogen ratio of 9.12:1 (20 mL of straw hydrolyzate, 0.2 g of shrimp and crab shell powder).

[0041] 5 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate, with a carbon-nitrogen ratio of 18.25:1 (20 mL of straw hydrolyzate, 0.1 g of shrimp and crab shell powder).

[0042] Each culture medium was sterilized at 108°C for 30 minutes and then inoculated with an overnight culture of Clostridium tyrobutyricum L319 (Accession No. GDMCC 62289) at a 5% inoculum level. The cultures were incubated at 37°C for 50 hours, with OD and growth rate measurements taken at regular intervals.

[0043] The results are as follows Figure 2 As shown, when 20 g / L of shrimp and crab shell powder was added and the C / N ratio of the rice straw / shrimp and crab shell culture medium was 4.56:1, the butyric acid concentration reached its highest level, 4.72 g / L. When 30 g / L of shrimp and crab shell powder was added and the C / N ratio of the rice straw / shrimp and crab shell culture medium was 3.04:1, the butyric acid concentration reached its lowest level, 4.24 g / L. The acetic acid concentration in each group showed an overall trend of first increasing and then decreasing, ultimately stabilizing at around 1 g / L. Based on this, the optimal C / N ratio of the rice straw / shrimp and crab shell culture medium was determined to be 4.56:1, which means adding 20 g of shrimp and crab shell powder per liter of rice straw hydrolyzate.

[0044] (4) Fermentation production of single-cell protein: The rice straw hydrolyzate obtained in step (1) was used as the sole carbon source, and the shrimp and crab shell powder processed in step (1) was used as the sole nitrogen source. The SSS culture medium was prepared according to a carbon-nitrogen ratio of 4.56:1, i.e., 20 g of shrimp and crab shell powder was added per liter of rice straw hydrolyzate. The other components of the SSS culture medium were the same as those in step (2).

[0045] Determination of protein content in shrimp and crab shell powder using an automated Kjeldahl nitrogen analyzer: Place 0.2 g (accurate to 0.001 g) of shrimp and crab shell powder into a digestion tube. Add 0.2 g of copper sulfate, 3 g of potassium sulfate, and 10 mL of concentrated sulfuric acid. Place the digestion tube in a graphite digester for digestion. Continue digestion for 1 hour after the digester temperature reaches 420°C, at which point the liquid in the digestion tube will appear light green and transparent. Remove the sample, cool it, and then automatically add liquid and distill it on an automated Kjeldahl nitrogen analyzer. The collected distillate is titrated and recorded to calculate the nitrogen and protein content of the sample. Three replicates are performed for each group.

[0046] To determine the bacterial protein content of Clostridium tyrobutyricum using a BCA kit, 200 μL of the bacterial culture was collected at 36 hours of serum bottle fermentation. The sample was centrifuged and treated with 0.05 mol / L NaOH solution at 100°C for 20 minutes to release intracellular proteins. Protein content was determined using a BCA kit from Nanjing Novozymes Biotech Co., Ltd., and protein concentration was calculated using a standard protein curve.

[0047] Amino acid determination method: Add 20 mL of the final fermentation broth to a 50 mL centrifuge tube and centrifuge at 8,000 rpm for 10 minutes. Remove the supernatant. Add 20 mL of sterile water, vortex to mix, centrifuge at 8,000 rpm for 10 minutes, and remove the supernatant. Repeat this process twice. Leave the bacterial slurry at the bottom of the centrifuge tube. Seal the tube with parafilm and pre-freeze at -80°C for 12 hours. Then, freeze-dry the tube in a vacuum freeze dryer overnight. Collect the dried bacterial powder for future use. Subsequent amino acid content determination will be performed by Shenzhen Huace Testing.

[0048] The protein content of Clostridium tyrobutyricum fermented in SSS medium and TGXY medium is as follows Figure 3 As shown in Figure A, when Clostridium tyrobutyricum reaches the stable phase of growth in SSS medium, the protein content of bacterial protein is 52.8%, while the protein content of bacterial protein in TGXY medium is 45.4%, the former is 16.3% higher than the latter. Therefore, the production of bacterial protein by fermentation of Clostridium tyrobutyricum in SSS medium has higher biomass and protein content, which is more conducive to achieving high-level production of bacterial protein of Clostridium tyrobutyricum. Figure 3 B It can be seen that the biomass of Clostridium tyrobutyricum in SSS medium is 6.4 g / L, while the biomass in TGXY medium is 5.7 g / L, the former is 13.1% higher than the latter.

[0049] Depend on Figure 4It can be seen that when Clostridium tyrobutyricum was grown in SSS medium, the proportion of aspartic acid, glutamic acid and glycine increased significantly compared with that in TGXY medium. The proportions of the three were 6.5%, 7.4% and 3.6%, respectively, which increased by 16.1%, 13.8% and 44%, respectively.

[0050] The comparison of nutrient content in the culture medium of the prior patent (application number 202210840433.2) is shown in Table 1.

[0051] Comparison Type Content in prior patents (g / 100 g) Content in the present invention (g / 100 g) Increase ratio (%) Nitrogen content 7.55 8.45 11.9 Protein content 47.2 52.8 11.9 Aspartic acid (ASP) 3.4 6.5 91.2 Glutamate (GLU) 4.2 7.4 76.2 Glycine (GLY) 1.5 3.6 140.0 Serine (SER) 1.5 2.8 86.7 Threonine (THR) 1.5 2.0 33.3 Arginine (ARG) 1.8 5.3 194.4 Alanine (ALA) 1.7 3.2 88.2 Tyrosine (TYR) 1.4 5.6 300.0 Valine (VAL) 1.6 5.4 237.5 Phenylalanine (PHE) 1.7 2.1 23.5 Isoleucine (ILE) 1.5 3.8 153.3 Leucine (LEU) 2.3 4.4 91.3 Lysine (LYS) 2.0 5.6 180.0 While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for cultivating microorganisms to produce single-cell protein, characterized in that: The single-cell protein is obtained by fermentation using a product of straw hydrolyzed by cellulase as a carbon source, shrimp and crab shells as a nitrogen source, and Clostridium tyrobutyricum as a microbial source.

2. The culture method according to claim 1, wherein The method for obtaining the carbon source is: The straw is subjected to acid treatment, and then cellulase is added under acidic conditions, and hydrolyzed at 50-57° C. for 24-48 hours, preferably at 57° C. for 48 hours, to obtain the carbon source.

3. The culture method according to claim 2, wherein The acid treatment conditions are as follows: adding the straw into the acid solution at a solid-liquid ratio of 5-10% w / v, preferably 10% w / v, immersing the straw, and then treating at 108-121° C. for 20-30 min, preferably 121° C. for 30 min.

4. The culture method according to claim 2, wherein The straw is rice straw.

5. The culture method according to claim 2, wherein The acid solution is a sulfuric acid solution, and the concentration of the acid solution is 0.02-0.05M, preferably 0.02M.

6. The culture method according to claim 2, wherein The acidic condition is pH=5.

7. The culture method according to claim 2, wherein The added amount of the cellulase is 0.5-2% w / v, preferably 1% w / v.

8. The culture method according to claim 1, wherein The shrimp and crab shells are discarded shrimp and crab shell powder.

9. The culture method according to claim 1, wherein The carbon source and the nitrogen source satisfy a carbon-nitrogen ratio of 4.56:

1.

10. The culture method according to claim 1, wherein The Clostridium tyrobutyricum is Clostridium tyrobutyricum L319, with a preservation number of GDMCC No: 62289.

Images