Terramycin mushroom dreg treatment and utilization method

Abstract

The invention discloses an oxytetracycline mushroom dreg treatment and utilization method. The method comprises the following steps: firstly, dehydrating mushroom dregs, mixing the dehydrated mushroom dregs with an acidic solution with the pH value of 3.0-4.0 in proportion, and carrying out drug removal pretreatment at 50-60 DEG C to destroy mycelia and degrade oxytetracycline; carrying out solid-liquid separation after pretreatment to obtain reagent-removed solid residues, and carrying out homogenizing and size mixing until the pH value is 6.5-7.5; then adding compound protease composed of neutral protease and alkaline protease according to the mass ratio of 1: 1-1: 2 for directional enzymolysis; after the enzymatic hydrolysate is subjected to solid-liquid separation, supernate is sequentially subjected to multi-stage membrane separation and purification steps such as microfiltration / ultrafiltration, 10kDa and 1kDa ultrafiltration membrane combination grading, nanofiltration desalination concentration and the like, and finally, a protein peptide product is obtained through drying. Optionally, a decoloring refining step may be added after nanofiltration. The invention also relates to a protein peptide product which is prepared by the method and has the oxytetracycline residue lower than 0.1 mg / kg and the molecular weight mainly distributed in 1-10kDa, and application of the protein peptide product. The method realizes high-value utilization of hazardous wastes, and has environmental and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of industrial solid waste resource processing, in particular to a terramycin bacterial residue processing and high-value utilization method, and particularly relates to a process for converting terramycin bacterial residue into protein peptide products. BACKGROUND

[0002] Terramycin is a broad-spectrum antibiotic, and a large amount of bacterial residue is produced in the fermentation production process. These bacterial residues contain rich organic substances such as proteins and polysaccharides, but also contain trace amounts of harmful substances such as terramycin and heavy metals, and are listed as hazardous waste. Traditional bacterial residue treatment methods such as landfill and incineration not only cause resource waste, but also have the risk of environmental secondary pollution.

[0003] At present, the resource utilization research of antibiotic bacterial residue mainly focuses on extracting residual antibiotics, producing feed protein or organic fertilizer, etc. However, direct use as feed protein has the safety hidden danger of antibiotic residue, and the product added value of composting treatment is low. Enzymatic hydrolysis technology can degrade macromolecular proteins into small molecular peptides and amino acids, improve their digestion and absorption rate and biological activity, and is an effective way for high-value utilization of protein resources. However, direct application of enzymatic hydrolysis technology to terramycin bacterial residue faces two major problems: first, the residual terramycin will inhibit the enzymatic hydrolysis efficiency, and may lead to antibiotic residue exceeding the standard in the product; second, the solid mycelium structure in the bacterial residue will hinder the full contact of the enzyme and the substrate, resulting in incomplete enzymatic hydrolysis.

[0004] Therefore, it is of great significance to develop a technology that can effectively remove terramycin residues, destroy the mycelium structure, and realize efficient and directional enzymatic hydrolysis to produce high-value protein peptide products, for solving the environmental protection pressure of pharmaceutical industry and realizing waste resource utilization. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide an efficient, safe and resourceful terramycin bacterial residue processing and utilization method. The method converts terramycin bacterial residue into high-value protein peptide products with low terramycin residue and rich small molecular active peptides by specific drug removal pretreatment combined with directional enzymatic hydrolysis and multi-stage membrane separation technology.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows: A terramycin bacterial residue processing and utilization method, characterized in that it comprises the following steps: a) Drug removal pretreatment: The terramycin mycelium residue is mechanically dewatered, and then mixed with a dilute hydrochloric acid or citric acid solution with a pH of 3.0-4.0 at a mass ratio of 2:1 to 4:1, and stirred at 50-60°C for 1-2 hours. This step aims to degrade the residual terramycin using acidic conditions, while also destroying the dense mycelium structure, making it loose and facilitating subsequent enzymatic hydrolysis. After the reaction is complete, solid-liquid separation is performed to obtain a drug-removed solid residue.

[0007] b) Homogenization and slurry preparation: The drug-removed solid residue obtained in step a) is mixed with water in a certain proportion, and homogenized to form a uniform slurry. Then, the slurry pH is adjusted to the neutral range (6.5-7.5) using a base such as sodium hydroxide solution, to create an optimal pH environment for subsequent enzymatic hydrolysis.

[0008] c) Directional enzymatic hydrolysis: A complex protease is added to the slurry obtained in step b). The complex protease is composed of a neutral protease (preferably trypsin) and an alkaline protease (preferably alkaline protease from Bacillus licheniformis or Bacillus subtilis) at a mass ratio of 1:1 to 1:2. The total amount of enzyme added is controlled at 0.5%-2.0% of the dry weight of the substrate (drug-removed solid residue). Enzymatic hydrolysis is carried out at a suitable temperature of 50-60°C for 2-4 hours, allowing the mycelium protein to be efficiently degraded into peptide fragments within the target molecular weight range. After the enzymatic hydrolysis is complete, the temperature is raised to 85-90°C for 10-15 minutes to inactivate the enzyme.

[0009] d) Solid-liquid separation: The mixture after enzyme inactivation is subjected to solid-liquid separation (such as centrifugation or filtration), and the supernatant rich in soluble protein peptides is collected, and the unhydrolyzed residue is discarded.

[0010] e) Multi-stage membrane separation and purification: The supernatant obtained in step d) is subjected to the following membrane treatment in sequence: Microfiltration or ultrafiltration: To remove small suspended particles and bacteria remaining in the supernatant.

[0011] Combined ultrafiltration fractionation: Sequentially use ultrafiltration membranes with molecular weight cut-offs of 10 kDa and 1 kDa for fractionation, and collect the target protein peptide components with molecular weights between 1-10 kDa. Peptides in this range generally have good biological activity and solubility.

[0012] Nanofiltration desalination and concentration: The target peptide components are subjected to desalination and concentration at the same time to obtain protein peptide concentrate with higher purity and lower salt content.

[0013] f) Drying: The protein peptide concentrate obtained in step e) is dried, preferably by spray drying, to obtain a powdered protein peptide product.

[0014] As a preferred embodiment of the present invention, a decolorization and purification step may be added between step e) and step f): the protein peptide solution concentrated by nanofiltration is decolorized by passing it through a macroporous adsorption resin column or activated carbon column to remove pigments and improve the color of the product.

[0015] This invention also protects the protein peptide product prepared by the above method. The oxytetracycline residue in the product is less than 0.1 mg / kg, which meets the relevant safety standards; the peptide molecular weight is mainly distributed between 1-10 kDa; the product is a water-soluble powder with a protein recovery rate of not less than 70%, and is rich in small molecule active peptides, which can be used in functional foods, health products or animal feed.

[0016] This invention further protects the application of the above method in the resource utilization of pharmaceutical industrial waste, especially in converting oxytetracycline bacterial residue into high-value-added protein peptide products.

[0017] The beneficial effects of this invention are as follows: 1. Highly efficient drug removal and cell wall disruption: Pretreatment with an acidic solution of a specific pH under gentle heating conditions can not only effectively degrade residual oxytetracycline (reducing it to below 0.1 mg / kg), but also disrupt the mycelial cell wall, significantly improving the efficiency of subsequent enzymatic hydrolysis.

[0018] 2. Targeted enzymatic hydrolysis: A specific ratio of neutral protease and alkaline protease is used to work synergistically to efficiently and directionally hydrolyze bacterial residue protein into target small molecule peptides with a main content of 1-10 kDa, resulting in products with high bioactivity.

[0019] 3. High purity and high recovery rate: Through multi-stage membrane separation and purification technology of microfiltration / ultrafiltration-combined ultrafiltration-nanofiltration, the target peptides are effectively fractionated, desalted and concentrated, resulting in high product purity and a protein recovery rate of over 70%.

[0020] 4. Safety and high added value: The final product has extremely low antibiotic residues, is safe and harmless, and is a small molecule protein peptide with nutritional and economic value far exceeding that of ordinary feed protein. It can be applied to higher-end functional foods, health products and other fields.

[0021] 5. Environmentally friendly and resource-efficient: The entire process achieves the harmlessness, reduction and resource utilization of hazardous waste, turning waste into treasure, solving the environmental problems of pharmaceutical companies, and meeting the requirements of green circular economy development. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of the method for treating and utilizing oxytetracycline bacterial residue according to the present invention. Detailed Implementation

[0023] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1 1. De-drying pretreatment: Take 1 kg of oxytetracycline bacterial residue (200 g dry basis) with a moisture content of approximately 80%, and centrifuge to remove water until the moisture content is 65%. Then mix it with 400 g of dilute hydrochloric acid solution with pH 3.5 (solid-liquid mass ratio approximately 2.5:1), and stir at 55℃ for 1.5 hours. After the reaction, centrifuge to obtain approximately 350 g of de-drying solid residue (wet basis).

[0025] 2. Homogenization and slurry preparation: Mix the above-mentioned de-medicated solid residue with 700g of water, homogenize, and then adjust the pH of the slurry to 7.0 with dilute NaOH solution.

[0026] 3. Targeted enzymatic hydrolysis: Add a complex protease (neutral protease: alkaline protease = 1:1.5) to the slurry, with the amount of enzyme added being 1.2% of the substrate dry weight (based on an initial dry basis of 200g). Hydrolyze at 55℃ for 3 hours. After hydrolysis, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme.

[0027] 4. Solid-liquid separation: Centrifuge the enzyme hydrolysate and collect approximately 900 mL of supernatant.

[0028] 5. Multistage membrane separation and purification: The supernatant is first passed through a 0.2 μm microfiltration membrane to remove suspended particles. Then it is passed sequentially through 10 kDa and 1 kDa ultrafiltration membranes, collecting 1-10 kDa permeate / retentate (depending on membrane configuration). Finally, it is desalted by nanofiltration and concentrated to approximately 100 mL.

[0029] 6. Drying: Spray dry the concentrate (inlet air temperature 180℃, outlet air temperature 85℃) to obtain 28.5g of light yellow powdered protein peptide product.

[0030] Example 2 1. De-drug pretreatment: Take 1 kg of oxytetracycline bacterial residue (220 g dry basis) with a moisture content of approximately 78%, and centrifuge to reduce the moisture content to 65%. Then mix it with 300 g of citric acid solution with pH 3.0 (solid-liquid mass ratio approximately 4:1) and stir at 60°C for 1 hour. After the reaction, centrifuge to obtain de-drug solid residue (wet basis, approximately 380 g).

[0031] 2. Homogenization and slurry preparation: Mix the above-mentioned de-treated solid residue with 1140g of water, homogenize, and then adjust the pH of the slurry to 6.8 with dilute NaOH solution.

[0032] 3. Targeted enzymatic hydrolysis: Add a complex protease (neutral protease: alkaline protease = 1:2) to the slurry, with the amount of enzyme added being 0.8% of the substrate dry weight (based on an initial dry basis of 220g). Hydrolyze at 60℃ for 2 hours. After hydrolysis, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme.

[0033] 4. Solid-liquid separation: Centrifuge the enzyme hydrolysate and collect approximately 1350 mL of supernatant.

[0034] 5. Multistage membrane separation, purification, and decolorization: The supernatant is first passed through a 0.2 μm microfiltration membrane to remove suspended particles. Then, it is sequentially passed through 10 kDa and 1 kDa ultrafiltration membranes, collecting the 1-10 kDa permeate / retentate (depending on membrane configuration). The solution is then decolorized using an activated carbon column, and finally desalted and concentrated to approximately 100 mL using a nanofiltration membrane.

[0035] 6. Drying: The concentrate was spray-dried (inlet air temperature 180℃, outlet air temperature 85℃) to obtain 26.1g of off-white protein peptide powder product.

[0036] Example 3 1. De-drying pretreatment: Take 1 kg of oxytetracycline bacterial residue (180 g dry basis) with a moisture content of approximately 82%, and centrifuge to reduce the moisture content to 65%. Then mix it with 500 g of dilute hydrochloric acid solution with pH 4.0 (solid-liquid mass ratio approximately 2:1), and stir at 50°C for 2 hours. After the reaction, centrifuge to obtain approximately 316 g of de-drying solid residue (wet basis).

[0037] 2. Homogenization and slurry preparation: Mix the above-mentioned de-medicated solid residue with 632g of water, homogenize, and then adjust the pH of the slurry to 7.5 with dilute NaOH solution.

[0038] 3. Targeted enzymatic hydrolysis: Add a complex protease (neutral protease: alkaline protease = 1:1) to the slurry, with the enzyme amount being 2.0% of the substrate dry weight (based on an initial dry basis of 180g). Hydrolyze at 50℃ for 4 hours. After hydrolysis, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme.

[0039] 4. Solid-liquid separation: Centrifuge the enzyme hydrolysate and collect approximately 800 mL of supernatant.

[0040] 5. Multistage membrane separation and purification: The supernatant is first passed through a 0.2 μm microfiltration membrane to remove suspended particles. Then it is passed sequentially through 10 kDa and 1 kDa ultrafiltration membranes, collecting 1-10 kDa permeate / retentate (depending on membrane configuration). Finally, it is desalted by nanofiltration and concentrated to approximately 100 mL.

[0041] 6. Drying: The concentrate was spray-dried (inlet air temperature 180℃, outlet air temperature 85℃) to obtain 29.8g of light yellow protein peptide powder product.

[0042] Comparative Example 1 Similar to Example 1, the difference is that the de-drug pretreatment in step 1 is omitted; instead, the dehydrated oxytetracycline residue is directly homogenized and slurryed, with subsequent steps identical to Example 1. The results showed a significant decrease in enzymatic hydrolysis efficiency, with a final product yield of only 15.2g. Furthermore, testing revealed a oxytetracycline residue content of 5.8mg / kg in the product, far exceeding safety standards.

[0043] Comparative Example 2 Similar to Example 1, except that in step 1, a near-neutral aqueous solution with a pH of 6.0 was used instead of an acidic solution for the drug removal pretreatment; other conditions were the same as in Example 1. The results showed that mycelial destruction was insufficient, and the degradation of oxytetracycline was poor (1.2 mg / kg residue), leading to incomplete subsequent enzymatic hydrolysis and low product yield (19.5 g) and protein recovery rate.

[0044] Comparative Example 3 Similar to Example 1, the difference lies in the targeted enzymatic hydrolysis in step 3, where only a single neutral protease is used, and the amount of enzyme added is the same as the total amount of enzyme in Example 1. Other steps are the same as in Example 1. The hydrolysis solution has a lower degree of hydrolysis, resulting in a lower proportion of the target small molecule peptides (1-10 kDa), thus reducing the product's functionality and value.

[0045] Comparative Example 4 Similar to Example 1, the difference lies in omitting the combined ultrafiltration and nanofiltration desalination steps in step 5, and directly spray-drying the supernatant after the initial solid-liquid separation. The resulting product is dark in color, has a salty taste, poor solubility, and contains a large amount of large molecular proteins and salts, with low purity of the target peptides and poor quality.

[0046] Table 1. Test results of the examples and comparative examples.

[0047] The data above shows that, compared with Example 1, the product of Comparative Example 1 (without de-drug treatment) had a oxytetracycline residue of up to 5.8 mg / kg, far exceeding the safety standard of 0.1 mg / kg, proving that the product is unsafe. At the same time, the enzymatic hydrolysis efficiency was low, and the yield and protein recovery rate decreased significantly. This indicates that de-drug pretreatment is not only the key to reducing antibiotic residues, but also a prerequisite for destroying the mycelial structure and ensuring the efficiency of subsequent enzymatic hydrolysis. Compared with Example 1, the de-drug and cell wall disruption effects of Comparative Example 2 (using neutral water) were not ideal, resulting in an excessive oxytetracycline residue (1.2 mg / kg) and a low product yield. This proves that a specific acidic environment (pH 3.0-4.0) is indispensable for the effective degradation of oxytetracycline and the loosening of the mycelial structure. Compared with Example 1, Comparative Example 3 (using a single enzyme) was acceptable in terms of yield and safety, but the proportion of its target small molecule peptides (1-10kDa) was significantly reduced. This indicates that the combination of neutral protease and alkaline protease in a specific ratio can produce a synergistic effect, hydrolyzing proteins into active peptides within the required molecular weight range more efficiently and directionally, thereby improving the functionality and added value of the product. Compared with Example 1, Comparative Example 4 (without membrane separation) yielded a higher total amount of solid product, but it contained a large amount of non-target macromolecular proteins, salts, and impurities, resulting in low purity of the target peptides and poor product color, taste, and solubility, leading to low quality. In contrast, the examples effectively removed impurities and enriched the target peptides through multi-stage membrane separation, resulting in high-purity, high-quality protein peptide products.

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A terramycin bacterial residue treatment and utilization method, characterized in that, The method comprises the following steps: a) drug removal pretreatment: the terramycin fungal residue is dehydrated, then mixed with a dilute hydrochloric acid or citric acid solution with a pH of 3.0-4.0 at a mass ratio of 2:1 to 4:1, reacted at 50-60°C for 1-2 hours to degrade the residual terramycin and destroy the mycelium structure, and then subjected to solid-liquid separation to obtain a drug removal solid residue; b) homogenization and slurry preparation: the drug removal solid residue obtained in step a) is mixed with water for homogenization, and the pH of the slurry is adjusted to 6.5-7.5; c) directional enzymolysis: a complex protease composed of neutral protease and alkaline protease at a mass ratio of 1:1 to 1:2 is added to the slurry obtained in step b), the total amount of enzyme is 0.5%-2.0% of the dry weight of the substrate, and the enzyme is hydrolyzed at 50-60°C for 2-4 hours, and then the enzyme is inactivated by heating; d) solid-liquid separation: the mixture after enzymolysis is subjected to solid-liquid separation, and the supernatant rich in protein peptides is collected; e) multi-stage membrane separation and purification: the supernatant obtained in step d) is sequentially subjected to microfiltration or ultrafiltration to remove suspended particles, subjected to fractional separation by combining ultrafiltration membranes with a molecular weight cutoff of 10 kDa and 1 kDa to collect the target protein peptide component with a molecular weight of 1-10 kDa, and then subjected to nanofiltration desalting and concentration to obtain a protein peptide concentrate; f) drying: the protein peptide concentrate obtained in step e) is dried to obtain a protein peptide product.

2. The method of claim 1, wherein, In step c), the neutral protease is trypsin, and the alkaline protease is an alkaline protease from Bacillus licheniformis or Bacillus subtilis.

3. The method of claim 1, wherein, Between steps e) and f), a decolorization and purification step is further included: the protein peptide solution after nanofiltration concentration is subjected to decolorization treatment by passing through a macroporous adsorption resin column or an activated carbon column.

4. The method of claim 1, wherein, In step f), the drying is spray drying.

5. A protein peptide product prepared by the method of any one of claims 1-4, wherein, The product has a terramycin residue of less than 0.1 mg / kg, and the peptide molecular weight is mainly distributed between 1-10 kDa.

6. The protein peptide product according to claim 5, characterized in that, The product is a powder with good water solubility, and can be used in the fields of functional food, health products or animal feed.

7. The protein peptide product according to claim 5, characterized in that, The protein recovery rate of the product is not less than 70%, and the product is rich in small molecule active peptides.

8. The use of a method according to any one of claims 1 to 4 for the recycling of pharmaceutical industry waste, characterized by, The terramycin fungal residue is converted into a high-value protein peptide product.

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