Method for preparing micromolecule polypeptide from municipal sludge and recycling micromolecule polypeptide

Abstract

The invention relates to a method for separating and preparing micro-molecule polypeptide from municipal sludge and recycling the micro-molecule polypeptide, which comprises the following steps: obtaining high-purity protein through the technological processes of concentration, purification and the like, further hydrolyzing to generate the micro-molecule polypeptide and recycling the micro-molecule polypeptide. The micromolecule polypeptide is prepared through the five stages of pretreatment, sludge pyrolysis, protein extraction and concentration, protein separation and purification and enzymolysis conversion, the micromolecule polypeptide with extremely high value can be obtained, and other generated by-products such as residual solid powder and residual nutrient solution can be used in the fields of building material industry, soil improvement and the like; and resource comprehensive utilization is realized.

Description

Technical Field

[0001] The present invention relates to the field of solid waste resource utilization, and particularly relates to a method for efficiently separating and preparing small molecule polypeptides from excess sludge through a hot alkali method combined with an enzymatic hydrolysis process to achieve high-value resource utilization of excess sludge. Background Art

[0002] Municipal sludge is an inevitable by-product in the sewage treatment process. If it is not reasonably treated and disposed of in time, it will cause serious secondary pollution to the environment. There are three main ways to dispose of municipal sludge: harmless disposal, reduction disposal, and resource utilization disposal. Among them, resource utilization mainly refers to the utilization method of becoming fertilizer after being disposed of by different technical means.

[0003] At present, the resource utilization rate of municipal sludge is extremely low, mainly limited by: the relatively high access threshold, strictly controlling the entry of municipal sludge-related products into farmland; the diverse sources of municipal sludge raw materials, and it is necessary to strictly control the raw materials (mainly controlling the content of heavy metals, Cr 6 + , Al, viruses, antibiotics, etc.) to meet the required standards for subsequent resource utilization; there are relatively few types of technologies for the resource utilization of municipal sludge that can be promoted and the maturity is relatively low.

[0004] Since municipal sludge is rich in organic matters such as proteins and polysaccharides, proteins can be extracted from the sludge and utilized to achieve high-value resource utilization of excess sludge. The organic matters such as proteins in municipal sludge are mainly stored in the active microorganisms in the sludge. It is necessary to first perform certain physical / chemical process treatment on the municipal sludge to break the microbial cell walls and release the high-value organic matters, and then separate the organic matters through process methods.

[0005] Chinese Patent Publication No. CN 114106081 A discloses a method for extracting protein from excess sludge. The specific operation steps are as follows: First, the excess sludge is adjusted with water to a concentration of 100 - 200 g / L; then, a composite enzyme preparation composed of glycosidase (α-amylase and β-amylase in a mass ratio of 1 - 5:1) and lipase in a mass ratio of 1 - 5:1 is added, and enzymolysis is carried out at 20 - 40 °C at 30 - 150 rpm for 0.5 - 2 hours; then, the enzymolysis solution is pressurized to 0.1 - 2 MPa, the pH is adjusted to 10 - 13, and stirring hydrolysis is carried out at 100 - 180 °C at 50 - 500 rpm for 1 - 5 hours; finally, the hydrolysis solution is pressure-filtered (pressure 0.8 - 2 MPa, time 0.5 - 4 hours), and evaporation concentration is carried out at 80 - 120 °C for 0.5 - 4 hours to obtain a 20% - 40% protein solution. There are certain problems in the above process: (1) When the temperature exceeds 100 °C, the Maillard reaction occurs between reducing sugar and amino acid or protein, generating pigments such as melanoidins and flavor substances. And this reaction causes a significant increase in the viscosity of the hydrolyzed solution after pyrolysis, partially forming a colloidal solution, thus reducing its fluidity and increasing the difficulty of subsequent pressure filtration separation; (2) In the hot hydrolysis process, to ensure the efficient dissolution of protein, the temperature is usually controlled at about 650 °C. Research shows that the introduction of an appropriate alkaline environment (pH 10 - 13) can significantly reduce the temperature required for cell wall cracking, thereby enhancing the intensity of the cell cracking reaction. Under this condition, the amount of protein released by cracking is sufficient to make up for the protein loss during hydrolysis and denaturation, and thus significantly improve the protein dissolution rate. However, considering the biological activity and structural stability of protein, on the premise that the protein dissolution rate is higher than the denaturation rate, too high a pyrolysis temperature may cause protein denaturation, so the temperature needs to be strictly controlled. In the patented process, although effective dissolution of protein is achieved in the temperature range of 100 - 180 °C, the problem of maintaining protein activity is not fully considered, and the process of maintaining protein activity has always been one of the key research and optimization directions in this field. (3) In the patented process, the concentrated and extracted protein solution contains a relatively high level of impurities and has not undergone further separation and purification treatment, which to a certain extent limits the application scope of protein products.

[0006] Small molecule polypeptides are in great demand, have high value, and good therapeutic effects in the fields of medicine, beauty, drugs, etc. Therefore, preparing small molecule polypeptides from protein can create greater economic value. However, there are not many high-quality protein sources that can be used to prepare small molecule polypeptides, and the cost of pure protein is expensive. Summary of the Invention

[0007] The object of the present invention is to propose a method for separating and preparing small molecule polypeptides from municipal sludge and carrying out resource utilization. By optimizing the hot alkali process and introducing a purification process, it is ensured that the separated protein has higher activity and purity, and the separated protein is hydrolyzed to generate small molecule polypeptides under optimized specific process conditions, and various valuable by-products are obtained in the process, so as to realize greater value of municipal sludge resource utilization.

[0008] To achieve the above object, the present invention provides a method for separating and preparing small molecule polypeptides from municipal sludge and carrying out resource utilization, including the following steps:

[0009] Step S1, pretreatment: strictly select municipal sludge and adjust the moisture content to 80% - 90%;

[0010] Step S2, sludge pyrolysis: high temperature stage: add quicklime to the adjusted municipal sludge to adjust the sludge pH to 12 - 13, put it into a reaction kettle for cracking for 0.5 - 1 h, and control the temperature in the reaction kettle at 80 - 90 °C under normal pressure; low temperature stage: then adjust the sludge pH to 11 - 12, gradually control the reaction kettle temperature to drop to 65 - 75 °C, and react for 1 - 4 h;

[0011] Step S3, protein extraction and concentration: carry out plate and frame pressure filtration on the sludge slurry after pyrolysis reaction to separate the extract and the residual solid powder, and carry out resource utilization on the residual solid powder; then concentrate the extract by vacuum distillation, and control the temperature at 30 - 60 °C and the pressure at 10 - 100 mmHg during the vacuum distillation process;

[0012] Step S4, protein separation and purification: gradually add ammonium sulfate with a saturation of 20% - 100% to the concentrated extract by salting out method, stir and then let it stand for 1 - 2 hours to obtain crude protein and the remaining nutrient solution, and carry out resource utilization on the remaining nutrient solution; then use Tris-HCl as the dialysis buffer to dialyze the crude protein for 4 - 48 hours to obtain a high-purity protein solution;

[0013] Step S5, enzymatic conversion: add 0.3% - 1% w / w plant protease to the high-purity protein solution, control the temperature at 35 - 50 °C, and enzymatically hydrolyze for 2 - 24 h to obtain small molecule polypeptides; the plant protease includes bromelain, papain, ficin or a mixture thereof.

[0014] Preferably, the resource utilization of the residual solid powder includes: being used as building materials, fertilizers for forest land and flowers, and raw materials for improving acidic soil.

[0015] Preferably, the residual solid powder has a moisture content of 20% to 30% w / w, a calcium content of 5% to 20% w / w, an organic matter content of 20% to 30% w / w, and a nitrogen content of 1% to 5% w / w.

[0016] Preferably, in step S5, the obtained small molecule polypeptide consists of 2 to 50 amino acids, the solution color is a light yellow colloidal solution, and the molecular weight is 202 to 4618 g / mol.

[0017] Preferably, resource utilization of the remaining nutrient solution includes: using it for plant growth regulation, promoting bacterial reproduction, improving fertilizer efficiency, and as a feed additive.

[0018] Preferably, the humic acid content in the remaining nutrient solution is 0.1-5 g / L, and the N content is 0.5-5 g / L.

[0019] Preferably, the concentration of Tris-HCl is 1 mol / L.

[0020] Preferably, the step of obtaining crude protein comprises: adding ammonium sulfate stepwise to the concentrated extract at saturations of 20%, 40%, 60%, 80%, and 100%, respectively, stirring at a speed of 50 to 100 rpm for 10 to 15 minutes, and then standing to separate layers, wherein the standing time is 1 to 2 hours in the low saturation stage of 20% to 60%, and the standing time is shortened to 0.5 hours in the high saturation stage of 80% to 100%, and the salting-out components with a saturation of 40% or above are collected to obtain crude protein.

[0021] Based on the above technical solution, the advantages of the present invention are:

[0022] The method of the present invention uses a hot alkaline method to break down sludge cells to release proteins, obtains high-purity proteins through over-concentration, purification and other process flows, and converts them into small molecule polypeptides by combining plant protein enzymatic hydrolysis technology, significantly improving the stability and application potential of the product.

[0023] This method extracts protein from excess sludge and enzymatically hydrolyzes it into small-molecule peptides. This method not only effectively solves sludge disposal issues and enables sludge recycling, but also provides a new approach for obtaining small-molecule peptides. In addition to obtaining highly valuable small-molecule peptides, other byproducts produced by this method, such as residual solid powder and excess nutrient solution, can be used in the building materials industry, soil improvement, and other fields, generating significant economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 Process flow chart of a method for separating and preparing small molecule polypeptides from municipal sludge and carrying out resource utilization. Specific implementation manners

[0026] The technical solution of the present invention will be further described in detail below through the accompanying drawings and embodiments.

[0027] The present invention provides a method for extracting proteins from municipal sludge by using a hot alkali method, obtaining high-purity proteins through process flows such as concentration and purification, and further hydrolyzing to generate small molecule polypeptides and carrying out resource utilization, that is: strictly selecting municipal sludge with low contents of heavy metals, Cr6+, Al, viruses, antibiotics, etc., and preparing small molecule polypeptides through five stages of pretreatment, sludge hot alkali, extraction and concentration of proteins, separation and purification of proteins, and enzymatic conversion.

[0028] As Figure 1 shown, the method for separating and preparing small molecule polypeptides from municipal sludge and carrying out resource utilization of the present invention includes the following steps:

[0029] Step S1, pretreatment: strictly select municipal sludge and adjust the water content to 80% - 90%. The strictly selected municipal sludge has low contents of heavy metals, Cr6+, Al, viruses, antibiotics, etc.

[0030] Step S2, sludge pyrolysis: High temperature stage: Add quicklime to the adjusted municipal sludge to adjust the sludge pH to 12 - 13, put it into a reaction kettle to crack for 0.5 - 1 h, and control the temperature in the reaction kettle at 80 - 90 °C under normal pressure; Low temperature stage: Then adjust the sludge pH to 11 - 12, and gradually control the reaction kettle temperature to drop to 65 - 75 °C and react for 1 - 4 h.

[0031] The present invention aims to achieve two main goals by controlling the pH value of the sludge between 11 and 13 in step S2. First, reduce the temperature required for cell cracking, so that the cell cracking reaction is more intense. At the same time, the amount of protein released by cracking is sufficient to compensate for the protein consumed in the hydrolysis and denaturation processes, thereby increasing the dissolution rate of the protein. Second, considering the activity and stability of the protein, the pH value should not exceed 13 to prevent adverse effects on the quality of the protein extracted subsequently.

[0032] With the increase of the system pH value, the cell lysis reaction is significantly enhanced. Especially under the condition of pH > 11, the amount of protein released by lysis is sufficient to compensate for the protein loss during hydrolysis and denaturation, thus significantly increasing the protein dissolution rate. By precisely regulating the pH range to 11 - 13, the present invention significantly improves the cell lysis efficiency and maintains the protein dissolution rate at a high level. This improvement not only minimizes the impact of protein loss during hydrolysis and denaturation on the protein dissolution rate, but also provides more reliable technical support for efficient protein extraction, further promoting the process optimization of protein resource utilization.

[0033] Furthermore, controlling the reaction time to 1.5 - 5 h is because the release of macromolecular substances from sludge lysis mainly occurs within the first 30 min, and when the reaction duration reaches 3 h, the ammonia nitrogen content is the highest and the total nitrogen content continues to increase. When the reaction reaches 5 h, the total nitrogen content almost remains unchanged and the ammonia nitrogen content is lower than that at 3 h. It is judged that when the reaction duration is 5 h, the lysis reaction of sludge is higher than the generation amount of ammonia nitrogen, and the protein in the sludge is not completely hydrolyzed into amino acids at this time. By controlling the thermo-alkali reaction time within 1.5 - 5 hours, the present invention significantly improves the flexibility of the process while ensuring the quality and quantity of protein extraction. Especially in the case of excessive sludge volume, a shorter reaction duration not only effectively maintains the protein dissolution efficiency, but also significantly improves the overall efficiency of sludge treatment, providing a more efficient and adaptable technical solution for protein resource utilization and sludge treatment.

[0034] Controlling the temperature in the reaction kettle at 65 - 95 °C under normal pressure is one of the key parameters. First, considering that the temperature above 100 °C will exacerbate the Maillard reaction, the temperature is controlled below 100 °C. Second, appropriately increasing the temperature can relieve the pressure of the alkaline environment required for cell lysis to ensure the efficiency of the lysis reaction.

[0035] For the sludge pyrolysis in step S2 of the present invention, a staged pyrolysis strategy is adopted, and an efficient protein extraction is achieved by establishing a temperature-pH co-regulation system. In the high-temperature stage (80 - 90 °C), the thermodynamic advantage is utilized to rapidly lyse microbial cells and instantaneously release proteins; then it is transferred to the low-temperature stage (65 - 75 °C), and by moderately reducing the reaction system temperature and pH value (11 - 12), the hydrolysis time is extended while maintaining the protein structure stability, thereby maximizing the dissolution efficiency on the premise of ensuring the protein biological activity.

[0036] Currently, the temperature range of hot alkali treatment commonly used in conventional technologies is 100 - 180 °C or even higher. However, under alkaline conditions, when the temperature exceeds 100 °C, the Maillard reaction will significantly intensify, resulting in an increase in impurities in the hydrolysis solution, an increase in the solution viscosity, and difficulty in effective separation. In addition, proteins are highly sensitive to temperature and are extremely prone to denaturation under high-temperature conditions, thereby affecting their functional properties. To address this problem, in the present invention, by controlling the reaction temperature within the range of 65 - 95 °C, the incidence of the Maillard reaction is significantly reduced, the generation of impurities is decreased, and at the same time, the natural activity of proteins is maximally maintained. This optimization provides a high-quality raw material basis for the subsequent preparation of bioactive small peptides and provides important technical support for the efficient utilization of protein resources.

[0037] Without affecting the protein dissolution rate, the present invention tries to control the reaction conditions and time to reduce the influence of temperature on protein activity; it avoids the situation where the Maillard reaction causes the hot alkali sludge solution environment to be colloidal, which provides convenience for the subsequent plate and frame pressure filtration to separate the extract.

[0038] Step S3, protein extraction and concentration: subject the sludge slurry after pyrolysis reaction to plate and frame pressure filtration to separate the extract and the residual solid powder, and utilize the residual solid powder for resource recovery; then concentrate the extract by vacuum distillation, and control the temperature at 30 - 60 °C and the pressure at 10 - 100 mmHg during the vacuum distillation process.

[0039] The present invention selects vacuum distillation to concentrate the extract. To ensure that the activity of proteins in the extract is not damaged, by reducing the liquid surface pressure, the boiling point of the liquid is decreased. The higher the degree of vacuum during vacuum reduction, the lower the boiling point of the liquid drops, and the faster the evaporation. By controlling the pressure, the demand for temperature during distillation is reduced, and the influence of temperature on protein activity is decreased.

[0040] Furthermore, the pH of the by-product residual solid powder after plate and frame pressure filtration is 8 - 11, the moisture content of the residual solid powder is 20% - 30% w / w, the calcium content is 5% - 20% w / w, the organic matter content is 20% - 30% w / w, and the N content is 1% - 5% w / w. Utilizing the residual solid powder for resource recovery includes: using it as a fertilizer for building materials, forest land, and flowers, and as a raw material for improving acidic soil.

[0041] Specifically, the residual solid powder of the by-product after plate-and-frame filtration contains abundant calcium, which is one of the main components of cement. The high-calcium sludge can be directly used as a raw material for cement production to replace some natural calcium sources such as limestone. The residual solid powder of the by-product can provide abundant organic matter to supply carbon sources and energy for soil microorganisms, and can promote the formation of soil aggregate structure, enhancing soil aeration, water retention and fertilizer retention capabilities. The residual solid powder of the by-product contains alkaline substances (such as calcium carbonate, calcium hydroxide, etc.), which can neutralize hydrogen ions (H+) in acidic soil, thereby increasing the pH value of the soil to make it close to neutral or weakly alkaline.

[0042] Step S4, protein separation and purification: Gradually add ammonium sulfate with a saturation of 20% - 100% to the concentrated extract by salting-out method, stir and then let it stand for 1 - 2 hours to obtain crude protein and the remaining nutrient solution, and recycle the remaining nutrient solution; then use Tris-HCl as the dialysis buffer to dialyze the crude protein for 4 - 48 hours to obtain a high-purity protein solution.

[0043] Specifically, gradient salting-out technology is used to selectively precipitate and purify the concentrated extract to obtain crude protein, and the specific operation is as follows:

[0044] Gradually increase the ammonium sulfate saturation in the concentrated extract (20% → 40% → 60% → 80% → 100%). After each gradient adjustment, stir at a low speed of 50 - 100 rpm for 10 - 15 minutes to ensure system homogenization, and then let it stand for stratification. Among them, at the low saturation stage (20% - 60%), let it stand for 1 - 2 hours; at the high saturation stage (80% - 100%), the standing time is shortened to 0.5 hour. Collect the salting-out components with a saturation of 40% and above to obtain a crude protein enrichment product.

[0045] In the present invention, protein is separated by gradient salting-out in step S4. One is to reduce non-specific precipitation caused by local supersaturation when dropping high-concentration ammonium sulfate, and the other is to achieve hierarchical removal of front-end impurities (such as polysaccharides, etc.), that is, at low-saturation ammonium sulfate (below 40% saturation), impurities such as polysaccharides and nucleic acids are preferentially removed by using solubility differences and charge effects.

[0046] When adding ammonium sulfate at each level, stir slowly (50 - 100 rpm) for 10 - 15 minutes. There are three reasons for this: Avoid mechanical damage to proteins: Protein molecules are prone to conformational changes or aggregation under high shear forces. Eddies and shear forces generated by high-speed stirring (>200 rpm) may cause protein denaturation and non-specific aggregation. Prevent resuspension of precipitates: The protein precipitates formed by salting out are mostly loose flocs. High-speed stirring will destroy the formed precipitate particles, resulting in partial redissolution of the target protein. Avoid local supersaturation: Slow stirring can ensure the uniform distribution of ammonium sulfate, avoid local supersaturation, and prevent non-specific precipitation.

[0047] The conventional process of extracting proteins with hot alkali generally stops at the concentration stage, which still contains a large amount of impurities such as humic acid. To address this problem, the present invention adds a salting-out process while maintaining the protein activity, achieving efficient separation and purification of proteins and obtaining highly pure active proteins. This improvement not only significantly enhances the quality of protein products but also provides important technical support for the refined utilization of resources. In addition, it lays a solid foundation for the subsequent preparation of active small molecule polypeptides by protein decomposition. The obtained small molecule polypeptides have broad application prospects in fields such as medical aesthetics. The separation and purification process of the present invention reduces the impact of impurities on the protein quality, expands the application space of the product, and provides favorable support for the subsequent preparation of active small molecule polypeptides.

[0048] The crude protein separated by the salting-out method is reversible and does not involve extreme pH, high temperature, or organic solvents, so it will not damage the higher-order structure of proteins. The present invention selects Tris-HCl as the dialysis buffer, considering its strong buffering capacity, suitability for neutral to weakly alkaline conditions, stable chemical properties, ease of preparation and storage, and non-toxicity to most biomolecules, ensuring the protein activity to the greatest extent.

[0049] Specifically, the dialysis buffer selected in the present invention is a 1 mol / L Tris-HCl solution, and its preparation process is as follows:

[0050] (1) Weigh 121.14 g of Tris base (molecular weight: 121.14 g / mol). Tris base is also known as tris(hydroxymethyl)aminomethane.

[0051] (2) Add the Tris base to about 800 mL of deionized water and stir until completely dissolved.

[0052] (3) Slowly add concentrated hydrochloric acid (HCl) dropwise to the solution. The amount of concentrated hydrochloric acid used is 10 - 70 ml. At the same time, monitor the pH value with a pH meter until the target pH of 7.0 - 9.0 is reached.

[0053] (4) Add deionized water to make the volume up to 1 L.

[0054] The remaining nutrient solution obtained after salting out, with the humic acid content in the remaining nutrient solution being 0.1 - 5 g / L and the N content being 0.5 - 5 g / L, can be used for plant growth regulation, promoting the reproduction of strains, improving fertilizer efficiency, and as a feed additive.

[0055] Specifically, the by - product remaining nutrient solution contains a large amount of humic acid, which has a stimulating effect on the growth and development of crops and their internal physiological metabolism, and can be used for plant growth regulation.

[0056] The by - product remaining nutrient solution obtained after salting out is rich in a large amount of humic acid and N. The active groups of humic acid can combine with nitrogen, reducing the volatilization of nitrogen fertilizer and prolonging the fertilizer efficiency. At the same time, it can also carry out complexation or chelation with iron and aluminum, reducing the formation of iron phosphate and aluminum phosphate, thereby reducing the fixation of phosphorus, increasing the mobility of phosphorus, and promoting root growth. In addition, humic acid can combine with potassium, reducing the lattice fixation of potassium, increasing the content of available potassium, promoting fruit swelling and coloring, and improving fruit quality. Through these effects, the resource utilization of the present invention can significantly improve the utilization rate of fertilizers.

[0057] The remaining nutrient solution obtained after salting out is rich in a large amount of humic acid and N, and can also provide carbon source, nitrogen source and energy for soil microorganisms, promoting the growth and activities of beneficial microorganisms.

[0058] It is rich in a large amount of humic acid and can also combine with minerals (such as calcium, magnesium, iron, zinc, etc.) in the feed to form complexes that are easily absorbed, improving the utilization rate of minerals in the feed.

[0059] Step S5, enzymatic hydrolysis and transformation: Add 0.3% - 1% w / w plant protease to the high - purity protein solution, control the temperature at 35 - 50 °C, and carry out enzymatic hydrolysis for 2 - 24 h to obtain small - molecule polypeptides; the plant protease includes bromelain, papain, ficin or a mixture thereof. The present invention uses municipal sludge as a raw material to extract high - purity protein and prepare small - molecule polypeptides, realizing greater value in the resource utilization of municipal sludge.

[0060] The present invention selects plant protease considering the rich resources of plant protease and its own wide specificity. The obtained small - molecule polypeptides are composed of 2 - 50 amino acids, the solution color is a light - yellow colloidal solution, the molecular weight is 202 - 4618 g / mol. For the small - molecule polypeptides composed of 2 - 50 amino acids, the enzymatic hydrolysis efficiency of one - time enzymatic hydrolysis is about 63%, and the product extraction rate does not increase much after secondary enzymatic hydrolysis, and the cost - effectiveness of production is too low.

[0061] Specifically, the enzymatic hydrolysis time is set to 2 - 24 h. Since short peptides are usually composed of 2 to 20 amino acid residues and long peptides are usually composed of more than 20 amino acid residues, the formation time of short peptides is usually shorter than that of long peptide chains, which is mainly attributed to the abundance of cleavage sites in short peptide chains. Since proteases can recognize and bind to these sites more frequently, the catalytic efficiency is significantly improved, thus accelerating the formation of short peptides. In contrast, the cleavage sites in long peptide chains are relatively sparse, and it is difficult for proteases to effectively approach and cleave, resulting in an extended reaction time. Therefore, the synthesis of short peptides is more advantageous kinetically, while the formation of long peptides is limited by the accessibility of cleavage sites and reaction kinetics.

[0062] The small molecule polypeptides generated by the present invention can be used in multiple industries such as medicine, cosmetics, and food. Currently, small molecule polypeptides are mostly directly extracted from natural organisms or obtained through enzymatic hydrolysis and artificial synthesis methods based on chemical synthetic peptide libraries and biosynthetic molecular peptide libraries. The present invention provides a new synthetic route and simultaneously realizes the high-value resource utilization of excess sludge.

[0063] The present invention extracts proteins from excess sludge and enzymatically hydrolyzes them into small molecule polypeptides. This method not only effectively solves the problem of sludge treatment and realizes the resource recycling of sludge, but also provides a new way to obtain small molecule polypeptides. In addition to obtaining small molecule polypeptides of extremely high value, other by-products generated, such as residual solid powders and remaining nutrient solutions, can be used in fields such as the building materials industry and soil improvement, realizing certain economic value.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications can still be made to the specific implementation manners of the present invention or equivalent replacements can be made to some technical features; without departing from the spirit of the technical solutions of the present invention, they should all be covered within the scope of the technical solutions claimed by the present invention.

Claims

1. A method for separating and preparing small molecule polypeptides from municipal sludge and carrying out resource utilization, characterized in that: It includes the following steps: Step S1, pretreatment: strictly select municipal sludge and adjust the moisture content to 80% - 90%; Step S2, sludge pyrolysis: high-temperature stage: add quicklime to the adjusted municipal sludge to adjust the sludge pH to 12 - 13, put it into the reaction kettle for cracking for 0.5 - 1 h, and control the temperature in the reaction kettle at 80 - 90 °C under normal pressure; low-temperature stage: then adjust the sludge pH to 11 - 12, gradually control the reaction kettle temperature to drop to 65 - 75 °C, and react for 1 - 4 h; Step S3, protein extraction and concentration: perform plate and frame filtration on the sludge slurry after pyrolysis reaction to separate the extract and the residual solid powder, and utilize the residual solid powder resourcefully; then concentrate the extract by vacuum distillation, and control the temperature at 30 - 60 °C and the pressure at 10 - 100 mmHg during the vacuum distillation process; Step S4, protein separation and purification: gradually add ammonium sulfate with a saturation of 20% - 100% to the concentrated extract by the salting-out method, stir and then let it stand for 1 - 2 hours to obtain crude protein and the remaining nutrient solution, and utilize the remaining nutrient solution resourcefully; then use Tris-HCl as the dialysis buffer to dialyze the crude protein for 4 - 48 hours to obtain a high-purity protein solution; Step S5, enzymatic hydrolysis conversion: add 0.3% - 1% w / w plant protease to the high-purity protein solution, control the temperature at 35 - 50 °C, and perform enzymatic hydrolysis for 2 - 24 h to obtain small molecule polypeptides; the plant protease includes bromelain, papain, ficin or a mixture thereof.

2. The method according to claim 1, wherein: Utilizing the residual solid powder resourcefully includes: using it as building materials, fertilizers for forest land and flowers, and raw materials for improving acidic soil.

3. The method according to claim 2, wherein: The residual solid powder has a moisture content of 20% - 30% w / w, a calcium content of 5% - 20% w / w, an organic matter content of 20% - 30% w / w, and an N content of 1% - 5% w / w.

4. The method according to claim 1, characterized in that: In step S5, the obtained small molecule polypeptides are composed of 2 - 50 amino acids, the solution color is a light yellow colloidal solution, and the molecular weight is 202 - 4618 g / mol.

5. The method according to claim 1, characterized in that: Utilizing the remaining nutrient solution resourcefully includes: using it for plant growth regulation, promoting the reproduction of strains, improving fertilizer efficiency, and as a feed additive.

6. The method according to claim 5, wherein: The remaining nutrient solution has a humic acid content of 0.1 - 5 g / L and an N content of 0.5 - 5 g / L.

7. The method according to claim 1, wherein: The concentration of the Tris-HCl is 1 mol / L.

8. The method according to claim 1, characterized in that: The steps for obtaining crude protein include: gradually add ammonium sulfate with saturations of 20%, 40%, 60%, 80%, and 100% to the concentrated extract, stir at a speed of 50 - 100 rpm for 10 - 15 minutes, and then let it stand for layering. Among them, let it stand for 1 - 2 hours at the low saturation stage of 20% - 60%, shorten the standing time to 0.5 hours at the high saturation stage of 80% - 100%, and collect the salting-out components with a saturation of 40% and above to obtain crude protein.

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