Method for harmless treatment of cattle and sheep hides

Abstract

The present application belongs to the technical field of animal leather waste treatment, and discloses a harmless treatment method for cattle and sheep leather, which comprises the following steps: pretreatment, in which the cattle and sheep leather raw material is cleaned and cut into leather pieces with a length-width size of 1-3 cm; pulse electric field treatment, in which the leather pieces spread and compacted by a compression roller device are subjected to PEF treatment; autocatalytic degradation, in which a staged temperature and pH control strategy is adopted, and the stage conversion is triggered based on the proportion of amino acid nitrogen in the total nitrogen in the liquid phase; solid-liquid separation, in which liquid products and solid residues are obtained; product treatment, in which the liquid products are dried into powder finished products, and the solid residues are composted into compost finished products. The present application replaces exogenous enzymes with PEF-activated endogenous zymogens, and realizes deep degradation of proteins by combining staged temperature control and process monitoring, with a conversion rate of up to 93% or more, a treatment cost of only about one fourth of that of traditional methods, and the quality of the finished products meeting the requirements of organic fertilizer standards.

Description

Technical Field

[0001] This invention belongs to the field of animal leather waste treatment technology. Specifically, it relates to a method for harmlessly treating bovine and sheep hides, and particularly to a method for harmlessly treating untanned bovine and sheep hide waste by activating endogenous zymogens with a pulsed electric field to synergistically break down cell walls. Background Technology

[0002] Animal leather processing, such as cowhide and sheepskin, generates a large amount of waste, including scraps and defective products. This waste is rich in collagen; direct disposal not only wastes resources but also pollutes the environment. Currently, common disposal methods include landfill, incineration, and resource utilization. Landfill and incineration, however, consume land resources and are prone to secondary pollution, and their use is gradually being restricted.

[0003] From the perspective of leather processing procedures, the main sources of cow and sheep hide waste include the following categories:

[0004] (1) Bare skin waste generated during the leather dehairing process: including bare skin scraps after lime / sodium sulfide dehairing, residual leather pieces after enzymatic dehairing, etc. This type of waste has not been tanned, retains the integrity of the skin tissue, and the endogenous enzyme progenitors (such as cathepsin B progenitor, D progenitor, L progenitor, etc.) are well preserved.

[0005] (2) Waste raw hides in the leather preparation section: including soaked hides, limed hides, delimed hides, softened hides and other raw hides that have undergone preliminary treatment. These wastes have undergone relatively light chemical treatment and some endogenous enzymes still have the potential to be activated.

[0006] (3) Waste leather after tanning and wet finishing: including chrome tanned leather, vegetable tanned leather, dyed leather, coated leather, etc. This type of waste has been cross-linked by tanning agents or treated at high temperature, and the endogenous enzyme activity has been irreversibly deactivated.

[0007] (4) Scrap materials from finished leather products: including cutting waste, defective products, etc. The composition of this type of waste is complex, including fiber fabrics, plastic parts, metal parts, etc., and the endogenous enzymes are basically inactivated.

[0008] In terms of resource utilization, traditional methods often employ exogenous enzymatic hydrolysis, which involves adding commercially available proteases (such as trypsin and papain) to waste hides to degrade proteins into small nitrogen-containing molecules through catalysis. However, this method has several technical drawbacks: first, the cost of adding exogenous enzymes is high, typically accounting for a large proportion of the processing cost; second, maintaining the activity of exogenous enzymes in the reaction system is difficult, as they are easily deactivated by factors such as temperature and pH; and third, the contact efficiency between exogenous enzymes and substrates is limited, making it difficult to achieve deep protein degradation, resulting in a generally low protein conversion rate. Furthermore, single-enzymatic hydrolysis is currently a commonly used biological treatment method, utilizing exogenous proteases such as subtilisin and trypsin to hydrolyze bovine and sheep hides. However, the stratum corneum and basement membrane of bovine and sheep hides form a dense barrier, making it difficult for enzyme molecules to penetrate into the hide's interior. This results in hydrolysis occurring only on the surface, with processing cycles lasting 24-48 hours and low production efficiency.

[0009] Existing technologies also include research on physical methods to assist in processing, such as pulsed electric field (PEF) technology. PEF technology is a novel non-thermal processing technology that uses a high-voltage pulsed electric field to treat biological tissues and has been widely used in food sterilization, cell wall disruption, and other fields. However, in the field of animal leather waste treatment, PEF technology is mainly used to assist in exogenous enzymatic hydrolysis and is usually only used as a means of cell wall disruption, failing to fully realize its potential to activate endogenous zymogens.

[0010] Therefore, how to utilize the resources of cattle and sheep hides to achieve low-cost enzymatic hydrolysis while improving the efficiency and depth of hydrolysis is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0011] The purpose of this invention is to provide a method for harmlessly treating bovine and mutton hides, so as to solve the technical problems of high cost and low protein conversion rate in the prior art.

[0012] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0013] A method for harmlessly treating cow and sheep hides includes the following steps:

[0014] (1) Pretreatment: Clean and cut the untanned raw cow and sheep hides into pieces with a length and width of 1-3 cm;

[0015] (2) Pulse electric field treatment: The leather block is subjected to pulse electric field treatment;

[0016] (3) Autocatalytic degradation: The leather pieces treated with pulsed electric field are subjected to autocatalytic degradation. No exogenous protease is added during the autocatalytic degradation process. A staged temperature and pH control strategy is adopted, and the stage conversion is triggered based on the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase.

[0017] (4) Solid-liquid separation: The autocatalytic degradation products are separated into liquid products and solid residues;

[0018] (5) Product processing: The liquid product is dried to obtain a powder product, and the solid residue is composted to obtain a compost product.

[0019] Furthermore, in step (2), the field strength of the pulsed electric field treatment is 20-40 kV / cm, the number of pulses is 10-30, the pulse width is 20-100 μs, the pulse frequency is 0.5-2 Hz, and the treatment temperature is 20-30℃.

[0020] Furthermore, in step (2), the electrode spacing for pulsed electric field treatment is 2-3 cm, and the leather block is placed between the electrodes in a single-layer flat manner.

[0021] Furthermore, in step (2), the processing medium for pulsed electric field treatment is deionized water, and the moisture content of the leather block is not less than 70%.

[0022] Furthermore, the phased temperature and pH control strategy described in step (3) includes:

[0023] First stage: Temperature 35-45℃, pH 5.5-6.5. When the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 30%, proceed to the next stage.

[0024] Second stage: Temperature 38-48℃, pH 5.8-6.8. When the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 55%, proceed to the next stage.

[0025] The third stage: temperature 42-50℃, pH 6.2-7.0, the reaction is terminated when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 85%.

[0026] Further, in step (3), the amino acid nitrogen content is sampled and tested every 30 minutes in each stage, and the reaction is terminated or the next stage is determined based on the test results.

[0027] Furthermore, in step (3), the material-to-water ratio for autocatalytic degradation is 1:5 to 1:10, and the stirring speed is 150-250 rpm.

[0028] Furthermore, in step (4), the solid-liquid separation is performed using a plate and frame filter press with a filtration pressure of 0.2 MPa and a filter cloth pore size of 8 μm.

[0029] Furthermore, in step (5), the liquid product is dried by spray drying, with an inlet air temperature of 190°C and an outlet air temperature of 85°C.

[0030] Furthermore, in step (5), the solid residue is mixed with cow dung and straw and then composted. The mass ratio of solid residue to cow dung is 1:1.5, the amount of straw added is 0.8 times the mass of solid residue, the composting cycle is 15-20 days, and the composting temperature is 55-65℃.

[0031] Furthermore, the pulsed electric field treatment described in this invention is used to activate endogenous zymogens in bovine and ovine hides. Bovine and ovine hides naturally contain various proteases, including pro-Cathepsin B, pro-Cathepsin D, and pro-Cathepsin L, which are inactive under normal physiological conditions. This invention uses pulsed electric field treatment to generate an electroporation effect, causing the zymogens to be released from the cells and activated. Simultaneously, the conformational changes in proteins caused by the pulsed electric field expose the active sites of the zymogens, promoting their conversion into active enzymes.

[0032] Furthermore, this invention uses amino acid nitrogen content as the stage switching threshold, which is determined as follows: The first stage threshold of 30% corresponds to the completion point of the collagen degradation stage. When the amino acid nitrogen generated by collagen degradation reaches 30% of the total nitrogen in the liquid phase, it indicates that the collagen degradation is sufficient and can proceed to the next stage; the second stage threshold of 55% corresponds to the completion point of the elastin degradation stage. When the amino acid nitrogen generated by elastin degradation reaches 55% of the total nitrogen in the liquid phase, it indicates that the elastin degradation is sufficient and can proceed to the deep enzymatic hydrolysis stage; the third stage threshold of 85% corresponds to the termination point of the deep enzymatic hydrolysis stage. When the amino acid nitrogen reaches 85% of the total nitrogen in the liquid phase, it indicates that the protein has achieved deep degradation, and the benefits of continuing the reaction decrease.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention utilizes a pulsed electric field to activate the endogenous zymogens in bovine and ovine hides, eliminating the need for any exogenous enzyme preparations and achieving low-cost enzymatic hydrolysis, significantly reducing processing costs. Simultaneously, the pulsed electric field treatment, combined with a staged temperature and pH control strategy, significantly improves protein conversion rate, achieving deep protein degradation. Furthermore, once the endogenous zymogens are activated, they can rapidly catalyze protein degradation under suitable temperature and pH conditions, greatly shortening the processing cycle. The resulting products are of excellent quality; the powdered product has a high total nitrogen content and low heavy metal content, meeting the standards for organic fertilizers; the composted product has a high organic matter content and is rich in nutrients. This invention eliminates the need for strong acids and alkalis, avoids secondary pollution, and realizes the resource utilization of bovine and ovine hide waste, converting waste into organic fertilizer and avoiding the environmental pollution caused by incineration and landfill, thus exhibiting significant environmental benefits. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0036] I. Explanation of Raw Material Sources and Identification Methods

[0037] 1. During the research process, the inventors discovered that the sources and states of bovine and sheepskin waste vary significantly, resulting in substantial differences in the retention state, activity state, salt content, pH, heavy metal content, tanning agent residue, and degradability of their endogenous enzyme progeny. For leather waste that has already been tanned or treated with high temperature and high acid / alkali, the pulsed electric field activation mechanism of this invention is no longer applicable because the endogenous enzyme progeny has been permanently destroyed or the enzyme active center has been chemically cross-linked. The technical solution of this invention is only applicable to the following raw materials: untanned raw hides such as bare hide waste after the leather dehairing process, water-soaked hides, limed hides, delimed hides, and softened hides. The raw materials should meet the following conditions:

[0038] (1) The collagen structure of the dermis remains intact without tanning agents;

[0039] (2) Without high-temperature (≥60℃) heat treatment, the zymogen did not undergo irreversible thermal denaturation;

[0040] (3) The pH range is between 5.0 and 9.0, and the enzyme activity is not chemically inhibited without treatment with strong acid (pH < 3) or strong base (pH > 12);

[0041] (4) The heavy metal content (calculated as Cr) is <100 mg / kg to ensure that the activation efficiency of the enzyme by the pulsed electric field is not affected by heavy metal ions.

[0042] 2. The raw material used in this embodiment of the invention is bovine raw hide scraps produced during the hair removal process of a tannery. Specific parameters are as follows:

[0043] (1) Appearance: grayish-white to light yellow blocks, with a thickness of 3 to 8 mm;

[0044] (2) pH value: 7.2 (10 g leather piece + 100 mL deionized water, stirred for 30 min and then measured);

[0045] (3) Heavy metal content (calculated as Cr): Not detected (<5 mg / kg);

[0046] (4) Endogenous enzyme retention status: The activity of cathepsin B progenitor, D progenitor, and L progenitor was detected to be above 85% of the activity of fresh skin.

[0047] 3. Those skilled in the art can determine whether the raw materials fall within the scope of this invention through the following characteristics:

[0048] (1) Appearance identification: Untanned leather is grayish-white or light yellow and has no metallic luster; Tanned leather is brownish-brown (chrome tanned) or yellowish-brown (vegetable tanned) and has a metallic or oily luster.

[0049] (2) Combustion identification: Take a small sample and burn it with an alcohol lamp. Untanned leather has a smell of burning hair when burning, and the ash is black and loose. Tanned leather has a smell of plastic or resin when burning, and the ash is hard and clumpy.

[0050] (3) Chemical identification: Take 1 g of sample and soak it in 10 mL of 10% formic acid solution. Untanned leather softens and dissolves within 1 hour; tanned leather remains intact within 24 hours.

[0051] II. Explanation of the mechanism of endogenous zymogen activation

[0052] As animal tissues, bovine and sheep hides naturally carry various protease systems, primarily including the cathepsins family. These enzymes exist in the dermis in zymogen form and require specific conditions to be activated. Only when bovine and sheep hides have not been tanned, subjected to high temperatures, or treated with strong acids or alkalis do their endogenous zymogens maintain their intact structure and activation potential. The main types of endogenous zymogens include the following (Table 1).

[0053] Table 1

[0054]

[0055] The high-voltage pulsed electric field (PEF) technology used in this invention activates endogenous zymogens through the following triple mechanism:

[0056] (1) Electroporation effect: The high-voltage pulsed electric field forms instantaneous microporous channels on the cortical cell membrane, which changes the permeability of the cell membrane, allowing the zymogen to be released from inside / between cells and come into contact with the substrate.

[0057] (2) Structural denaturation and conformational activation: The high electric field intensity generated by the pulsed electric field can cause reversible changes in protein conformation, expose the active site of the zymogen, remove the shielding of the zymogen inhibitory peptide, and promote the conformational transformation of the zymogen into the active enzyme.

[0058] (3) Free radicals and oxidative activation: The ionization effect under the action of pulsed electric field can generate a small amount of reactive oxygen free radicals, which oxidize the inhibitory peptide of prozymogen, promote its degradation, and activate the active center of prozymogen.

[0059] For tanned or high-temperature treated cattle and sheep hides, the above activation mechanism no longer applies because the endogenous enzyme progenitors have been permanently destroyed or the enzyme active centers have been chemically cross-linked.

[0060] According to the verification results of the present invention, the key parameter range for activating endogenous enzyme progenitors in bovine and ovine hides is 20-40 kV / cm and 10-30 pulses. Within this range, the enzyme activity is increased by ≥3 times, which can meet the process requirements of autocatalytic degradation.

[0061] III. Basis for Determining Stage Switching Thresholds

[0062] This invention uses 30% / 55% / 85% of the total nitrogen in the liquid phase as the stage switching threshold, and the determination is based on the following:

[0063] (1) Basis for determining the first-stage threshold of 30%: The degradation target is collagen (accounting for 60-70% of the total protein in bovine and ovine hides). Under mild conditions (35-45℃, pH 5.5-6.5), it first denatures. Cathepsins B and L first attack the non-helical regions of collagen, producing polypeptide fragments with larger molecular weights. When the amino acid nitrogen accounts for about 30% of the total nitrogen in the liquid phase, it indicates that the initial stage of collagen degradation has been completed. When it is below 30%, it indicates that the collagen degradation is not yet sufficient.

[0064] (2) Basis for determining the second-stage threshold of 55%: The degradation targets are elastin (accounting for 10-15% of the total protein in bovine and ovine hides) and some incompletely degraded collagen. The cross-linking structure of elastin is more complex, requiring higher temperatures and a slightly acidic pH for effective degradation. When amino acid nitrogen accounts for about 55% of the total nitrogen in the liquid phase, it indicates that the collagen degradation products have been basically degraded and the elastin degradation has entered a critical turning point.

[0065] (3) Basis for determining the 85% threshold in the third stage: The degradation targets are residual stubborn proteins and peptides with large molecular weights. Under conditions of higher temperature and near-neutral pH, multiple endogenous enzymes form a cascade amplification effect, working together to completely degrade the peptides into free amino acids. When amino acid nitrogen accounts for more than 85% of the total nitrogen in the liquid phase, it indicates that the protein has achieved deep degradation, and the benefits of continuing the reaction are diminishing.

[0066] By tracking and detecting the reaction process of Examples 1-3, the rationality of the threshold setting was verified: when the amino acid nitrogen content reached 30%, the reaction proceeded to the second stage; when it reached 55%, the reaction proceeded to the third stage; and when it reached 85%, the reaction was terminated. The threshold setting matched the actual process of protein degradation.

[0067] IV. Key Implementation Conditions for Pulse Electric Field Processing

[0068] The pulsed electric field treatment of this invention can stably activate endogenous enzyme progenitors in bovine and ovine hides, depending on the synergistic effect of the following key implementation conditions:

[0069] (1) Electric field parameters: field strength 20-40 kV / cm, number of pulses 10-30, pulse width 20-100 μs, pulse frequency 0.5-2 Hz. This parameter range is the optimal range that can effectively activate endogenous zymogens, which has been verified by a large number of experiments.

[0070] (2) Treatment medium: Deionized water is used as the treatment medium, and the conductivity is controlled below 100 μS / cm to avoid the ineffective dissipation of electric field energy in the medium;

[0071] (3) Leather piece condition: The length and width of the leather piece are controlled within 1-3 cm, and the moisture content is not less than 70% to ensure that the leather piece has sufficient electrical conductivity and electric field penetration;

[0072] (4) Filling method: Before treatment, the leather pieces soaked in deionized water are compacted and spread out by a pressure roller device to form a uniform thin layer with a thickness of 1-1.5 cm. The electrode spacing is set to 2-3 cm. The leather pieces are laid flat between the electrodes in a single layer. Stacking or overlapping is not allowed to ensure that the electric field can penetrate the entire leather layer uniformly and avoid the electric field shielding effect.

[0073] (5) Temperature rise control: The processing temperature is controlled at 20-30℃. The temperature rise caused by the pulse electric field treatment is controlled within 5℃ through the cooling system to avoid the inactivation of enzymes due to high temperature.

[0074] Example 1

[0075] 1. Pretreatment

[0076] Take 100 kg of raw cowhide scraps from the dehairing process at a tannery, rinse them with running water for 10 minutes to remove surface dirt, bloodstains, and sand, keeping the water temperature at 20℃. Cut the cleaned cowhide into pieces 1-3 cm in length and width using a cutting machine to obtain pre-treated pieces for later use.

[0077] 2. Pulse electric field processing

[0078] The pretreated leather pieces were placed in the PEF treatment chamber of the pulsed electric field treatment device, and soaked in deionized water with a moisture content controlled at 75%. Before treatment, the soaked leather pieces were compacted and spread using a pressure roller device to form a uniform thin layer with a thickness controlled at 1.25 cm. The electrode spacing was set to 2.5 cm, and the leather pieces were placed between the electrodes in a single-layer flat arrangement. The pulsed electric field parameters were set as follows: field strength 30 kV / cm, number of pulses 20, pulse width 50 μs, pulse frequency 1 Hz, and treatment temperature 25℃. The pulsed electric field treatment device was started to pretreat the leather pieces for 20 seconds, resulting in PEF-treated leather pieces.

[0079] 3. Autocatalytic degradation

[0080] The PEF-treated leather blocks were transferred to a 50 L constant-temperature reaction vessel, and deionized water was added, with a material-to-water ratio controlled at 1:7 (mass ratio). The stirring device was started, and the speed was controlled at 200 rpm. A staged control strategy based on the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase was adopted to trigger the phase transition.

[0081] (1) First stage (collagen degradation): temperature 37℃, pH 6.0, sample every 30 minutes to detect amino acid nitrogen content; when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 30%, proceed to the next stage;

[0082] (2) Second stage (elastin degradation): Temperature 42℃, pH 6.2, continue to take samples every 30 minutes; when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 55%, proceed to the next stage;

[0083] (3) Third stage (deep enzymatic hydrolysis): temperature 47℃, pH 6.8, continue to take samples every 30 minutes; when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 85%, the reaction is terminated;

[0084] Total reaction time: 4.0 hours.

[0085] 4. Solid-liquid separation

[0086] The reaction product was introduced into a plate and frame filter press, and the filtration pressure was controlled at 0.2 MPa. The filter cloth pore size was 8 μm, and filtration continued until the filtrate was clear. Liquid product and solid residue were obtained.

[0087] 5. Product processing

[0088] (1) Liquid product processing

[0089] The liquid product is dried in a spray drying tower with the following parameters set: inlet air temperature 190℃, outlet air temperature 85℃, feed rate 100 mL / h, and atomization pressure 0.3 MPa, to obtain the finished organic fertilizer powder.

[0090] (2) Solid residue treatment

[0091] Solid residue was mixed with cow dung and straw for composting, with a mass ratio of solid residue to cow dung of 1:1.5 and straw added at 0.8 times the mass of solid residue. The composting period was 18 days, during which the compost was turned 4 times, and the composting temperature was controlled at 55-65℃. After the compost matured, organic fertilizer was obtained.

[0092] Example 2

[0093] Steps 1 (pretreatment) and 4-5 (solid-liquid separation and product processing) in this embodiment are exactly the same as in Example 1. The only difference is:

[0094] 1. Step 2 (Pulse Electric Field Processing)

[0095] The pulsed electric field parameters were set as follows: field strength 20 kV / cm, number of pulses 10, pulse width 20 μs, pulse frequency 0.5 Hz, and processing temperature 20℃. The electrode spacing was 2 cm, the thickness of the compacted leather block was 1 cm, and the moisture content was controlled at 70%. Other operations were the same as in Example 1.

[0096] 2. Step 3 (Autocatalytic Degradation)

[0097] The material-to-water ratio is 1:5, and the stirring speed is 150 rpm.

[0098] (1) First stage: Temperature 35℃, pH 5.5, when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 30%, proceed to the next stage;

[0099] (2) Second stage: Temperature 38℃, pH 5.8, when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 55%, proceed to the next stage;

[0100] (3) Third stage: Temperature 42℃, pH 6.2, the reaction is terminated when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 85%;

[0101] The total reaction time is 5.5 hours.

[0102] Example 3

[0103] Steps 1 (pretreatment) and 4-5 (solid-liquid separation and product processing) in this embodiment are exactly the same as in Example 1. The only difference is:

[0104] 1. Step 2 (Pulse Electric Field Processing)

[0105] The pulsed electric field parameters were set as follows: field strength 40 kV / cm, number of pulses 30, pulse width 100 μs, pulse frequency 2Hz, and processing temperature 30℃. The electrode spacing was 3 cm, the thickness of the compacted leather block was 1.5 cm, and the moisture content was controlled at 80%. Other operations were the same as in Example 1.

[0106] 2. Step 3 (Autocatalytic Degradation)

[0107] The material-to-water ratio is 1:10, and the stirring speed is 250 rpm.

[0108] (1) First stage: Temperature 45℃, pH 6.5, when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 30%, proceed to the next stage;

[0109] (2) Second stage: Temperature 48℃, pH 6.8, when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 55%, proceed to the next stage;

[0110] (3) Third stage: Temperature 50℃, pH 7.0, the reaction is terminated when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 85%;

[0111] Total reaction time: 3 hours.

[0112] Comparative Example 1

[0113] Steps 1 (pretreatment) and 4-5 (solid-liquid separation and product processing) in this comparative example are exactly the same as in Example 1. The only difference is that steps 2 (pulsed electric field treatment)-3 (autocatalytic degradation) in Example 1 are replaced with exogenous enzymatic hydrolysis.

[0114] The pretreated leather blocks were transferred to a constant-temperature reaction vessel, and deionized water was added at a material-to-water ratio of 1:7. Commercially available trypsin was added at 5% of the leather block mass. The reaction conditions were set as follows: temperature 40℃, pH 7.0, stirring speed 200 rpm, and reaction time 24 hours. During the reaction, samples were taken every 2 hours to determine the proportion of amino acid nitrogen to total nitrogen in the liquid phase.

[0115] Comparative Example 2

[0116] Step 1 (pretreatment) and steps 3-5 (autocatalytic degradation, solid-liquid separation, and product treatment) of this comparative example are exactly the same as those of Example 1. The only difference is that when performing step 2 (pulsed electric field treatment), the field strength of the pulsed electric field is set to 10 kV / cm, and all other aspects are the same as those of Example 1.

[0117] Comparative Example 3

[0118] Step 1 (pretreatment) and steps 3-5 (autocatalytic degradation, solid-liquid separation, and product treatment) of this comparative example are exactly the same as those of Example 1. The only difference is that when performing step 2 (pulsed electric field treatment), the number of pulses of the pulsed electric field is set to 5; all other steps are the same as those of Example 1.

[0119] Comparative Example 4

[0120] Steps 1 (pretreatment), 2 (pulsed electric field treatment), and 4-5 (solid-liquid separation and product treatment) of this comparative example are exactly the same as those of Example 1. The only difference is that in step 3 (autocatalytic degradation), the same temperature and pH conditions as in the three stages of Example 1 are used, but the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase is not monitored during the process. Instead, the reaction is directly terminated after a fixed reaction time of 3 hours (1 hour each for the first, second, and third stages).

[0121] Comparative Example 5

[0122] Steps 1 (pretreatment), 2 (pulsed electric field treatment), and 4-5 (solid-liquid separation and product treatment) of this comparative example are exactly the same as those of Example 1. The only difference is that in step 3 (autocatalytic degradation), a strategy of non-stage adjustment is adopted, that is, the temperature is kept constant at 40°C, the pH is kept constant at 6.5, and the reaction time is 4 hours.

[0123] Comparative Example 6

[0124] Steps 1 (pretreatment) and 3-5 (autocatalytic degradation, solid-liquid separation, and product treatment) of this comparative example are exactly the same as those of Example 1. The only difference is that step 2 (pulsed electric field treatment) is omitted, meaning the pretreated leather is directly subjected to autocatalytic degradation without pulsed electric field pretreatment. All other operations are the same as in Example 1.

[0125] Data Analysis and Comparison

[0126] To objectively evaluate the technical effects of the present invention, the samples processed in Examples 1-3 and Comparative Examples 1-6 were subjected to the following tests.

[0127] 1. Enzyme activity (U / g)

[0128] Take 1 g of the untreated leather sample and add 5 mL of pH 6.0 phosphate buffer (0.1 mol / L). Sonicate the sample in an ice bath for 5 minutes (300 W, 1 s interval, 1 s interval). Centrifuge at 10,000 rpm for 10 minutes and collect the supernatant as the enzyme solution. Take 100 μL of the enzyme solution, add 900 μL of pH 6.0 phosphate buffer, and then add 50 μL of Z-Phe-Arg-MCA fluorescent substrate (10 μmol / L). Incubate at 37℃ for 30 minutes and detect the fluorescence intensity using a fluorescence spectrophotometer (excitation wavelength 380 nm, emission wavelength 460 nm). The enzyme activity unit is defined as the amount of enzyme required per gram of leather sample to hydrolyze the substrate to produce 1 μmol of MCA per minute at 37℃ and pH 6.0, defined as 1 U / g.

[0129] 2. Protein conversion rate (%)

[0130] Protein conversion rate refers to the proportion of protein in the hide that is enzymatically converted into soluble nitrogenous substances during the autocatalytic degradation step. Take 1 g of samples before and after the reaction, and determine the total nitrogen content using the Kjeldahl method. The formula for calculating protein conversion rate is: Protein conversion rate = (Total nitrogen content of sample after reaction - Total nitrogen content of sample before reaction) × 100%.

[0131] 3. Amino acid nitrogen content (%)

[0132] After the reaction, the amino acid nitrogen content of the sample was determined by ninhydrin colorimetric-spectrophotometric method. 1 mL of sample supernatant was taken, and 1 mL of ninhydrin reagent (2% ninhydrin + 0.8% ninhydrin hydrazine + 20% pyridine) and 2 mL of phosphate buffer (pH 8.0) were added. The mixture was boiled in a water bath for 15 minutes, cooled to room temperature, and the absorbance at 570 nm was measured using a UV spectrophotometer. The amino acid nitrogen concentration was calculated based on the standard curve, and then the proportion of amino acid nitrogen to total nitrogen in the liquid phase was calculated.

[0133] 4. Processing cost (RMB / ton)

[0134] The processing costs include: water and electricity consumption, consumables (filter cloth, centrifuge tubes, etc.), equipment depreciation, and labor costs. The total cost is calculated based on a processing volume of 100 kg of raw materials, and then converted to yuan / ton.

[0135] 5. Quality inspection of finished products obtained from product processing

[0136] (1) Liquid products (powder products)

[0137] 1) Moisture content: Take 5 g of the finished product sample and dry it in an oven at 105℃ until constant weight, and calculate the percentage of weight loss.

[0138] 2) pH value: Take 5 g of the finished product sample, add 50 mL of deionized water, stir for 30 minutes, and use a pH meter to measure the pH value of the supernatant.

[0139] 3) Total nitrogen content: Take 0.5 g of sample, add concentrated sulfuric acid and catalyst (copper sulfate: potassium sulfate = 1:10) for digestion, and then use a Kjeldahl nitrogen analyzer to determine the amount of ammonia released and calculate the total nitrogen content.

[0140] 4) Heavy metal content: Take 0.5 g of sample, digest with nitric acid-hydrofluoric acid, and then determine the absorbance values ​​of lead and arsenic using an atomic absorption spectrometer. Calculate the content based on the standard curve.

[0141] (2) Solid residue (composted product)

[0142] 1) Organic matter content: The sample was dried at 105℃ to constant weight, and then placed in a muffle furnace at 600℃ for 4 hours. The percentage difference in mass before and after ignition was calculated.

[0143] 2) pH value: Mix the sample with deionized water at a ratio of 1:10, shake for 30 minutes, and measure the pH value of the supernatant using a pH meter.

[0144] 3) Total nitrogen, phosphorus and potassium content: Total nitrogen was determined by the Kjeldahl method (samples were digested with concentrated sulfuric acid before determination); phosphorus was determined by the ammonium vanadate method (samples were digested with nitric acid-perchloric acid, and a yellow complex was formed with ammonium vanadate under acidic conditions, and the absorbance was measured at 400 nm); potassium was determined by the flame photometry method (samples were digested with nitric acid-perchloric acid, and the potassium ion concentration was measured using a flame photometer).

[0145] 4) Fecal coliform count: Take 10 g of sample, add 90 mL of sterile physiological saline, shake for 30 minutes, and take the filtrate for culture and counting.

[0146] 5) Take 100 g of sample and use the centrifugal flotation method to detect Ascaris eggs and calculate the mortality rate.

[0147] Table 2

[0148]

[0149] As shown in Table 2, Examples 1-3 significantly outperformed Comparative Examples 1-5 in the aforementioned indicators, demonstrating superior treatment effects. After PEF treatment, the enzyme activity in Examples 1-3 increased by 3.05 to 7.13 times, the protein conversion rate reached 93.1% to 97.5%, and the proportion of amino acid nitrogen in the liquid phase remained stable at 85.1% to 87.3% of the total liquid nitrogen. This reflects the synergistic effect of activating endogenous zymogens through pulsed electric field pretreatment, staged temperature and pH control, and triggering phase transitions based on the proportion of amino acid nitrogen in the liquid phase to the total liquid nitrogen, resulting in multi-dimensional deep enzymatic hydrolysis of the proteins within the tissue. The lower enzyme activity increase and lower protein conversion rate corroborate each other, indicating that when the PEF field strength and pulse number do not reach the threshold, the activation effect of endogenous zymogens is limited, leading to insufficient protein degradation.

[0150] Comparative Example 1, using a traditional exogenous enzymatic hydrolysis method, achieved a protein conversion rate of only 72.3%, a difference of approximately 20 percentage points compared to the examples. This indicates that relying solely on exogenous enzymes is insufficient for achieving deep protein degradation, because the activity of exogenous enzymes is difficult to maintain after addition, and they cannot achieve the same level of contact with the substrate as endogenous enzymes.

[0151] Comparative Examples 2 and 3 deviated from the technical solution of this invention in terms of PEF field strength and pulse number, respectively. The enzyme activity increases by only 1.96 times and 2.33 times, respectively, and the protein conversion rates also decreased to 68.7% and 71.2%. This result clearly shows that there is a synergistic effect between PEF field strength and pulse number; insufficient values ​​of either parameter will restrict the activation effect of endogenous zymogens.

[0152] Although the PEF parameters for Comparative Example 4 were the same as those for Example 1 (field strength 30 kV / cm, number of pulses 20), the protein conversion rate was only 82.5% because a fixed 3-hour termination was used instead of process monitoring. This indicates that simply determining the process parameters is insufficient; it is still necessary to accurately determine the reaction endpoint by detecting the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase.

[0153] Comparative Example 5 also used the compliant PEF parameters, but because it used a single pH condition (pH 6.5) instead of staged regulation, the protein conversion rate was 88.7%, lower than the 96.2% of Example 1. This indicates that the staged temperature and pH control strategy can provide the optimal reaction conditions for the degradation characteristics of different types of proteins (collagen, elastin, etc.), and is the key to achieving deep enzymatic hydrolysis.

[0154] The results of Comparative Example 6 further demonstrate the necessity of the PEF treatment step: In bovine and ovine hides without PEF treatment, even with the same phased control strategy, the endogenous enzyme activity was barely increased (only 1.09-fold), the protein conversion rate was only 58.2%, and the proportion of amino acid nitrogen in the liquid phase to total nitrogen was only 52.3%, all far lower than in Example 1. This fully demonstrates that PEF pretreatment is a necessary prerequisite for activating endogenous zymogens; without PEF treatment, endogenous zymogens cannot be effectively activated, and autocatalytic degradation cannot occur.

[0155] The processing cost of Examples 1-3 is only RMB 260 to 320 per ton, which is significantly lower than RMB 1,200 per ton of Comparative Example 1. This is because the present invention does not require the addition of exogenous enzymes and can achieve efficient degradation by relying solely on PEF to activate endogenous enzymes.

[0156] Table 3

[0157]

[0158] Table 3 shows that the quality of the powdered product directly reflects the depth and thoroughness of protein degradation. The powdered products obtained in Examples 1-3 are significantly better than those in Comparative Examples 1-5. The moisture content of Examples 1-3 ranges from 5.9% to 7.8%, the pH value is maintained in a weakly acidic range of 6.3 to 6.7, the total nitrogen content is as high as 11.8% to 13.1%, and the heavy metal content (lead 7.5 to 9.1 mg / kg, arsenic 1.9 to 2.3 mg / kg) all meet the requirements of the organic fertilizer standard (NY / T 525-2021). This reflects the synergistic effect of activating endogenous zymogens through PEF and controlling temperature and pH in stages, resulting in the deep enzymatic hydrolysis of proteins into free amino acids, and the high nitrogen content and stable quality of the obtained products.

[0159] The powder products of Comparative Examples 1-3 had high moisture content (8.5% to 9.2%), alkaline pH (7.0 to 7.2), low total nitrogen content (8.6% to 9.8%), and high heavy metal content (lead 11.8 to 12.3 mg / kg, arsenic 3.2 to 3.5 mg / kg). This indicates that the PEF parameters were not up to standard or that the traditional exogenous enzymatic hydrolysis method resulted in insufficient protein degradation and poor product quality. Comparative Example 4, due to insufficient reaction time, showed incomplete protein degradation, and the product still contained a certain amount of large nitrogenous molecules. Comparative Example 5, lacking staged control, failed to completely hydrolyze some difficult-to-degrade proteins. The total nitrogen content of both examples was 10.2% and 10.8%, respectively, higher than Comparative Examples 1-3, but still different from the examples. This demonstrates that process monitoring and staged control strategies have a positive effect on improving product quality.

[0160] Comparative Example 6, lacking PEF treatment, exhibited the least complete protein degradation, resulting in a powder product with a moisture content as high as 9.5%, a pH value of 7.3 (slightly alkaline), a total nitrogen content of only 7.8%, and the highest heavy metal content (lead 13.2 mg / kg, arsenic 3.8 mg / kg). This further demonstrates the decisive impact of the PEF treatment step on product quality.

[0161] Table 4

[0162]

[0163] Table 4 shows that the compost quality of the solid residue reflects the value of waste resource utilization and also the sanitary and safety effects of harmless treatment. The compost products obtained in Examples 1-3 are significantly better than those in the comparative example. The organic matter content of Examples 1-3 is maintained at a high level of 45.2% to 51.2%, the pH value is controlled within a suitable range of 6.5 to 7.0, and the total nitrogen, phosphorus, and potassium content reaches 5.2% to 6.3%, all of which meet the requirements of the organic fertilizer standard (NY / T 525-2021). The fecal coliform count of the compost products in Examples 1-3 is less than 100 CFU / g, and the mortality rate of Ascaris eggs reaches 97.8% to 99.2%, indicating that the high temperature conditions (55-65℃) during the composting process effectively kill pathogenic microorganisms and parasite eggs, achieving the goal of harmless treatment.

[0164] Comparative analysis revealed that the organic matter content of the compost from the example was approximately 10 to 15 percentage points higher than that of Comparative Examples 1-3. This difference may be due to the more thorough protein degradation in the example, resulting in a lower content of difficult-to-degrade crude protein in the residue and a higher proportion of organic matter readily available for microbial utilization. Composting is essentially a process of microbial decomposition of organic matter; the higher the bioavailability of organic matter in the residue, the better the compost quality. The total nitrogen, phosphorus, and potassium content of the compost from the example was approximately 2 to 3 percentage points higher than that of Comparative Examples 1-3. This indicates that the process in the example is more conducive to retaining nitrogen from the raw materials in the residue. The reason for this is that the endogenous enzymes activated by PEF, while catalyzing protein degradation, also promote the transfer of nitrogen-containing small molecules to the liquid phase; while the undigested residue is enriched with mineral elements such as phosphorus and potassium. Both work synergistically in composting to jointly increase nutrient content. The compost quality of Comparative Examples 4 and 5 also falls between that of the example and Comparative Examples 1-3, further confirming the impact of process monitoring and phased control on residue quality.

[0165] Comparative Example 6, lacking PEF treatment, exhibited the least complete protein degradation, resulting in the worst compost quality. Its organic matter content was only 32.5%, total nitrogen, phosphorus, and potassium content was only 2.8%, fecal coliform count reached a high of 250 CFU / g, and the ascarid egg mortality rate was only 78.5%. This fully demonstrates that the PEF treatment step is a crucial prerequisite for the resource utilization and harmless treatment of bovine and sheepskin waste.

[0166] In summary, this invention achieves highly efficient and harmless treatment of untanned bovine and ovine hide waste through the synergistic effects of pulsed electric field pretreatment to activate endogenous zymogens, staged temperature and pH control, and triggering phase transitions based on the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase. Compared with traditional exogenous enzymatic hydrolysis methods, this invention does not require the addition of exogenous enzymes, and the processing cost is only about one-quarter of that of traditional methods. Simultaneously, the protein conversion rate is as high as 93% to 98%, the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase remains stable at over 85%, and the quality of the resulting powder and compost products meets the standards for organic fertilizers. The high-temperature conditions during composting effectively kill pathogenic microorganisms and parasite eggs, achieving the goal of harmless treatment. PEF field strength (20 to 40 kV / cm) and pulse number (10 to 30 pulses) are key parameters affecting the activation effect of endogenous zymogens; staged control strategies and process monitoring are necessary means to achieve deep protein enzymatic hydrolysis. This invention is applicable to the resource utilization of untanned raw hides such as bare hides after the dehairing process, water-soaked hides, limed hides, delimed hides, and softened hides, and has significant economic and environmental benefits.

Claims

1. A method for harmlessly treating cow and sheep hides, characterized in that, Includes the following steps: (1) Pretreatment: Clean and cut the untanned raw cow and sheep hides into pieces with a length and width of 1-3 cm; (2) Pulse electric field treatment: The leather block is subjected to pulse electric field treatment; (3) Autocatalytic degradation: The leather pieces treated with pulsed electric field are subjected to autocatalytic degradation. No exogenous protease is added during the autocatalytic degradation process. A staged temperature and pH control strategy is adopted, and the stage conversion is triggered based on the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase. (4) Solid-liquid separation: The autocatalytic degradation products are separated into liquid products and solid residues; (5) Product processing: The liquid product is dried to obtain a powder product, and the solid residue is composted to obtain a compost product.

2. The method according to claim 1, characterized in that, In step (2), the field strength of the pulsed electric field treatment is 20-40 kV / cm, the number of pulses is 10-30, the pulse width is 20-100 μs, the pulse frequency is 0.5-2 Hz, and the treatment temperature is 20-30℃.

3. The method according to claim 1, characterized in that, In step (2), the electrode spacing for pulsed electric field treatment is 2-3 cm, and the leather block is placed between the electrodes in a single-layer flat manner.

4. The method according to claim 1, characterized in that, In step (2), the treatment medium for pulsed electric field treatment is deionized water, and the moisture content of the leather block is not less than 70%.

5. The method according to claim 1, characterized in that, The phased temperature and pH control strategy described in step (3) includes: First stage: Temperature 35-45℃, pH 5.5-6.

5. When the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 30%, proceed to the next stage. Second stage: Temperature 38-48℃, pH 5.8-6.

8. When the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 55%, proceed to the next stage. The third stage: temperature 42-50℃, pH 6.2-7.0, the reaction is terminated when the proportion of amino acid nitrogen in the liquid phase to the total nitrogen in the liquid phase reaches 85%.

6. The method according to claim 5, characterized in that, Step (3): Sample the amino acid nitrogen content every 30 minutes in each stage, and determine whether to proceed to the next stage or terminate the reaction based on the test results.

7. The method according to claim 1, characterized in that, In step (3), the material-to-water ratio for autocatalytic degradation is 1:5 to 1:10, and the stirring speed is 150-250 rpm.

8. The method according to claim 1, characterized in that, In step (4), solid-liquid separation is performed using a plate and frame filter press with a filtration pressure of 0.2 MPa and a filter cloth pore size of 8 μm.

9. The method according to claim 1, characterized in that, In step (5), the liquid product is dried by spray drying, with an inlet air temperature of 190°C and an outlet air temperature of 85°C.

10. The method according to claim 1, characterized in that, In step (5), the solid residue is mixed with cow dung and straw and then composted. The mass ratio of solid residue to cow dung is 1:1 to 1:2, the amount of straw added is 0.5 to 1 times the mass of solid residue, the composting cycle is 15-20 days, and the composting temperature is 55-65℃.

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