Application of bacterium-enzyme synergistic process in leather-making aldehyde tanning solid waste treatment

Abstract

The invention relates to treatment of tanning solid waste, in particular to application of a bacterium-enzyme synergistic process in tanning aldehyde solid waste treatment. According to the method, the fermentation liquor containing enterobacter asparagi and stenotrophomonas maltophilia, the alkaline protease and the alkaline lipase are used for synergistically degrading the tanning aldehyde tanning solid waste, so that the problems that high-temperature treatment is needed by enzymolysis in the prior art, and the added value of a degradation product is low due to small molecular weight are effectively solved; the method has the advantages of short treatment period, no need of high-temperature pretreatment, high added value of the product due to high molecular weight and the like.

Description

Technical Field

[0001] The invention relates to the treatment of leather-making solid waste, and in particular to the application of a bacterial enzyme synergistic process in the treatment of leather-making aldehyde tanning solid waste. Background Art

[0002] Macromolecular proteins are widely used in many fields such as medicine, food, and cosmetics. For example, in the medical field, macromolecular proteins such as collagen and gelatin are used for wound repair, tissue engineering, drug carriers, etc.; in the food field, macromolecular proteins are used as functional ingredients to improve the nutritional value, taste and stability of food; in the cosmetic field, macromolecular proteins have moisturizing and anti-aging effects, and are deeply loved by consumers. Compared with small molecule amino acids, macromolecular proteins have more complex structures and higher biological activity. This makes macromolecular proteins have irreplaceable advantages in certain specific application fields. For example, in the medical field, macromolecular proteins can better simulate biological tissues and promote cell regeneration and repair; in the cosmetic field, macromolecular proteins can form a stable protective film and provide better moisturizing and anti-aging effects. As people pay more attention to health, environmental protection and sustainable development, the demand for macromolecular proteins is also increasing.

[0003] Aldehyde tanning is the use of aldehyde compounds (such as formaldehyde, glutaraldehyde, etc.) as tanning agents to tan leather and fur. Formaldehyde can block the amino groups on the side chains of collagen polypeptide chains and form a mesityle bridge between two polypeptide chains, so that the adjacent amino groups between the peptide chains are "stitched" to produce a tanning effect. This tanning method makes the leather have good sweat resistance and water washing resistance, and has a certain ability to resist acids, alkalis, oxidants and reducing agents. Between aldehyde tanned leather and finished products, trimming and shaving will be carried out, resulting in a large amount of leather scraps. At present, leather scraps are mainly treated by incineration or landfilling. The enzyme method is a method of using the catalytic action of enzymes to decompose macromolecular substances. It has a certain decomposition effect on protein and fat molecules and is a more environmentally friendly treatment method. However, due to the complexity of the cross-linked structure between leathers, it is usually difficult to completely open this cross-linked structure by the enzyme method. In order to better degrade leather solid waste, in the early stage of enzymatic treatment, it is necessary to use a combination of multiple methods to treat it, such as chemical hydrolysis, high-temperature heating, etc.

[0004] The problems existing in the above-mentioned prior art are: first, chemical hydrolysis will introduce acid, alkali and other reagents, causing secondary processing problems; second, the high-temperature heating process accelerates the hydrolysis process of skin collagen under high temperature conditions. High temperature can intensify the movement of water molecules, thereby more easily destroying the chemical bonds in the raw materials. However, enzymatic hydrolysis after high-temperature environment treatment will cause the original spatial structure of the protein to be destroyed, destroying the hydrogen bonds and hydrophobic interactions in the protein, causing the three-dimensional structure of the protein to change and make it lose its activity. Ultimately, the protein is over-hydrolyzed and degraded into a small molecule amino acid mixture, which is used in water-soluble fertilizers, feed additives and other industries, and its product added value is low.

[0005] As Enterobacter abies can form a symbiotic relationship with plant roots, promote the absorption and utilization of nutrients by plants, and thus improve the yield and quality of crops; As Enterobacter abies also has certain application potential in the field of microbial fertilizers. In addition, As Enterobacter abies has the ability to inhibit the growth and reproduction of pathogens, which helps to reduce the occurrence and spread of plant diseases. Therefore, As Enterobacter abies is generally used as an additive to microbial fertilizers or prepared into microbial fertilizer products. There are currently no reports on its application in the treatment of leather solid waste.

[0006] The existing literature searched by the applicant includes:

[0007] Patent documents with application numbers 202410899303.5 and 202410929134.5 both use Enterobacter abies in the field of biocontrol agents; the article "Isolation and Identification of a Bacteria Causing Increased Wort Turbidity and Research on Turbidity-Causing Substances" published in "China Food Additives" applies Enterobacter abies to food additives to increase wort turbidity.

[0008] The patent document with publication number CN114230855A treats leather solid waste and recovers wool fibers, but this method only uses enzyme preparations for treatment, and cannot open the cross-linking between the skins, resulting in only the effect of depilation, and the skin part cannot be further processed. The patent document with publication number CN108018327B destroys the aldehyde and protein binding structure by heating treatment, and the reduction in product molecular weight and energy consumption caused by the heating process are unavoidable. The patent document with application number 2024100831456 is a patent application submitted by the applicant. In the patent document, one of the degradation raw materials is solid waste from fresh leather from leather making, and the raw material has not undergone aldehyde tanning process. Fresh leather is raw hide that has not been tanned, and its collagen fibers are in a relatively loose state; aldehyde tanned leather is usually tanned with formaldehyde or other aldehyde substances. Formaldehyde reacts with the collagen amino groups in the leather to form a cross-linked structure, which enhances the stability and physical properties of the leather, making its structure more stable and more difficult to degrade; second, the fresh leather undergoes high-temperature sterilization during the processing process, that is, heat-bacteria treatment, which will inevitably reduce the molecular weight of the hydrolyzed protein. Therefore, the molecular weight of the enzymatic hydrolysis product obtained is small and the added value of the enzymatic hydrolysis product is low; third, a single strain is used, and its degradation time is 4 days, which is time-consuming and prone to the risk of bacterial contamination and energy loss.

[0009] The applicant has not found any patent document reporting the same or similar to the present application. Summary of the invention

[0010] The purpose of the present invention is to provide an application of a bacterial-enzyme synergistic process in the treatment of aldehyde-tanned solid waste from leather making. The process of the present invention can quickly and effectively hydrolyze aldehyde-tanned leather that has not been treated at high temperature into an enzymatic hydrolysis product rich in macromolecular proteins. The enzymatic hydrolysis product has a high added value due to its wide application field.

[0011] The main technical concept of the present invention is:

[0012] The application of bacterial enzyme synergistic process in the treatment of leather aldehyde tanning solid waste includes the following process steps:

[0013] A. Pretreatment of leather solid waste

[0014] According to the mass ratio of leather solid waste treated by aldehyde tanning process to water = 20-30: 500-1000, the leather solid waste treated by aldehyde tanning process is added into water and soaked for 3-5 hours, and then the leather solid waste is removed and the water is controlled, and the leather solid waste is selected from one of sheepskin solid waste, cowhide solid waste and rabbit skin solid waste;

[0015] B. Preparation of strain fermentation broth

[0016] Enterobacter asburiae and Stenotrophomonas maltophilia are inoculated into sterile culture medium containing carbon source and nitrogen source respectively, and fermentation broth containing Enterobacter asburiae and fermentation broth containing Stenotrophomonas maltophilia are prepared after shaking culture; Enterobacter asburiae is named in Latin as Enterobacter asburiae, and its preservation number is CGMCC No.28900; Stenotrophomonas maltophilia is named in Latin as Stenotrophomonas maltophilia, and its preservation number is CGMCC No.28626;

[0017] C. Preparation of composite bacterial solution

[0018] The fermentation liquid containing Enterobacter asphyxia and the fermentation liquid containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation liquid containing Enterobacter asphyxia to the fermentation liquid containing Stenotrophomonas maltophilia of 2.5 to 3.5:1 by mass;

[0019] D. Bacterial enzyme synergistic hydrolysis

[0020] D1, the leather solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C are mixed in a ratio of 1:8-10 by mass, and 1.5%-1.9% sodium chloride by volume weight and 0.8-1.2 mmol / L potassium chloride are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured for 35-40 hours at a pH of 7.5-8.5, a temperature of 40°C-50°C, and a rotation speed of 80-120 rpm;

[0021] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 3.5‰ to 4.5‰ to the reaction system after oscillation culture in step D1, and oscillate and hydrolyze for 3 to 5 hours at a temperature of 40°C to 50°C and a rotation speed of 80 to 120 rpm.

[0022] The Enterobacter asburiae in the present invention is isolated from the soil around the tannery solid waste in Xinji leather-making area, Hebei Province. It can ferment to produce lipase. After physiological and biochemical reactions and molecular biological identification, it was determined to be Enterobacter asburiae. Its Latin name is Enterobacter asburiae. The applicant has deposited it in the General Microbiology Center of China National Microbiological Culture Collection on November 8, 2023, with a deposit number of CGMCC No. 28900; the Latin name of the Stenotrophomonas maltophilia in the present invention is Stenotrophomonas maltophilia, which can ferment to produce protease. The applicant has deposited it in the General Microbiology Center of China National Microbiological Culture Collection on October 13, 2023, with a deposit number of CGMCC No. 28626; the address of the preservation institution is located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, abbreviated as CGMCC.

[0023] The specific process steps and process conditions of the present invention also include:

[0024] The fermentation broth containing Enterobacter asphodeloides is obtained by inoculating Enterobacter asphodeloides in a sterile culture medium containing a carbon source and a nitrogen source and culturing the culture medium and the culture conditions. The culture medium and the culture conditions can be further optimized on the premise of satisfying the growth of the bacteria and the acquisition of secondary metabolites, without departing from the essence of the present invention. The preferred technical implementation means is that the preparation of the fermentation broth containing Enterobacter asphodeloides in step B is to inoculate Enterobacter asphodeloides in a sterile culture medium at an inoculation amount of 3% to 5%, and to obtain the result after oscillating and culturing for 10 to 14 hours under the conditions of natural pH, temperature of 35° C. to 40° C., and rotation speed of 150 to 200 rpm. The sterile culture medium is composed of the following components:

[0025] Yeast powder 4-6 g / L; peptone 8-12 g / L; olive oil 2-4 g / L; sodium chloride 4-6 g / L; the balance is water.

[0026] The fermentation broth containing Stenotrophomonas maltophilia is obtained by inoculating Stenotrophomonas maltophilia into a sterile culture medium containing a carbon source and a nitrogen source and culturing the culture medium under shaking conditions. The culture medium and culture conditions can be further optimized on the premise of satisfying bacterial growth and obtaining secondary metabolites, without departing from the essence of the present invention. The preferred technical implementation means is that the preparation of the fermentation broth containing Stenotrophomonas maltophilia in step B is to inoculate Stenotrophomonas maltophilia into a sterile culture medium at an inoculation amount of 3% to 5%, and culturing the culture medium under shaking conditions of natural pH, 35° C. to 40° C., and 150 to 200 rpm for 10 to 14 hours. The sterile culture medium is composed of the following components:

[0027] Yeast powder 0.5-1.5 g / L; casein 4-6 g / L; sodium chloride 4-6 g / L; the balance is water.

[0028] In order to further improve the technical effect of the bacterial enzyme synergistic treatment of leather solid waste treated by the aldehyde tanning process in the present invention, the enzyme preparation in the present invention preferably adopts the following technical scheme, the alkaline lipase activity in step D2 is 1500U / g~2000U / g; the reaction temperature is 30℃~60℃, and the reaction pH=7~10.

[0029] In step D2, the activity of alkaline protease is 700000 U / g to 800000 U / g, the reaction temperature is 20° C. to 60° C., and the reaction pH is 8 to 12.

[0030] The applicant needs to explain that the method of the present invention can also be used to process fresh leather, because the fresh leather has not been treated with the aldehyde tanning process, and therefore the treatment is easier and faster. However, under the same conditions, the content of macromolecular protein in the hydrolyzate obtained is lower than that in the leather solid waste treated with the aldehyde tanning process. Therefore, the above improvements do not deviate from the essence of the present invention.

[0031] The substantial features and significant technical advances of the present invention are:

[0032] 1. The present invention adopts bacterial enzymes to synergistically degrade aldehyde tanning solid waste from leather making. First, a bacterial strain capable of degrading fat and protein is adopted to achieve the effect of opening the cross-linking between the leathers of the aldehyde tanning solid waste through bacterial strain degradation; second, the synergistic treatment of bacterial enzymes can degrade the aldehyde tanning solid waste in a relatively short time while maintaining the macromolecular biological activity of the protein after degradation; third, the obtained enzymatic hydrolysate rich in macromolecular proteins has great application prospects in the industries of biomedicine, tissue engineering, protein spinning, cosmetics, etc., and can significantly improve its industrial added value compared with small molecule hydrolysate.

[0033] 2. The present invention uses bacterial enzymes to synergistically degrade organic matter in leather aldehyde tanning solid waste. First, it can convert it into harmless or low-toxic substances, thereby reducing the pollutant content in wastewater and reducing the difficulty and cost of wastewater treatment; second, during the treatment process, the metabolic activities of microorganisms will only produce carbon dioxide. Compared with the existing heat treatment method, the treatment process of the present invention has low energy consumption, simple equipment requirements, smaller waste gas emissions and environmental impact, and is safe and pollution-free; third, it can make up for the problems of low enzyme activity of microbial enzymes and fewer enzyme types. The introduced strains can resist the influence of other bacteria. Compared with single enzyme preparation treatment, it can effectively delay system corruption and reduce the risk of disease transmission. Fourth, the treatment cycle is short, which improves efficiency and reduces costs, providing a new direction for the resource treatment of leather solid waste.

[0034] The Latin name of Enterobacter asburiae in the present invention is Enterobacter asburiae, and the applicant has deposited it in the General Microbiology Center of China National Microorganism Culture Collection Administration on November 8, 2023, with a deposit number of CGMCC No. 28900; the Latin name of Stenotrophomonas maltophilia in the present invention is Stenotrophomonasmaltophilia, and the applicant has deposited it in the General Microbiology Center of China National Microorganism Culture Collection Administration on October 13, 2023, with a deposit number of CGMCC No. 28626; the address of the deposit institution is located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, referred to as CGMCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a comparative schematic diagram of the fat hydrolysis circle of the Enterobacter aspergillus of the present invention and other strains.

[0036] Figure 2 It is a comparative schematic diagram of the protein degradation hydrolysis circle of the Stenotrophomonas maltophilia of the present invention and other strains.

[0037] Figure 3 These are the results of affinity tests on Enterobacter aspergillus and Stenotrophomonas maltophilia in the present invention.

[0038] Figure 4 It is a schematic diagram of the before and after comparison results of hydrolyzing sheepskin solid waste treated by an aldehyde tanning process using the method of the present invention.

[0039] Figure 4 The results show that when the method of the present invention is used to hydrolyze the sheepskin solid waste treated by the aldehyde tanning process, there is no solid matter in the hydrolyzed product, which indicates that the method of the present invention can form a good hydrolysis effect on the sheepskin solid waste treated by the aldehyde tanning process.

[0040] Figure 5It is the electrophoresis band diagram of the product obtained by the enzymatic hydrolysis method and the method of the present invention.

[0041] Lanes 1, 2, 3, and 4 in the figure are samples of the product obtained by the enzymatic hydrolysis method, and the protein bands are almost invisible. Lanes 5, 6, and 7 are samples of the product obtained by the method of the present invention, and their molecular weight is close to 140 kDa.

[0042] Figure 6 It is a schematic diagram of the antibacterial effect of the composite bacterial solution in the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with embodiments, but is not intended to be limiting of the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims, and any replacement of equivalent means made based on the description of the present invention shall not depart from the protection scope of the present invention.

[0044] Example 1

[0045] In order to verify the difference in the ability of lipase produced by the Enterobacter aspergillus of the present invention and existing strains to hydrolyze fat, the applicant inoculated the Enterobacter aspergillus of the present invention and some existing strains into the sterilized fermentation medium of the fat-degrading strain, and measured the hydrolysis zone and colony diameter. Figure 1 . Among them, ZF1 is Enterobacter aspergillus in the present invention, ZF2 is Brevundimonas defective, ZF3 is Stenotrophomonas maltophilia, ZF4 is Bacillus subtilis, ZF16 is Bacillus licheniformis, ZF18 is Pseudomonas, ZF20 is Delftia, and ZF22 is Stenotrophomonas. The specific method is: take a single colony point to the fat-degrading strain culture medium, culture it at a temperature of 30°C for 48 hours, and measure the hydrolysis circle and colony diameter with a vernier caliper. The fermentation medium of the fat-degrading strain is composed of the following raw materials: 5 g / L yeast powder; 10 g / L peptone; 3 g / L olive oil; 5 g / L sodium chloride; the balance is water; the pH is natural, and 15% to 20% agar is added to the solid culture medium. The obtained results of the ratio of the hydrolysis circle diameter to the colony diameter of the fat-degrading strain are shown in Table 1.

[0046] Table 1 Ratio of hydrolysis zone diameter to colony diameter of fat-degrading strains (H / C)

[0047] Strain number Ratio of hydrolysis zone diameter to colony diameter (H / C) ZF1 2.63 ZF2 1.09 ZF3 1.98 ZF4 1.25 ZF16 1.13 ZF18 1.03 ZF20 1.92 ZF22 1.75

[0048] The lipase activity was tested according to GB / T 23535-2009 "Lipase Preparations". The lipase activity produced by the fat-degrading strains was shown in Table 2.

[0049] Table 2 Results of enzyme activity test of lipolysis strains

[0050] Strain number Enzyme activity (U) ZF1 60.3 ZF2 10.7 ZF3 38.1 ZF4 25.7 ZF16 27.3 ZF18 16.2 ZF20 32 ZF22 20.6

[0051] Depend on Figure 1 From the results shown in Table 1 and Table 2, it can be seen that the ratio of the hydrolysis zone diameter to the colony diameter and the enzyme activity value measured by the Enterobacter asphaltenes in the present invention are the highest, indicating that the lipase produced by the Enterobacter asphaltenes in the present invention has the highest enzyme activity.

[0052] Example 2

[0053] In order to verify the difference in the ability of the protease produced by the Stenotrophomonas maltophilia in the present invention and the existing strains to hydrolyze proteins, the applicant inoculated the Stenotrophomonas maltophilia in the present invention and some existing strains into sterilized milk agar medium, and measured the hydrolysis zone and colony diameter. Figure 2 . Wherein D1 is the Stenotrophomonas maltophilia of the present invention, D2 is Stenotrophomonas, D6 is Bacillus, D8 is Bacillus licheniformis, D9 is Bacillus amyloliquefaciens, and D15 is Bacillus subtilis. The specific method is: use a sterilized toothpick to pick a single colony point on a milk agar medium, culture it at a temperature of 37°C for 48 hours, use a vernier caliper to measure the colony hydrolysis circle and colony diameter, and the obtained hydrolysis circle diameter to colony diameter ratio (H / C) is shown in Table 1. The milk agar medium is composed of the following raw materials: 20 g / L skim milk powder, 20 g / L agar, the balance is water, and the pH is natural. The preparation method is as follows: weigh 20 g / 500 ml skim milk powder, prepare with distilled water, and sterilize at 105°C for 15 minutes; weigh 20 g / 500 ml agar, prepare with distilled water, and sterilize at 121°C for 20 minutes. When the culture medium cools to 60°C, mix the skim milk powder and agar, pour into a flat plate and set aside.

[0054] Table 3 Ratio of hydrolysis zone diameter to colony diameter (H / C) of each colony

[0055] Strain number Ratio of hydrolysis zone diameter to colony diameter (H / C) D1 3.20 D2 2.83 D6 3.25 D8 2.50 D9 2.33 D15 1.50

[0056] The strain is inoculated into a protein-degrading strain fermentation medium at an inoculation rate of 3% to 5%, and the fermentation liquid is obtained after shaking culture for 10 to 14 hours under the conditions of natural pH, temperature of 35°C to 40°C, and rotation speed of 150 to 200 rpm. The fermentation supernatant is obtained after centrifugation to separate the bacterial cells and the protease activity is detected. The protein-degrading strain fermentation medium consists of the following components:

[0057] Yeast powder 5 g / L; peptone 10 g / L; sodium chloride 5 g / L; the balance is water; pH natural.

[0058] The protease activity was tested according to GB / T 23527-2009 "Protease Preparations". The protease activity produced by the protein-degrading strains was shown in Table 4.

[0059] Table 4 Results of protease activity test of protein-degrading strains

[0060] Strain number Enzyme activity (U) D1 125.33 D2 68.31 D6 78.15 D8 53.21 D9 40.66 D15 35.87

[0061] Depend on Figure 2 From the results shown in Table 1 and Table 2, it can be seen that the ratio of the hydrolysis zone diameter to the colony diameter measured by the Stenotrophomonas maltophilia in the present invention is higher, and the enzyme activity value is the highest. The protease produced by the Stenotrophomonas maltophilia in the present invention is stronger and the protease activity produced is higher.

[0062] Example 3

[0063] In order to verify the compatibility of Enterobacter aspergillus and Stenotrophomonas maltophilia in the present invention, the applicant conducted the following experiment: Stenotrophomonas maltophilia in the present invention and Enterobacter aspergillus were mixed at OD 600nm Under the conditions, culture for about 0.8 to 1.0 hours, evenly spread 100 microliters of Stenotrophomonas maltophilia bacterial solution on the solid culture medium. The solid culture medium is formulated with the same ratio as the protein-degrading strain fermentation medium in Example 2 and 15% to 20% agar is added. Then place the Oxford cup on the culture medium and press it tightly. Add 100 microliters of Enterobacter asphodeloides bacterial solution to the cup and culture it upright at 37°C for 24 hours. Take another solid culture medium, use an inoculation loop to pick single colonies of Enterobacter asphodeloides and Stenotrophomonas maltophilia, cross-draw lines on the culture medium in pairs, and conduct antagonism experiments. No transparent spots appear, and the bacteria can grow normally, which means they are compatible. The specific results are shown in Figure 3 The results of the antagonism experiment show that the Enterobacter aestivum and Stenotrophomonas maltophilia in the present invention are compatible with each other.

[0064] Example 4

[0065] In order to compare the molecular weight of the product obtained by hydrolyzing the sheepskin solid waste treated by the aldehyde tanning process by the enzymatic method and the method of the present invention, the applicant conducted the following experiment:

[0066] Preparation of enzymatic hydrolysis product (control): Sodium chloride in a volume-to-weight ratio of 1.5% to 1.9%, potassium chloride in a volume-to-weight ratio of 0.8 to 1.2 mmol / L, lipase in a volume-to-weight ratio of 3.5‰ to 4.5‰, and protease in a volume-to-weight ratio of 3.5‰ to 4.5‰ were added to the raw material prepared in step A of the present invention, and oscillated and hydrolyzed for 3 to 5 hours at a temperature of 40°C to 50°C and a rotation speed of 80 to 120 rpm, and the enzymatic hydrolysis product was obtained by centrifugal filtration.

[0067] The relative molecular weight of the enzyme protein was determined by SDS-PAGE electrophoresis with 5% concentrated gel and 20% separated gel. The results are as follows: Figure 5In the figure, lanes 1, 2, 3, and 4 are samples of the product obtained by the enzymatic hydrolysis method, and the protein bands are almost invisible. Lanes 5, 6, and 7 are samples of the product obtained by the method of the present invention, and their molecular weight is close to 140 kDa. Compared with the 25 kDa protein obtained in the publication number CN108018327B, the molecular weight is larger, and there are fewer impure protein bands, which is easy to separate and process, and has good application prospects and high added value.

[0068] Example 5

[0069] In order to verify the antibacterial effect of the composite bacterial solution in the present invention, the applicant conducted the following experiments:

[0070] The fermentation liquid containing Enterobacter aspergillus and the fermentation liquid containing Stenotrophomonas maltophilia were compounded into a composite bacterial liquid according to the ratio of the mass fraction of the fermentation liquid containing Enterobacter aspergillus to the fermentation liquid containing Stenotrophomonas maltophilia = 3:1; the composite bacterial liquid was centrifuged to filter out the bacterial bodies, and 2 ml of the sterile composite bacterial liquid was evenly applied to the left part of the solid culture medium; the hydrolyzate after 12 hours of aldehyde tanned leather hydrolysis using the method of the present invention was diluted with sterile distilled water for 10 6 times, take 50 microliters and evenly spread on both sides of the culture medium, and culture it upright at 37°C for 24 hours. The solid culture medium is prepared in the same proportion as the fermentation medium of the protein-degrading strain in Example 2 and 15% to 20% agar is added. Figure 6 As shown, the number of colonies in the left culture medium is less than that in the right culture medium, indicating that the composite bacterial solution prepared by the present invention can reduce the growth of miscellaneous bacteria.

[0071] Example 6

[0072] The application of bacterial enzyme synergistic process in the treatment of leather aldehyde tanning solid waste includes the following process steps:

[0073] A. Pretreatment of leather solid waste

[0074] According to the mass ratio of leather solid waste treated by aldehyde tanning process to water = 20-30: 500-1000, the sheepskin solid waste treated by aldehyde tanning process is added into water and soaked for 3-5 hours, then removed and water is controlled, and the leather solid waste is sheepskin solid waste;

[0075] B. Preparation of strain fermentation broth

[0076] Enterobacter asburiae and Stenotrophomonas maltophilia are inoculated into sterile culture medium containing carbon source and nitrogen source respectively, and fermentation broth containing Enterobacter asburiae and fermentation broth containing Stenotrophomonas maltophilia are prepared after shaking culture; Enterobacter asburiae is named in Latin as Enterobacter asburiae, and its preservation number is CGMCC No.28900; Stenotrophomonas maltophilia is named in Latin as Stenotrophomonas maltophilia, and its preservation number is CGMCC No.28626;

[0077] C. Preparation of composite bacterial solution

[0078] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 3:1 by mass;

[0079] D. Bacterial enzyme synergistic hydrolysis

[0080] D1. Mix the leather solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C in a ratio of 1:8-10 by mass, add 1.77% sodium chloride by volume to the mixture and 1 mmol / L potassium chloride to prepare a reaction system, and culture the reaction system under the conditions of pH=8, temperature of 45°C and rotation speed of 100 rpm for 38 hours;

[0081] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 4‰ and 4‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 45°C and a rotation speed of 100 rpm for 4 hours.

[0082] The fermentation broth containing Enterobacter asphodeloides in step B is prepared by inoculating Enterobacter asphodeloides in a sterile culture medium at an inoculation rate of 3% to 5%, and shaking culture for 10 to 14 hours at a natural pH, a temperature of 35° C. to 40° C., and a rotation speed of 150 to 200 rpm. The sterile culture medium is composed of the following components:

[0083] Yeast powder 4-6 g / L; peptone 8-12 g / L; olive oil 2-4 g / L; sodium chloride 4-6 g / L; the balance is water.

[0084] The fermentation broth containing Stenotrophomonas maltophilia in step B is prepared by inoculating Stenotrophomonas maltophilia in a sterile culture medium at an inoculum amount of 3% to 5%, and shaking culture for 10 to 14 hours under the conditions of natural pH, temperature of 35° C. to 40° C., and rotation speed of 150 to 200 rpm. The sterile culture medium consists of the following components:

[0085] Yeast powder 0.5-1.5 g / L; casein 4-6 g / L; sodium chloride 4-6 g / L; the balance is water.

[0086] In step D2, the activity of alkaline lipase is 1500 U / g to 2000 U / g; the reaction temperature is 30° C. to 60° C., and the reaction pH is 7 to 10.

[0087] In step D2, the activity of alkaline protease is 700000 U / g to 800000 U / g, the reaction temperature is 20° C. to 60° C., and the reaction pH is 8 to 12.

[0088] The final hydrolysis rate using the method of this embodiment is 96.3%.

[0089] Example 7

[0090] The difference between this embodiment and embodiment 6 is that:

[0091] C. Preparation of composite bacterial solution

[0092] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 2.5:1 by mass;

[0093] D. Bacterial enzyme synergistic hydrolysis

[0094] D1, the leather solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C are mixed in a ratio of 1:8-10 by mass, and 1.5% sodium chloride and 0.8 mmol / L potassium chloride are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured at pH = 7.5, 40°C, and 80 rpm for 35 hours;

[0095] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 3.5‰ and 3.5‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 40°C and a rotation speed of 80 rpm for 3 hours.

[0096] The rest of the method is the same as that in Example 6.

[0097] The final hydrolysis rate using the method of this embodiment is 90.3%.

[0098] Example 8

[0099] The difference between this embodiment and embodiment 6 is that:

[0100] C. Preparation of composite bacterial solution

[0101] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 3.5:1 by mass;

[0102] D. Bacterial enzyme synergistic hydrolysis

[0103] D1, the leather solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C are mixed in a ratio of 1:8-10 by mass, and sodium chloride with a volume weight ratio of 1.9% and potassium chloride with an amount of 1.2 mmol / L are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured at pH = 8.5, a temperature of 50° C., and a rotation speed of 120 rpm for 40 hours;

[0104] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 4.5‰ and 4.5‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 50°C and a rotation speed of 120 rpm for 5 hours.

[0105] The rest of the method is the same as that in Example 6.

[0106] The final hydrolysis rate using the method of this example is 95.4%.

[0107] The effects of sheepskin solid waste treatment before and after the treatment in Examples 6 to 8 are as follows: Figure 4 As shown; the electrophoresis bands and molecular weight distribution results of the hydrolyzates obtained in Examples 6 to 8 are as follows Figure 5 And the accompanying drawings are shown.

[0108] Example 9

[0109] The difference between this embodiment and embodiment 6 is that:

[0110] A. Pretreatment of leather solid waste

[0111] According to the mass ratio of leather solid waste treated by aldehyde tanning process to water = 20-30: 500-1000, the leather solid waste treated by aldehyde tanning process is added into water and soaked for 3-5 hours, and then the leather solid waste is taken out and the water is controlled, and the leather solid waste is cowhide solid waste;

[0112] C. Preparation of composite bacterial solution

[0113] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 3:1 by mass;

[0114] D. Bacterial enzyme synergistic hydrolysis

[0115] D1. Mix the pre-treated cowhide solid waste obtained in step A and the composite bacterial solution prepared in step C in a ratio of 1:8-10 by mass, add 1.77% sodium chloride by volume to the mixture and 1 mmol / L potassium chloride to prepare a reaction system, and culture the reaction system under the conditions of pH=8.0, 45°C, and 100 rpm for 38 hours;

[0116] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 4‰ and 4‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 45°C and a rotation speed of 100 rpm for 4 hours.

[0117] The rest of the method is the same as in Example 6.

[0118] The final hydrolysis rate using the method of this embodiment is 98.3%.

[0119] Example 10

[0120] The difference between this embodiment and embodiment 6 is that:

[0121] A. Pretreatment of leather solid waste

[0122] According to the mass ratio of leather solid waste treated by aldehyde tanning process to water = 20-30: 500-1000, the leather solid waste treated by aldehyde tanning process is added into water and soaked for 3-5 hours, and then the leather solid waste is removed and the water is controlled, and the leather solid waste is rabbit skin solid waste;

[0123] C. Preparation of composite bacterial solution

[0124] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 3:1 by mass;

[0125] D. Bacterial enzyme synergistic hydrolysis

[0126] D1, the rabbit skin solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C are mixed in a ratio of 1:8-10 by mass, and sodium chloride with a volume weight ratio of 1.77% and potassium chloride with an amount of 1 mmol / L are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured at pH = 8.0, a temperature of 45° C., and a rotation speed of 100 rpm for 38 hours;

[0127] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 4‰ and 4‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 45°C and a rotation speed of 100 rpm for 4 hours.

[0128] The rest of the method is the same as in Example 6.

[0129] The final hydrolysis rate using the method of this example is 91.5%.

[0130] Embodiment 11

[0131] The difference between this embodiment and embodiment 6 is that:

[0132] A. Pretreatment of leather solid waste

[0133] According to the weight ratio of leather solid waste not treated by aldehyde tanning process to water = 20-30: 500-1000, the leather solid waste not treated by aldehyde tanning process is added into water and soaked for 3-5 hours, then the leather solid waste is removed and the water is controlled, and the leather solid waste is fresh sheepskin;

[0134] C. Preparation of composite bacterial solution

[0135] The fermentation broth containing Enterobacter asphyxia and the fermentation broth containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation broth containing Enterobacter asphyxia to the fermentation broth containing Stenotrophomonas maltophilia of 3:1 by mass;

[0136] D. Bacterial enzyme synergistic hydrolysis

[0137] D1, the fresh sheepskin solid waste obtained after the pretreatment in step A: the composite bacterial liquid prepared in step C are mixed in a ratio of 1:8-10, and sodium chloride with a volume weight ratio of 1.77% and potassium chloride with an amount of 1 mmol / L are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured for 38 hours under the conditions of pH=8.0, temperature of 45°C, and rotation speed of 100 rpm;

[0138] D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 4‰ and 4‰ respectively to the reaction system after shaking culture in step D1, and shake and hydrolyze at a temperature of 45°C and a rotation speed of 100 rpm for 4 hours.

[0139] The rest of the method is the same as in Example 6.

[0140] The final hydrolysis rate using the method of this embodiment is 98.5%.

Claims

1. Application of bacterial enzyme synergistic process in the treatment of aldehyde tanning solid waste from leather making, characterized in that The process steps include: A. Pretreatment of leather solid waste According to the mass ratio of leather solid waste treated by aldehyde tanning process to water = 20-30: 500-1000, the leather solid waste treated by aldehyde tanning process is added into water and soaked for 3-5 hours, and then the leather solid waste is removed and the water is controlled, and the leather solid waste is selected from one of sheepskin solid waste, cowhide solid waste and rabbit skin solid waste; B. Preparation of strain fermentation broth Enterobacter asburiae and Stenotrophomonas maltophilia are inoculated into sterile culture medium containing carbon source and nitrogen source respectively, and fermentation broth containing Enterobacter asburiae and fermentation broth containing Stenotrophomonas maltophilia are prepared after shaking culture; Enterobacter asburiae is named in Latin as Enterobacter asburiae, and its preservation number is CGMCC No.28900; Stenotrophomonas maltophilia is named in Latin as Stenotrophomonas maltophilia, and its preservation number is CGMCC No.28626; C. Preparation of composite bacterial solution The fermentation liquid containing Enterobacter asphyxia and the fermentation liquid containing Stenotrophomonas maltophilia prepared in step B are compounded to prepare a composite bacterial liquid according to a ratio of the fermentation liquid containing Enterobacter asphyxia to the fermentation liquid containing Stenotrophomonas maltophilia of 2.5 to 3.5:1 by mass; D. Bacterial enzyme synergistic hydrolysis D1, the leather solid waste obtained after the pretreatment in step A and the composite bacterial solution prepared in step C are mixed in a ratio of 1:8-10 by mass, and 1.5%-1.9% sodium chloride by volume weight and 0.8-1.2 mmol / L potassium chloride are added to the mixture to prepare a reaction system, and the reaction system is shaken and cultured for 35-40 hours at a pH of 7.5-8.5, a temperature of 40°C-50°C, and a rotation speed of 80-120 rpm; D2. Add alkaline lipase and alkaline protease in a volume-to-mass ratio of 3.5‰ to 4.5‰ to the reaction system after oscillation culture in step D1, and oscillate and hydrolyze for 3 to 5 hours at a temperature of 40°C to 50°C and a rotation speed of 80 to 120 rpm.

2. The application of the bacterial enzyme synergistic process according to claim 1 in the treatment of aldehyde tanning solid waste from leather making is characterized in that The fermentation broth containing Enterobacter aspergillus in step B is prepared by inoculating Enterobacter aspergillus in a sterile culture medium at an inoculation rate of 3% to 5%, and shaking culture for 10 to 14 hours at a natural pH, a temperature of 35°C to 40°C, and a rotation speed of 150 to 200 rpm. The sterile culture medium is composed of the following components: composition: Yeast powder 4-6 g / L; peptone 8-12 g / L; olive oil 2-4 g / L; sodium chloride 4-6 g / L; the balance is water.

3. The application of the bacterial enzyme synergistic process according to claim 1 in the treatment of aldehyde tanning solid waste from leather making is characterized in that The fermentation broth containing Stenotrophomonas maltophilia in step B is prepared by inoculating Stenotrophomonas maltophilia into a sterile culture medium at an inoculum amount of 3% to 5%, and shaking culture for 10 to 14 hours at a natural pH, a temperature of 35° C. to 40° C., and a rotation speed of 150 to 200 rpm. The sterile culture medium is composed of the following components: composition: Yeast powder 0.5-1.5 g / L; casein 4-6 g / L; sodium chloride 4-6 g / L; the balance is water.

4. The application of the bacterial enzyme synergistic process according to claim 1 in the treatment of aldehyde tanning solid waste from leather making is characterized in that In step D2, the activity of alkaline lipase is 1500U / g-2000U / g; the reaction temperature is 30°C-60°C, and the reaction pH is 7-10.

5. The application of the bacterial enzyme synergistic process in the treatment of aldehyde tanning solid waste from leather making according to claim 1 is characterized in that In step D2, the activity of alkaline protease is 700000 U / g to 800000 U / g, the reaction temperature is 20° C. to 60° C., and the reaction pH is 8 to 12.

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