阅读说明：本技术 基于离子液体双水相系统从铬鞣革屑中提取蛋白质的方法 (Method for extracting protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system ) 是由 陈朱琦 罗放 赵钦地 蔡伽怡 王思淇 于 2021-09-24 设计创作，主要内容包括：本发明涉及基于离子液体双水相系统从铬鞣革屑中提取蛋白质的方法,属于铬鞣革屑资源化利用技术领域。将亲水性离子液体、螯合剂和水加入铬鞣革屑中,充分混匀后,得到两相分层溶液；其中上相为离子液体与蛋白质富集相,下相为铬与螯合剂富集相；将两相分层溶液进行分离,向上相分离物中加入蛋白质沉淀剂,使上相分离物中的蛋白沉淀下来,即从铬鞣革屑中脱铬并提取出蛋白质。本发明能高效且有效的从铬鞣革屑中提取出重金属污染物含量小于10mg/kg的蛋白质产物,且收率大于60％,在不使用挥发性有机物、无需高温加热的绿色条件下,实现铬鞣革屑高值化、无害化、资源化利用。(The invention relates to a method for extracting protein from chrome tanned leather scraps based on an ionic liquid aqueous two-phase system, belonging to the technical field of resource utilization of the chrome tanned leather scraps. Adding hydrophilic ionic liquid, a chelating agent and water into the chrome tanned leather scraps, and fully and uniformly mixing to obtain a two-phase layered solution; wherein the upper phase is an ionic liquid and protein-enriched phase, and the lower phase is a chromium and chelating agent-enriched phase; separating the two-phase layered solution, adding protein precipitant into the upper phase, and precipitating protein from the upper phase, i.e. removing chromium from the chrome tanned leather scraps and extracting protein. The method can efficiently and effectively extract the protein product with the heavy metal pollutant content of less than 10mg/kg from the chrome tanning scraps, has the yield of more than 60 percent, and realizes high-valued, harmless and resource utilization of the chrome tanning scraps under the green conditions of no use of volatile organic matters and no need of high-temperature heating.)

1. The method for extracting protein from chrome tanned leather scraps by dechromization based on the ionic liquid aqueous two-phase system is characterized by comprising the following steps:

(1) adding hydrophilic ionic liquid, a chelating agent and water into the chrome tanned leather scraps, and fully and uniformly mixing to obtain a two-phase layered solution; wherein the upper phase is a hydrophilic ionic liquid and a protein enrichment phase, and the lower phase is a chromium ion and chelating agent enrichment phase; the mass ratio of the chelating agent to the water is more than or equal to 5:6, and the mass ratio of the hydrophilic ionic liquid to the chrome tanning crumbs is less than or equal to 1: 4;

(2) and (2) separating the two-phase layered solution obtained in the step (1), adding a protein precipitator into the upper phase separated matter, and precipitating the protein in the upper phase separated matter, namely removing chromium from the chrome tanned leather scraps and extracting the protein.

2. The method for dechromization extraction of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 1, characterized in that after the protein in the upper phase separated material is precipitated, a mixed solution of hydrophilic ionic liquid and protein precipitator is left, the mixed solution is evaporated and separated to separate the protein precipitator and the hydrophilic ionic liquid, and the separated hydrophilic ionic liquid can be reused in step (1).

3. The method for dechromization extraction of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 1 or 2, characterized in that the hydrophilic ionic liquid is quaternary ammonium salt ionic liquid or imidazole ionic liquid.

4. The method for dechromization extraction of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 3, wherein the quaternary ammonium salt ionic liquid is tetrabutyl ammonium hydroxide, tetrabutyl ammonium chloride or tetrabutyl ammonium acetate; the imidazole ionic liquid is 1-butyl-3-methylimidazole chloride salt, 1-ethyl-3-methylimidazole acetate or 1-butyl-3-methylimidazole methanesulfonate.

5. The method for dechromisation of protein from chrome tanned leather shavings based on ionic liquid aqueous two-phase system according to claim 1 or 2, wherein the chelating agent is tartrate or ethylenediamine tetraacetic acid.

6. The method for dechromization of protein from chrome tanned leather shavings based on an ionic liquid two aqueous phase system of claim 5, wherein the tartrate salt is potassium L-tartrate or sodium potassium tartrate.

7. The method for dechromization extraction of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 1, wherein the protein precipitant is at least one of acetone, ethanol, methanol, acetic acid, formic acid and ammonium sulfate.

8. The method for dechromization of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 1, wherein the step (1) further comprises adding a hydrating agent to the chrome tanned leather scraps.

9. The method for dechromization of protein from chrome tanned leather shavings based on an ionic liquid two aqueous phase system of claim 8, wherein the hydrating agent is phosphate, sodium hydroxide, potassium hydroxide or dithizone.

10. The method for dechromization extraction of protein from chrome tanned leather scraps based on ionic liquid aqueous two-phase system according to claim 1, wherein in the step (1), the fully mixing mode is stirring, and the stirring time is more than 2.5 h.

Technical Field

The invention belongs to the technical field of resource utilization of chrome tanned leather scraps, and particularly relates to a method for extracting protein from chrome tanned leather scraps based on an ionic liquid aqueous two-phase system.

Background

The annual output of leather in China is the front in the world, and the technology mainly applied to the production of leather in China at present is a chrome tanning method, namely a large amount of trivalent chromium salt is used for tanning raw leather, so that the raw leather becomes tough and soft and becomes the leather. Although the method is quick and effective and the produced leather product has excellent quality, a large amount of solid waste with high chromium content, namely chrome tanning leather scraps, is produced in the tanning process, the chromium content of the chrome tanning leather scraps is different from 2 to 3 percent, and the chromium content of the chrome tanning leather scraps far exceeds the chromium heavy metal limit value specified in the related waste standard. The chrome tanned leather scraps are classified as dangerous waste, a great amount of chrome tanned leather scraps are generated in the Chinese tanning industry every year, the requirements of harmless resource treatment of the chrome tanned leather scraps and no secondary pollution in the treatment process are increased day by day. The current industrialized technology for treating chrome tanned leather scraps is an acid/alkali hydrolysis method, and the method comprises the steps of firstly using an acidic or alkaline solution to soak and pretreat the chrome tanned leather scraps for a long time, and then heating the chrome tanned leather scraps under an acid/alkaline condition to hydrolyze the chrome tanned leather scraps so as to separate chromium elements crosslinked with collagen in the chrome tanned leather scraps. However, the method can not separate out protein products with chromium content less than 10mg/kg, and a large amount of acid/alkali wastewater is easily generated in the treatment process, thereby easily causing secondary pollution to the environment. The hydrolysis process in the method also needs to heat the hydrolysate for a long time, and the treatment process has high energy consumption and is not environment-friendly.

Disclosure of Invention

The invention solves the technical problems that the chromium content of protein separated from the chrome tanning scraps is higher, and a large amount of acid/alkali wastewater is easily generated in the treatment process, thereby causing pollution in the prior art. Aiming at the requirements of the existing situation and the defects of the prior art, the invention provides a chromium salt and protein separation technology based on an ionic liquid aqueous two-phase system, which can separate collagen and chromium salt at room temperature in one step and recover a protein product with the chromium content of less than 10 mg/kg. Because the method does not use volatile organic solvent and does not need high-temperature heating, industrial waste gas is hardly generated; the method does not need heating, so expensive high-temperature reaction equipment is not needed; because this scheme innovation has used the two aqueous phase separation technologies based on ionic liquid, compare in traditional art, this technique can reach the effect of chromium and collagen high efficiency separation.

According to the purpose of the invention, the method for extracting protein from chrome tanned leather scraps by dechroming based on the ionic liquid aqueous two-phase system comprises the following steps:

(1) adding hydrophilic ionic liquid, a chelating agent and water into the chrome tanned leather scraps, and fully and uniformly mixing to obtain a two-phase layered solution; wherein the upper phase is a hydrophilic ionic liquid and a protein enrichment phase, and the lower phase is a chromium ion and chelating agent enrichment phase; the mass ratio of the chelating agent to the water is more than or equal to 5:6, and the mass ratio of the hydrophilic ionic liquid to the chrome tanning crumbs is less than or equal to 1: 4;

(2) and (2) separating the two-phase layered solution obtained in the step (1), adding a protein precipitator into the upper phase separated matter, and precipitating the protein in the upper phase separated matter, namely removing chromium from the chrome tanned leather scraps and extracting the protein.

Preferably, after the protein in the upper phase separator is precipitated, a mixed solution of the hydrophilic ionic liquid and the protein precipitator is left, the mixed solution is subjected to evaporation separation, so that the protein precipitator is separated from the hydrophilic ionic liquid, and the separated hydrophilic ionic liquid can be reused in the step (1).

Preferably, the hydrophilic ionic liquid is quaternary ammonium salt ionic liquid or imidazole ionic liquid.

Preferably, the quaternary ammonium salt ionic liquid is tetrabutylammonium hydroxide, tetrabutylammonium chloride or tetrabutylammonium acetate; the imidazole ionic liquid is 1-butyl-3-methylimidazole chloride salt, 1-ethyl-3-methylimidazole acetate or 1-butyl-3-methylimidazole methanesulfonate.

Preferably, the chelating agent is tartrate or ethylenediaminetetraacetic acid.

Preferably, the tartrate salt is potassium L-tartrate or sodium potassium tartrate.

Preferably, the protein precipitant is at least one of acetone, ethanol, methanol, acetic acid, formic acid, and ammonium sulfate.

Preferably, the step (1) further comprises adding a hydrating agent to the chrome tanning waste.

Preferably, the hydrating agent is a phosphate, sodium hydroxide, potassium hydroxide or dithizone.

Preferably, in the step (1), the fully mixing mode is stirring, and the stirring time is more than 2.5 h.

Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) the acid/alkali hydrolysis method used for treating the chrome tanned leather scraps at present can not obtain a protein product with the chromium content of less than 10mg/kg, and the invention can obtain the protein product with the chromium content of less than 10mg/kg on the premise of not generating highly toxic hexavalent chromium, thereby filling the blank in the fields of harmless and recycling of the chrome tanned leather scraps to a certain extent. The technical scheme has simple and convenient operation flow, does not need heating treatment, can theoretically recycle the reagent, meets the requirements of green chemical process, is environment-friendly, has large scale production potential and has huge potential social benefit.

(2) At present, aiming at the traditional process of chrome tanned leather scraps, an acid/alkali hydrolysis method cannot separate out a protein product with the purity of less than 10mg/kg of chromium, and the subsequent application of the product is greatly limited. However, the invention can dissolve collagen in the chrome tanned leather scraps through an ionic liquid-based aqueous two-phase system, chelate chrome, and finally completely remove pollutants through natural phase separation and layering, wherein the protein product can reach the chrome content of less than 10 mg/kg.

(3) When lyophilic ions (such as phosphate anions and tartrate anions) are added into the aqueous solution of the chrome tanned leather scraps, water molecules close to the ions are in an electrical contraction state, a hydrogen bond network of water is enhanced at the stage, and the hydrogen bond network of water cannot form cavities around bulky organic cations, such as tetrabutyl ammonium radicals, imidazole cations and the like, so that an aqueous two-phase system can be effectively formed. In the system, the lower phase is preferably a tartrate-rich phase, and tartrate anions have strong chelating capacity for trivalent chromium, so that the trivalent chromium can be effectively removed from a protein macromolecule side chain and chelated, and then enters the lower phase.

(4) The reason why the mass ratio of the chelating agent to the water is 5:6 or more, the mass of the chrome tanned leather scraps accounts for 30% or less of the mass of the ionic liquid, and the mass ratio of the chelating agent to the water is 5:6 or more is that: the greater the concentration of chelant, the higher the rate of chelant partitioning into the lower phase, when the concentration reaches 5:6, more than 99.9 percent of the chelating agent enters the lower phase, so that the trivalent chromium concentration of the upper phase can be maintained at an extremely low level, thereby achieving the purpose of separating chromium element from protein, and the mass ratio of the chromium tanning powder to the ionic liquid is less than or equal to 1:4, because the ionic liquid can effectively and rapidly dissolve the chromium tanning powder only when the ratio is less than 1: 4.

(5) At present, a large amount of acidic/alkaline wastewater is generated after a hydrolysis step in the traditional process for chrome tanning leather scraps, the chemical wastewater easily causes secondary pollution to the environment, and subsequent treatment steps are complex and cannot be recycled. The ionic liquid and the protein precipitator used in the invention can be recycled through subsequent separation and purification steps, and can be recycled, thereby avoiding the discharge of a large amount of chemical wastewater.

(6) When the traditional alkali hydrolysis method and the traditional acid hydrolysis method are used for treating the chrome tanned leather scraps, the optimal dechromization effect is achieved by long-time heating treatment, but the high-temperature heating treatment is carried out under the acid/alkali condition for a long time, so that the corrosion resistance requirement on a reactor is high, and the problems of overhigh energy consumption and secondary pollution of generated waste gas to the environment exist. The ionic liquid used in the invention has low vapor pressure and non-volatility, and the technical scheme can be carried out at room temperature, so the energy consumption is low, no waste gas is discharged, the method is environment-friendly, and the method meets the requirements of green chemical processes.

Drawings

FIG. 1 is a schematic diagram of a main process flow for dechroming and extracting protein based on an ionic liquid aqueous two-phase system.

FIG. 2 is a graph of the chromium content of the protein product as a function of time with agitation.

FIG. 3 shows the difference between the chromium content of the protein product before and after the addition of the hydrating agent.

FIG. 4 shows the amino acid profile of the protein product obtained in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

FIG. 1 is a schematic diagram of a main process flow for dechroming and extracting protein based on an ionic liquid aqueous two-phase system. The method for extracting protein based on ionic liquid aqueous two-phase system dechromization specifically comprises the following steps:

(1) putting the ionic liquid, water, the chelating agent and the chrome tanning leather scraps into a mixing stirrer, stirring for 6-10 hours at a certain temperature, stopping stirring, and naturally forming two-phase layering, wherein the upper layer is an ionic liquid and collagen enriched phase, and the lower layer is a chromium and chelating agent phase; the ionic liquid comprises but is not limited to quaternary ammonium salt ionic liquid and imidazole ionic liquid which have solubility to protein;

(2) separating the two-phase layered solution obtained in the last step, adding a protein precipitator into the upper phase separator to precipitate protein, and separating the mixture to obtain a mixture of a solid protein product and a liquid ionic liquid and the protein precipitator; the protein precipitating agent comprises one or more of acetone, ethanol, methanol, acetic acid, formic acid, ammonium sulfate and the like, and is characterized by being capable of precipitating the protein in the ionic liquid;

(3) and (3) carrying out evaporation separation operation on the mixed liquid of the ionic liquid and the protein precipitator obtained in the last step, separating the protein precipitator from the ionic liquid, and recycling the reagent.

Preferably, step (1) further comprises adding a hydrating agent, including but not limited to at least one of phosphate, alkali compound and dithizone, which can promote natural phase separation and delamination with the ionic liquid.

Example 1

The invention relates to a method for extracting protein from chrome tanned leather scraps by dechroming based on an ionic liquid aqueous two-phase system, which comprises the following steps:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, and L-potassium tartrate (having a chelating agent and a hydrating agent) were mixed with ultrapure water in a ratio of tetrabutylammonium hydroxide: water: l-potassium tartrate: chrome tanned leather bits 4: 6: 5: 1;

(2) fully stirring the mixture for 8 hours at the temperature of 30 ℃;

(3) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(4) adding mixed solution of acetone and absolute ethyl alcohol (protein precipitant) into the yellowish supernatant to obtain protein product precipitate;

(5) carrying out three times of filtering and washing on the protein product, and drying for 2h at the temperature of 60 ℃ to obtain a final product;

(6) and (3) distilling and separating the mixed liquid of the ionic liquid and the protein precipitator, distilling the protein precipitator into gas at 80 ℃, condensing and recovering to obtain the purified ionic liquid and the purified protein precipitator, and recycling.

(7) The protein product was tested by ICP-OES and the Cr content was 3.33 mg/kg.

Example 2

The invention relates to a method for extracting protein from chrome tanned leather scraps by dechroming based on an ionic liquid aqueous two-phase system, which comprises the following steps:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, potassium sodium tartrate (chelating agent), potassium phosphate (hydrating agent) were mixed with ultrapure water in the ratio of tetrabutylammonium hydroxide: water: potassium sodium tartrate: potassium phosphate: chrome tanned leather bits 4: 6: 2: 1: 1;

(2) fully stirring the mixture for 8 hours at the temperature of 30 ℃;

(3) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(4) adding acetic acid and ethanol mixed solution (protein precipitant) into the supernatant to obtain protein product precipitate;

(5) and (3) filtering and washing the protein product for three times, and drying for 2 hours at the temperature of 60 ℃ to obtain the final product.

(6) The protein product was tested by ICP-OES and the Cr content was 1.74 mg/kg.

FIG. 4 shows the amino acid profile of the protein product obtained in example 2, demonstrating that the major component of the product is protein.

Example 3

The invention relates to a method for extracting protein from chrome tanned leather scraps by dechroming based on an ionic liquid aqueous two-phase system, which comprises the following steps:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, and L-potassium tartrate (having a chelating agent and a hydrating agent) were mixed with ultrapure water in a ratio of tetrabutylammonium hydroxide: water: l-potassium tartrate: chrome tanned leather bits 4: 6: 5: 1;

(2) fully stirring the mixture for 8 hours at the temperature of 30 ℃;

(3) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(4) adding mixed solution of acetone and absolute ethyl alcohol (protein precipitant) into the yellowish supernatant to obtain protein product precipitate;

(5) carrying out three times of filtering and washing on the protein product, and drying for 2h at the temperature of 60 ℃ to obtain a final product;

(6) and (3) distilling and separating the mixed liquid of the ionic liquid and the protein precipitator, distilling the protein precipitator into gas at 80 ℃, condensing and recovering to obtain the purified ionic liquid and the purified protein precipitator, and recycling.

(7) The protein product was tested by ICP-OES and the Cr content was 6.11 mg/kg.

Example 4

The invention relates to a method for extracting protein from chrome tanned leather scraps by dechroming based on an ionic liquid aqueous two-phase system, which comprises the following steps:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, potassium tartrate (chelating agent) were mixed with ultrapure water in the ratio of tetrabutylammonium hydroxide: water: potassium tartrate: chrome tanned leather bits 6: 6: 5: 1;

(2) fully stirring the mixture for 8 hours at the temperature of 30 ℃;

(3) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(4) adding acetic acid and ethanol mixed solution (protein precipitant) into the supernatant to obtain protein product precipitate;

(5) and (3) filtering and washing the protein product for three times, and drying for 2 hours at the temperature of 60 ℃ to obtain the final product.

(6) The protein product is detected by ICP-OES, and the Cr content is 5.32 mg/kg.

Comparative example 1

This example is a comparative experiment carried out to demonstrate the necessity of stirring time for chromium removal, the specific embodiment being:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, potassium sodium tartrate (chelating agent), potassium phosphate (hydrating agent) were mixed with ultrapure water in the ratio of tetrabutylammonium hydroxide: water: potassium sodium tartrate: potassium phosphate: chrome tanned leather bits 4: 6: 2: 1: 1;

(2) fully stirring the mixture for 1h, 2.5h and 4h at the temperature of 30 ℃;

(3) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(4) adding acetic acid and ethanol mixed solution (protein precipitant) into the supernatant to obtain protein product precipitate;

(5) carrying out three times of filtering and washing on the protein product, and drying for 2h at the temperature of 60 ℃ to obtain a final product;

(6) this comparative example is shown in fig. 2, comparative example 1, and shows that when the reaction time is insufficient, chromium crosslinked with collagen cannot be completely chelated and removed.

Comparative example 2

This example is a comparative experiment conducted to demonstrate the necessity of a hydrating agent, or the use of a sufficient amount of a chelating agent to replace the hydrating agent, for chromium removal, and the specific embodiments are:

(1) tetrabutylammonium hydroxide (a quaternary ammonium salt ionic liquid), chrome tanning waste, potassium sodium tartrate (chelating agent) and ultrapure water were mixed in a ratio of tetrabutylammonium hydroxide: water: potassium sodium tartrate: chrome tanned leather bits 4: 6: 2: 1, fully stirring the mixture for 1 hour, 2.5 hours and 4 hours at the temperature of 30 ℃;

(2) standing the mixture until the mixture is naturally split into an upper layer and a lower layer, wherein the upper layer is light yellow clear liquid, the lower layer is dark green clear liquid, and taking the light yellow clear liquid on the upper layer for the next operation;

(3) adding acetic acid and ethanol mixed solution (protein precipitant) into the supernatant to obtain protein product precipitate;

(4) carrying out three times of filtering and washing on the protein product, and drying for 2h at the temperature of 60 ℃ to obtain a final product;

(5) this comparative comparison is shown in FIG. 3, comparative example 2, and shows that when the chelating agent is not added in sufficient amounts without the addition of the hydrating agent, incomplete phase separation can result in chromium incorporation into the protein product, resulting in purification failure.

The heavy metal measuring method comprises the following steps: the chromium content in the protein product in the technical scheme is detected according to a determination method in GB5009.123-2014 determination of chromium in food safety national standard food.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.