Method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by combining acid treatment with chitosan flocculation and application
阅读说明：本技术 酸处理结合壳聚糖絮凝回收鱼糜漂洗液中的肌浆蛋白的方法及应用 (Method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by combining acid treatment with chitosan flocculation and application ) 是由 黄琪琳 周文燕 龙斯宇 熊善柏 刘念 张茜 于 2020-12-28 设计创作，主要内容包括：本发明涉及一种酸处理结合壳聚糖絮凝回收鱼糜漂洗液中的肌浆蛋白的方法及其应用,鱼肉置于蒸馏水中漂洗,离心,上清液为鱼糜漂洗液；鱼糜漂洗液中加入壳聚糖溶液,先调节pH至2.0-4.0,再用NaOH溶液调节pH至6.0-9.0,静置、离心,沉淀即为肌浆蛋白与壳聚糖的共絮凝物。本发明相对于传统壳聚糖絮凝法,酸处理可以提高壳聚糖对肌浆蛋白的回收率一倍以上,其回收的肌浆蛋白与壳聚糖共絮凝物无需分离,呈现良好的可纺性,可直接应用于静电纺丝制作肌浆蛋白纳米纤维膜,此电纺纤维膜具有强的表面疏水性、机械性能及热稳定性。本发明絮凝回收肌浆蛋白节能降耗,减少了鱼糜漂洗废水的处理成本和压力,解决了鱼糜工业化生产中副产物的回收和高值化利用的难题。(The invention relates to a method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by combining acid treatment with chitosan flocculation and application thereof, wherein fish is placed in distilled water for rinsing and centrifuging, and the supernatant is minced fillet rinsing liquid; adding chitosan solution into the minced fillet rinsing liquid, firstly adjusting the pH to 2.0-4.0, then adjusting the pH to 6.0-9.0 by NaOH solution, standing, centrifuging, and precipitating to obtain the co-flocculate of the sarcoplasmic protein and the chitosan. Compared with the traditional chitosan flocculation method, the acid treatment can improve the recovery rate of the chitosan to the sarcoplasmic protein by more than one time, the recovered sarcoplasmic protein and chitosan co-flocculate does not need to be separated, the good spinnability is presented, the acid treatment can be directly applied to electrostatic spinning to prepare the sarcoplasmic protein nano fiber membrane, and the electrospun fiber membrane has strong surface hydrophobicity, mechanical property and thermal stability. The invention can save energy and reduce consumption, reduce the treatment cost and pressure of the minced fillet rinsing wastewater, and solve the problems of recovery and high-value utilization of byproducts in the industrialized production of the minced fillet.)
1. The method for recovering the sarcoplasmic proteins in the minced fillet rinsing liquid by acid treatment and chitosan flocculation is characterized by comprising the following steps:
s1: preparing a minced fillet rinsing liquid: adding distilled water into fish meat for rinsing, centrifuging, and taking supernatant as minced fillet rinsing liquid;
s2: adding chitosan solution into the minced fillet rinsing liquid, and adjusting the pH value to 6.0-9.0;
s3: standing, centrifuging, and precipitating to obtain the co-flocculate of sarcoplasmic protein and chitosan.
2. The method of claim 1, wherein in step S1, the solid-to-liquid ratio of fish meat to distilled water is 1 (1.0-3.5) (g/mL), the centrifugation conditions are: 8000-11000g, 4 ℃, 12-18 min.
3. The method as claimed in claim 1, wherein in step S2, the deacetylation degree of chitosan is 85-98%, and the amount of chitosan added in the minced fish rinsing solution is 150-400 mg/L.
4. The method of claim 1, wherein in step S2, the pH is adjusted to 2.0-4.0, and then adjusted to 6.0-9.0 with NaOH solution.
5. The method of claim 4, wherein in step S2, the pH is adjusted to 3.0, and then adjusted to 7.0 with NaOH solution.
6. The method of claim 5, wherein in the step S2, the concentration of the NaOH solution is 1.0-6.0 mol/L.
7. The method of claim 1, wherein in step S3, the temperature of the still standing in the water bath is 10-35 ℃, and the centrifugation conditions are as follows after the still standing time is 30-300 min: 3500-4500r/min, 8-15 min.
8. Use of sarcoplasmic proteins recovered by the process of any of claims 1 to 7 for the manufacture of sarcoplasmic proteins electrospun nanofibrous membranes, characterized in that said use comprises the steps of:
freeze-drying the co-flocculate of the sarcoplasmic protein and the chitosan, dissolving the freeze-dried sarcoplasmic protein and the chitosan in hexafluoroisopropanol, and magnetically stirring to obtain a spinning solution;
carrying out electrostatic spinning on the spinning solution, and receiving the fiber membrane by rolling aluminum foil paper;
and drying the fiber membrane to remove residual organic solvent to obtain the sarcoplasmic protein electrospun nanofiber membrane.
9. The use according to claim 8, wherein the step 2) electrospinning conditions are as follows: electrostatic spinning is carried out at the temperature of 13-27 ℃, the humidity of 30-65%, under the conditions that electrostatic voltage of 15-22KV, spinning speed of 0.4-1.8mL/h and distance between a spinning nozzle and a receiving plate is 10-18 cm.
10. The use of claim 8, wherein the drying conditions of step 3) are as follows: naturally drying at room temperature or drying in a vacuum drying oven at 35-60 deg.C.
Technical Field
The invention belongs to the technical field of food, and relates to a method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by combining acid treatment and chitosan flocculation and application thereof.
Background
The minced fillet product is very high in nutritional value and tender mouthfeel due to the fact that the minced fillet product is rich in high-quality protein and amino acid, is widely popular worldwide, and the demand of the minced fillet product is continuously increased all the year round. By 2019, the total amount of processed aquatic products in China is 2171.41 ten thousand tons, wherein the yield of minced fillet and minced fillet products is about 139.40 ten thousand tons. The silver carp has high yield, low price and fresh and tender meat quality, and is a main freshwater fish for producing surimi. However, in the production process of minced fillet, a large amount of water is needed to rinse the fish for multiple times to remove the residual impurities such as blood, lipid, water-soluble protein, sarcolemma and the like, so that on one hand, the water-soluble sarcoplasmic protein accounting for 20-40% of the fish protein is lost in the rinsing process, and the serious waste of resources is caused; on the other hand, a large amount of rinsing wastewater rich in protein is generated and needs to be treated, otherwise, eutrophication of the water body is caused. Therefore, if the sarcoplasmic proteins in the minced fillet rinsing liquid can be fully recovered, the utilization rate of the sarcoplasmic proteins and the added value of fish products can be improved, organic pollutants in the rinsing wastewater can be reduced, the treatment cost is reduced, and therefore good economic value and environmental benefit are generated.
The chitosan is a natural linear high molecular polymer, the main chain of the chitosan contains abundant amino groups, the chitosan can be protonated in an acid medium, the typical cationic polyelectrolyte characteristic is shown, and the chitosan is endowed with good flocculation performance. In addition, chitosan has good biocompatibility and degradability, so that the chitosan is widely used in wastewater treatment. However, chitosan has poor solubility in neutral or alkaline aqueous solutions, which prevents optimal use of chitosan, and therefore, the recovery rate of the single chitosan flocculation of sarcoplasmic proteins in the minced fillet rinsing solution is low, namely about 40%. Therefore, how to improve the flocculation effect of chitosan on sarcoplasmic proteins and improve the protein recovery rate becomes a problem to be solved urgently.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by combining acid treatment and chitosan flocculation and application thereof.
The scheme of the invention is as follows:
a method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by acid treatment and chitosan flocculation, which comprises the following steps:
s1: preparing a minced fillet rinsing liquid: adding distilled water into fish meat for rinsing, centrifuging, and taking supernatant as minced fillet rinsing liquid;
s2: adding chitosan solution into the minced fillet rinsing liquid, and adjusting the pH value to 6.0-9.0;
s3: standing, centrifuging, and precipitating to obtain the co-flocculate of sarcoplasmic protein and chitosan.
Preferably, in the step S1, the solid-to-liquid ratio of the fish meat to the distilled water is 1 (1.0-3.5) (g/mL), and the centrifugation conditions are as follows: 8000-11000g, 4 ℃, 12-18 min.
Preferably, in the step S2, the deacetylation degree of chitosan is 85-98%, and the addition amount of chitosan in the minced fillet rinsing liquid is 150-400 mg/L.
Preferably, in step S2, the pH is adjusted to 2.0-4.0, and then adjusted to 6.0-9.0 with NaOH solution.
Further preferably, in step S2, the pH is adjusted to 3.0, and then adjusted to 7.0 by NaOH solution.
Still more preferably, in the step S2, the concentration of the NaOH solution is 1.0-6.0 mol/L.
Preferably, in step S3, the temperature of standing in the water bath is 10 to 35 ℃, the time of standing is 30 to 300min, and the centrifugation conditions are as follows: 3500-4500r/min, 8-15 min.
The sarcoplasmic protein recovered by the method is used for manufacturing the sarcoplasmic protein electrostatic spinning nanofiber membrane, and the application comprises the following steps:
1) freeze-drying sarcoplasmic protein and chitosan, dissolving in hexafluoroisopropanol, magnetically stirring,
obtaining a spinning solution;
2) carrying out electrostatic spinning on the spinning solution, and receiving the fiber membrane by rolling aluminum foil paper;
3) and drying the fiber membrane to remove residual organic solvent to obtain the sarcoplasmic protein electrospun nanofiber membrane.
Preferably, the electrostatic spinning conditions of step 2) are as follows: electrostatic spinning is carried out at the temperature of 13-27 ℃, the humidity of 30-65%, under the conditions that electrostatic voltage of 15-22KV, spinning speed of 0.4-1.8mL/h and distance between a spinning nozzle and a receiving plate is 10-18 cm.
Preferably, the drying conditions in step 3) are as follows: naturally drying at room temperature or drying in a vacuum drying oven at 35-60 deg.C.
The invention has the beneficial effects that:
1. compared with the traditional chitosan flocculation method, the acid treatment can improve the recovery rate of the chitosan to the sarcoplasmic proteins by more than one time, the action mechanism of the invention is derived from the protonation of the chitosan in an acid solution with positive charge, the invention has good solubility and can be fully dissolved with the sarcoplasmic proteins; when the pH value is increased, protein molecules are gradually dissociated into an electric negative state, and are easily and rapidly subjected to electrostatic attraction with the positively charged chitosan to be flocculated and settled. The sarcoplasmic protein and chitosan co-flocculate recovered by the invention does not need to be separated, has good spinnability, can be directly applied to electrostatic spinning to prepare the sarcoplasmic protein nano fiber membrane, and the electrospun fiber membrane has strong surface hydrophobicity, mechanical property and thermal stability. The invention can save energy and reduce consumption, reduce the treatment cost and pressure of the minced fillet rinsing wastewater, and solve the problems of recovery and high-value utilization of byproducts in the industrialized production of the minced fillet.
2. The acid treatment can improve the effect of flocculating the protein by the chitosan and also can well improve the removal of the chemical oxygen demand in the minced fillet rinsing liquid.
3. The acid treatment can obviously improve the recovery efficiency of the sarcoplasmic protein, and greatly reduces the energy consumption compared with the direct freeze drying method. Compared with the sarcoplasmic protein recovered by a single chitosan flocculation method, the sarcoplasmic protein flocculated by acid treatment and chitosan has good spinnability.
Drawings
FIG. 1 recovery of sarcoplasmic proteins by single chitosan flocculation;
FIG. 2 recovery of sarcoplasmic proteins by acid treatment in combination with chitosan flocculation;
FIG. 3 influence of pH on protein recovery;
FIG. 4 effect of chitosan dosage on protein recovery;
FIG. 5 effect of temperature on protein recovery;
FIG. 6 effect of time on protein recovery;
FIG. 7 is a scanning electron micrograph of different concentrations of flocculated sarcoplasmic protein electrospun membranes;
FIG. 8 contact angles of flocculated and directly freeze-dried sarcoplasmic protein fibrous membranes;
FIG. 9 mechanical properties of flocculated and directly freeze-dried sarcoplasmic protein fibrous membranes;
FIG. 10 DSC curves of chitosan powder and sarcoplasmic protein fibrous membranes of different recovery modes.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.
Example 1
1 materials and reagents
The farmer market of Huazhong agriculture university of silver carp; chitosan, Beijing Kulaibo science and technology Limited, is dissolved in 1% by mass of acetic acid to prepare a 1% by mass chitosan solution (1 wt% chitosan solution) when in use.
2 method of experiment
2.1 preparation of surimi rinsing liquid[21]
Knocking dizzy of fresh silver carp, removing head, tail, skin and internal organs, manually picking meat, mincing with a conditioner, and weighing fish meat. Rinsing fish meat with distilled water at a ratio of 1:3 (mass: volume, g/mL), centrifuging at 4 deg.C for 15min under 10000g, collecting supernatant as minced fillet rinsing water, and storing at 4 deg.C for use.
2.2 recovery of sarcoplasmic proteins by Single Chitosan flocculation
And (3) adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and sequentially adding 1.2mL of 1 wt% chitosan solution with deacetylation degrees of 85%, 90% and 95% into the test tube. The pH was subsequently adjusted to 7.0 with 3mol/L NaOH solution. Standing in a water bath kettle at 20 deg.C for 90min, centrifuging at 4000r/min for 10min, and collecting supernatant to measure protein concentration.
2.3 recovery of sarcoplasmic proteins by acid treatment in combination with Chitosan flocculation
And (3) adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and sequentially adding 1.2mL of 1 wt% chitosan solution with deacetylation degrees of 85%, 90% and 95% into the test tube. The pH was adjusted to 3.0, followed by 3mol/L NaOH solution to 7.0. Standing in a water bath kettle at 20 deg.C for 90min, centrifuging at 4000r/min for 10min, and collecting supernatant to measure protein concentration.
2.4 Single factor test
2.4.1 Effect of pH on protein recovery
And (3) taking 10 centrifugal tubes, adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and adding 1.2mL of 1 wt% chitosan solution into the test tubes respectively. The pH was adjusted to 3.0, followed by adjusting the pH with 3mol/L NaOH solution to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 in that order. Standing in a water bath kettle at 20 deg.C for 90min, centrifuging at 4000r/min for 10min, and collecting supernatant to measure protein concentration.
2.4.2 Effect of Chitosan dosage on protein recovery
Taking 6 centrifugal tubes, adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and then sequentially adding 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL of 1 wt% chitosan solution (counting the dosage of chitosan in minced fillet rinsing water to be 150, 200, 250, 300, 350 and 400mg/L in sequence). The pH was adjusted to 3.0, followed by 3mol/L NaOH solution to 7.0. Standing in water bath at 20 deg.C for 90min, centrifuging at 4000r/min for 10min, collecting supernatant, and measuring protein concentration
2.4.3 Effect of temperature on protein recovery
And (3) taking 7 centrifugal tubes, adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and adding 1.2mL of 1 wt% chitosan solution into the test tubes respectively. The pH was adjusted to 3.0, followed by sequential adjustment of the pH to 7.0 with 3mol/L NaOH solution. Standing in water bath at 10, 20, 30, 40, 50, 60, and 70 deg.C for 90min, centrifuging at 4000r/min for 10min, and collecting supernatant to determine protein concentration.
2.4.4 Effect of time on protein recovery
And (3) taking 7 centrifugal tubes, adding 40mL of minced fillet rinsing liquid into each centrifugal tube, and adding 1.2mL of 1 wt% chitosan solution into the test tubes respectively. The pH was adjusted to 3.0, followed by 3mol/L NaOH solution to 7.0. Standing in a water bath kettle at 20 deg.C for 30, 60, 90, 120, 150, 180, 210min, centrifuging at 4000r/min for 10min, and collecting supernatant to measure protein concentration.
2.5 response surface optimization test
On the basis of a single-factor test, Box-Bechnken center combination optimization Design is carried out by using Design-expert software, the total recovery rate of final protein is used as a test index, and a response surface test of three-factor three-level is carried out on 3 factors possibly having synergistic effect in chitosan amount, pH value and temperature. Wherein the levels of-1, 0, 1 of the three factors are respectively set as: the pH value of the A factor is 6.5, 7.0 and 7.5; the temperature of the factor B is 10, 20 and 30 ℃; the dosage of the C-factor chitosan is 250, 300 and 350 mg/L.
2.6 recovery of protein
Protein concentration was determined according to the forskol method (standard curve regression equation: y 0.0023x +0.0751, R2 0.9931) and protein recovery was calculated as follows:
in the formula: c0The concentration of protein in the original minced fillet rinsing liquid is mg/mL; c1Protein concentration in supernatant after flocculation, mg/mL.
3 results and analysis
3.1 recovery of sarcoplasmic proteins by Single Chitosan flocculation
The results of single chitosan flocculation to recover sarcoplasmic proteins are shown in FIG. 1. The recovery rates of the sarcoplasmic proteins recovered by the three deacetylation degrees of chitosan are as follows in sequence: the degree of deacetylation is more than 95% and more than 90% and more than 85%. The increase of the deacetylation degree can lead to the increase of amino groups, so that the density of positive charges is increased after protonation, the charge neutralization effect is improved, and the chitosan has a better flocculation effect. Therefore, the subsequent experiments selected chitosan with a degree of deacetylation of 95%. The recovery rate of the sarcoplasmic proteins obtained by the single chitosan flocculation method with the deacetylation degree of 95 percent is lower and is only about 35 percent.
3.2 recovery of sarcoplasmic proteins by acid treatment in combination with Chitosan flocculation
The results of acid treatment combined with the chitosan flocculation method for recovering sarcoplasmic proteins are shown in FIG. 2. The acid treatment can obviously improve the flocculation effect of the chitosan as a single flocculant, and the recovery rate of the sarcoplasmic proteins reaches up to 81.54 percent. Compared with a single chitosan flocculation method, the protein recovery rate is improved by 46.02%. It can be seen that the acid treatment (pH adjusted to 3.0) plays a crucial role in the recovery of surimi rinse solution protein by chitosan flocculation, because the acid-treated chitosan is positively charged by protonation, has good water solubility, and is fully miscible with sarcoplasmic protein; then the pH value is increased, the protein molecules are gradually dissociated into an electric negative state, and the electric negative state and the positively charged chitosan are quickly subjected to electrostatic attraction to flocculate and are jointly settled. Meanwhile, the isoelectric point of the protein is precipitated.
3.3 Single factor Experimental results and analysis
3.3.1 Effect of pH on protein recovery
The effect of pH on protein recovery is shown in figure 3. The pH value has a more obvious influence on the chitosan flocculation process. When the pH value is 4.5-7.0, the recovery rate increases along with the increase of the pH value, reaches the maximum value at the pH value of 7.0, and then gradually decreases. It is possible that at lower pH values, the protein species, this ampholyte, is positively charged in acidic solution, while the chitosan is positively charged by protonation of the chitosan's amino groups in acidic solution, the electrostatic repulsion effect of both conferring relative dynamic stability to the system, resulting in lower recovery; when the pH is more than the optimum pH of 7.0, the protein is solubilized in an alkaline environment, and the recovery rate is lowered.
3.3.2 Effect of Chitosan dosage on protein recovery
The effect of chitosan dosage on protein recovery is shown in figure 4. With the increase of the addition amount of chitosan, the protein recovery rate tended to increase first and then decrease, reaching a maximum value at a chitosan amount of 300 mg/L. It is possible that the reason is that as the chitosan content increases, chitosan and protein are able to undergo effective charge neutralization, bridging and electrostatic interaction to aggregate sedimentation, and the flocculation effect is optimal when the solution as a whole is electrically neutral. However, the amount of chitosan is too much, and the flocculation recovery rate of protein tends to be reduced.
3.3.3 Effect of temperature on recovery of sarcoplasmic proteins by Chitosan flocculation
The effect of temperature on protein recovery is shown in figure 5. When the temperature is below 30 ℃, the recovery rate does not change significantly with temperature, and there is a relatively high recovery rate at 20 ℃. When the temperature exceeds 30 ℃, the recovery rate of the protein tends to increase, and the possible reason is that the denaturation temperature of the sarcoplasmic proteins is about 40 ℃, the protein is thermally denatured due to high temperature, and the spatial structure is destroyed to destabilize and precipitate. Overall, it is not reasonable to trade a large increase in energy consumption for a small increase in recovery, so the optimum temperature for the flocculation process should be chosen to be 20 ℃.
3.3.4 Effect of time on recovery of sarcoplasmic proteins by Chitosan flocculation
The effect of time on protein recovery is shown in figure 6. With increasing flocculation time, protein recovery first tended to increase, reaching a maximum at 90min, after which recovery decreased. The possible reason is that chitosan is not sufficiently chelated with protein and flocculates to form a precipitate at the initial stage of flocculation; and the flocculation time is too long, the chitosan-protein aggregate is unstable and the depolymerization phenomenon occurs, so that the recovery rate is reduced.
3.4 response surface optimization experiment results and analysis
According to the result of the single-factor experiment, the flocculation time is set to 90 min. The pH, the temperature and the chitosan content are respectively set as three investigation factors, the Box-Bechnken center combination optimization Design of Design-expert software is adopted, the total recovery rate of the protein is taken as an investigation index for carrying out the test, and the result is shown in Table 1. Performing multiple quadratic regression fitting on the data in the table 1 to obtain a regression equation:
Y＝84.08-1.13A-0.12B+0.24C-0.17AB+1.08AC-0.082BC-0.072B2-0.39C2。
TABLE 1 Box-Bechnken test design and results
As can be seen from Table 2, the A and AC terms have a very significant effect on the experimental results by variance test and significance analysis (P)<0.01). Model Pr>F has a very significant effect on the experimental results (P)<0.01), the mismatching term Pr>F value of 0.1064, no significant effect on the experiment (P)>0.05), which shows that the model has good fitting degree and can accurately simulate the variation in response. Coefficient of determination R of model20.9201, the equation is relatively good in test fitting degree and small in error; properly modified R to increase the reliability of model prediction2The adj value is 0.8401, indicating that the model can interpret 84.01% response value changes, demonstrating that the model can be used to fit the relationship between experimental independent variables and response values.
TABLE 2 analysis of variance results of regression equation
Note that is significant (P <0.05) and is very significant (P < 0.01).
As can be seen from the equation, the coefficient of the quadratic term is negative, indicating that the equation has a maximum value. The optimal prediction condition obtained by analyzing the response surface design expert software is as follows: the predicted protein recovery rate is 85.72% when the pH is 6.5, the temperature is 29.51 ℃ and the dosage of chitosan is 250 mg/L.
4 conclusion
According to the model optimization result and the actual conditions, the optimal recovery process of the sarcoplasmic proteins is determined as follows: the pH value is 6.5, the temperature is 30 ℃, the dosage of chitosan is 250mg/L, the flocculation time is 90min, and the recovery rate of the obtained protein under the condition is 85.23%.
Example 2
A method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by acid treatment and chitosan flocculation, which comprises the following steps:
s1: preparing a minced fillet rinsing liquid: taking fish meat, adding distilled water for rinsing according to the mass: volume (g/mL) ratio of 1:3, centrifuging, and taking supernatant as minced fillet rinsing liquid;
s2: adding a chitosan solution into the minced fillet rinsing liquid, and adjusting the pH value to 7.0;
s3: standing, centrifuging, and precipitating to obtain the co-flocculate of sarcoplasmic protein and a little chitosan.
Preferably, in the step S1, the solid-to-liquid ratio of the fish meat to the distilled water is 1:3(g/mL), the centrifugation conditions are: 10000g, 4 ℃, 15 min.
Preferably, in the step S2, the deacetylation degree of chitosan is 95%, and the addition amount of chitosan in the minced fillet rinsing liquid is 250 mg/L.
Preferably, in step S2, the pH is adjusted to 3.0, and then adjusted to 7.0 by NaOH solution.
Preferably, in the step S2, the concentration of the NaOH solution is 3 mol/L.
Preferably, in step S3, after placing the mixture in a water bath kettle at 20 ℃ and standing for 90min, the centrifugation conditions are as follows: 4000r/min, 10 min.
Chemical demand (COD) measurement result
The results of chemical oxygen demand measurement of the rinse solution are shown in Table 3. As can be seen from Table 3, the chemical demand (COD) of the minced fish rinse after flocculation treatment in example 2 was changed from 674.55 mg.L-1The temperature is reduced to 179.98 mg.L-1The COD value removal rate reaches 73.32 percent. Therefore, the acid treatment can obviously reduce the chemical oxygen demand in the minced fillet rinsing liquid while improving the protein flocculation effect of chitosan.
TABLE 3 COD measurement results of surimi rinses
The recovered coprocellate of sarcoplasmic proteins and chitosan is used for making sarcoplasmic proteins electrospun nanofibrous membrane, the application comprising the steps of:
1) freeze-drying the co-flocculate of the sarcoplasmic protein and the chitosan, dissolving the freeze-dried co-flocculate in hexafluoroisopropanol to prepare solutions with the mass fractions of 2%, 4%, 6%, 8% and 10%, and magnetically stirring the solutions for 24 hours at room temperature to obtain a spinning solution;
2) the spinning solution is subjected to electrostatic spinning at 25 ℃, the humidity is 45%, 20KV electrostatic voltage is used, the spinning speed is 0.8mL/h, and the distance between a spinning nozzle and a receiving plate is 14cm, and an aluminum foil paper is used for receiving a fiber membrane in a rolling mode;
3) and (3) drying the fibrous membrane for 48 hours in vacuum at 40 ℃ to obtain the sarcoplasmic protein electrostatic spinning nanofiber membrane.
Scanning Electron Microscope (SEM) observation of flocculated sarcoplasmic protein electrospun membranes
Example 2 the flocculated sarcoplasmic proteins were used to make sarcoplasmic proteins electrospun nanofiber membranes (flocculated sarcoplasmic proteins electrospun membranes, FL-FSP for short), it can be seen from fig. 7 that at lower concentrations, the solution viscosity was lower and the surface tension was high, the fibers were difficult to form, and the fiber surface was accompanied by a large number of beads, as shown by SEM at 2% and 4%. When the concentration is increased, the viscosity is increased and the surface tension is reduced, so that balanced opposition among the surface tension, the viscosity and the electrostatic repulsion is formed, and thus, flat and uniform fibers are obtained, and no beading is generated on the surface, as shown by a 6% SEM. The solution viscosity was too high, the rheology was poor and spinning was difficult as shown by the 10% SEM at 8%. Therefore, it was subsequently decided to use a protein dope concentration of 6%.
The results of SEM show that the sarcoplasmic proteins obtained by acid treatment combined with chitosan flocculation can be directly used for electrostatic spinning film formation without separating the chitosan remained in the proteins, thereby reducing the consumption of manpower and material resources.
Contact Angle test results
The protein electricity of the flocculated muscle pulp is prepared by adopting the concentration of 6 percent of protein spinning solutionSpun membrane (FL-FSP) compared to directly freeze-dried sarcoplasmic protein electrospun membrane (FD-FSP). The preparation method of the muscle plasma protein electro-spinning membrane by direct freeze drying comprises the following steps: rinsing fish meat with 1:3 (mass: volume, g/mL) distilled water, and then washing at 8000r min-1Centrifuging at 4 deg.C for 15min, freezing the supernatant in-80 deg.C refrigerator, and freeze drying to obtain directly freeze-dried sarcoplasmic protein. Then the protein is dissolved in hexafluoroisopropanol to prepare a spinning solution with the concentration of 8 percent, and the direct freeze-dried sarcoplasmic protein electrospun membrane (FD-FSP) is prepared by electrostatic spinning under the conditions of 25 ℃, the humidity of 45 percent, the electrostatic voltage of 20KV, the spinning speed of 0.8mL/h and the distance between a spinning nozzle and a receiving plate of 14 cm.
The contact angle measurement results of the sarcoplasmic protein electrospun membranes in different recovery modes are shown in fig. 8, the contact angle of the directly freeze-dried sarcoplasmic protein electrospun membrane is 97.0 +/-0.9 degrees, and the contact angle of the flocculated sarcoplasmic protein electrospun membrane is 124.1 +/-1.5 degrees, which is probably because after the sarcoplasmic protein is treated by acid, the obtained flocculated sarcoplasmic protein electrospun membrane has rough surface and larger surface energy, so that the contact angle is increased, and the surface hydrophobicity of the fibrous membrane is improved.
Analysis of mechanical Properties
The mechanical properties of the sarcoplasmic protein electrospun fibrous membrane are shown in fig. 9, the elastic modulus and tensile strength of the direct freeze-dried sarcoplasmic protein fibrous membrane are 77.5MPa and 1.0MPa, respectively, while the elastic modulus and tensile strength of the flocculated sarcoplasmic protein fibrous membrane are 29.0MPa and 0.4MPa, respectively, which may be caused by the damage of acid treatment to the protein structure. The elongation at break of the direct freeze-dried and flocculated sarcoplasmic protein fibers was 4.6% and 4.8%, respectively, and the results showed that the acid treatment did not negatively affect the elongation at break of the protein fiber membrane.
Differential Scanning Calorimetry (DSC)
When the sarcoplasmic proteins reach a certain temperature, irreversible deformation (contraction) occurs and the molecules are transformed from an extended helical to a disordered coiled state. The higher the denaturation temperature of the sarcoplasmic proteins, the more stable their internal structure and the better the thermal stability. The thermodynamic behavior of chitosan powder, FD-FSP, FL-FSP fibrous membranes was studied by Differential Scanning Calorimetry (DSC). The DSC of fig. 10 shows that chitosan powder has an endothermic peak at 148.4 ℃, corresponding to the melting point (Tm) of chitosan. The FD-FSP fiber and the FL-FSP fiber show similar endothermic peaks, the melting point of the FD-FSP fiber is 167.0 ℃, the melting point of the FL-FSP fiber membrane is 173.0 ℃, and the endothermic peak value is increased, which shows that the heat stability of the sarcoplasmic protein electrospun membrane is high, and the flocculated sarcoplasmic protein membrane is superior to the direct freeze-dried sarcoplasmic protein membrane.
Compared with freeze drying, the flocculation process saves energy and reduces consumption, and the flocculation recovery of the sarcoplasmic proteins reduces the treatment cost and pressure of the minced fillet rinsing wastewater, and solves the problems of recovery and high-value utilization of byproducts in the industrialized production of the minced fillet.
Example 3
A method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by acid treatment and chitosan flocculation, which comprises the following steps:
s1: preparing a minced fillet rinsing liquid: adding distilled water into fish meat for rinsing, centrifuging, and collecting supernatant as minced fillet rinsing liquid;
s2: adding a chitosan solution into the minced fillet rinsing liquid, and adjusting the pH to 6.5;
s3: standing, centrifuging, and precipitating to obtain sarcoplasmic protein and chitosan cochlegmation.
Preferably, in the step S1, the solid-to-liquid ratio of the fish meat to the distilled water is 1:2(g/mL), the centrifugation conditions are as follows: 9000g, 4 ℃ for 12 min.
Preferably, in step S2, the deacetylation degree of chitosan is 90%, and the addition amount of chitosan in the minced fish rinse solution is 300 mg/L.
Preferably, in step S2, the pH is adjusted to 3.0, and then adjusted to 6.5 with NaOH solution.
Preferably, in the step S2, the concentration of the NaOH solution is 2.5 mol/L.
Preferably, in step S3, the temperature of standing in the 20 ℃ water bath is 15 ℃, the time of standing is 85min, and the conditions of centrifugation are as follows: 3500r/min, 8 min.
The sarcoplasmic protein prepared by the method is used for manufacturing the sarcoplasmic protein electrostatic spinning nanofiber membrane, and the application comprises the following steps:
1) freeze-drying the co-flocculate of the sarcoplasmic protein and the chitosan, dissolving the freeze-dried sarcoplasmic protein and the chitosan in hexafluoroisopropanol, and magnetically stirring to obtain a spinning solution;
2) carrying out electrostatic spinning on the spinning solution, and receiving the fiber membrane by rolling aluminum foil paper;
3) the fiber membrane is dried to remove residual organic solvent.
Preferably, the electrostatic spinning conditions of step 2) are as follows: electrostatic spinning is carried out at 23 ℃ and humidity of 40 percent under the conditions of electrostatic voltage of 18KV, spinning speed of 0.6mL/h and distance between a spinning nozzle and a receiving plate of 12 cm.
Preferably, the drying conditions in step 3) are as follows: naturally drying at room temperature.
Example 4
A method for recovering sarcoplasmic proteins in minced fillet rinsing liquid by acid treatment and chitosan flocculation, which comprises the following steps:
s1: preparing a minced fillet rinsing liquid: adding fish meat into distilled water for rinsing, and centrifuging to obtain supernatant as minced fillet rinsing liquid;
s2: adding a chitosan solution into the minced fillet rinsing liquid, and adjusting the pH to 8.0;
s3: standing, centrifuging, and precipitating to obtain the co-flocculate of sarcoplasmic protein and a little chitosan.
Preferably, in the step S1, the solid-to-liquid ratio of the fish meat to the distilled water is 1:1(g/mL), the centrifugation conditions are: 11000g, 4 ℃, 18 min.
Preferably, in the step S2, the deacetylation degree of chitosan is 90-98%, and the addition amount of chitosan in the minced fillet rinsing liquid is 150 mg/L.
Preferably, in step S2, the pH is adjusted to 3.0, and then adjusted to 8.0 with NaOH solution.
Preferably, in the step S2, the concentration of the NaOH solution is 2.5-3.5 mol/L.
Preferably, in step S3, the temperature of standing in the 20 ℃ water bath is 25 ℃, the time of standing is 95min, and the conditions of centrifugation are as follows: 4500r/min, 12 min.
The sarcoplasmic protein prepared by the method is used for manufacturing the sarcoplasmic protein electrostatic spinning nanofiber membrane, and the application comprises the following steps:
1) the precipitation is that sarcoplasmic protein and chitosan are dissolved in hexafluoroisopropanol after the co-flocculate is freeze-dried, and the spinning solution is obtained after magnetic stirring;
2) carrying out electrostatic spinning on the spinning solution, and receiving the fiber membrane by rolling aluminum foil paper;
3) the fiber membrane was vacuum dried to remove residual organic solvent.
Preferably, the electrostatic spinning conditions of step 2) are as follows: electrostatic spinning is carried out at 27 ℃ and humidity of 0 percent under the conditions of electrostatic voltage of 22KV, spinning speed of 1.2mL/h and distance between a spinning nozzle and a receiving plate of 16 cm.
Preferably, the drying conditions in step 3) are as follows: dried in a vacuum oven at 45 ℃ for 50 hours.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.