CN112279885A - Method for preparing functional hydrolysate by degrading egg shell membrane by steam explosion technology - Google Patents
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Abstract
The invention discloses a method for preparing functional hydrolysate by degrading eggshell membrane by using steam explosion technology, which comprises the steps of firstly placing the eggshell membrane in a steam explosion machine reactor, carrying out pressure maintaining treatment for 0.5-5 min under the condition that the pressure is 0.5-2.2 MPa, then carrying out instantaneous pressure relief, collecting a steam explosion sample, adding the steam explosion sample into NaOH solution, carrying out oscillation reaction for 0.5-4 h under the condition that the temperature is 25-60 ℃, filtering to obtain filtrate, and carrying out freeze drying to obtain the functional hydrolysate. Compared with the existing reduction method and enzymolysis method, the steam explosion assisted dilute alkali solution degradation method has the advantages of simple operation process, short treatment time, high efficiency and easy industrial production.
Description
Technical Field
The invention relates to the field of egg byproduct processing, in particular to a method for preparing a functional hydrolysate by degrading an egg shell membrane by using a steam explosion technology.
Background
Egg shells exist in large quantities as a by-product of human life, and the egg shell membranes attached in the egg shells are particularly easy to cause great environmental pollution. In fact, eggshell membrane and the main by-products of the animal products processing industry: the horn, hoof, skin and hair are potential protein and amino acid resources. The eggshell membrane contains about 90% protein, 2% carbohydrate and 3% liposome. The proteins in the shell membrane are mainly keratin, collagen (most of I, V, X type), complex protein and the like, and the keratin and the collagen are contained in a large amount. If the discarded eggshell membranes are fully utilized, the economic benefit can be increased, and the pollution to the environment can be avoided.
The shell membrane treatment methods reported in the prior literature mainly comprise an acid-base treatment method, a reduction method, an enzyme method and the like,
1. the acid-alkali treatment method firstly uses acid pretreatment to swell the natural shell membrane protein, and then the natural shell membrane protein is dissolved at a certain temperature and alkali concentration, so that the prepared protein has good solubility and high extraction rate. However, this method is relatively environmentally unfriendly, and the generated acid vapor and waste liquid have a great adverse effect on human bodies and the environment.
2. The reduction method utilizes a reducing agent to reduce disulfide bonds in eggshell membrane protein molecules into sulfydryl, so that soluble protein is obtained, but the sulfydryl compound reducing agent has large pungent smell and high cost.
3. The enzyme method has the advantages of high reaction speed, good water solubility, no environmental pollution, mild reaction conditions and stable physicochemical properties, and is an advanced method at present. However, the method is long in time consumption, and the enzyme preparation is expensive and difficult to be applied industrially.
The steam explosion technology is a typical physical processing method, and the principle of the method is that high-pressure steam is pressed into a gap inside a material in a high-pressure container, and pressure is released instantly after pressure maintaining, so that the steam in the container does work outwards through expansion, shock waves are generated to mechanically shear the material, a compact structure among material components is damaged, and the material is dissociated. It is known that there are patents which apply steam explosion technology to the treatment of raw materials such as lignocellulose, tartary buckwheat bran, etc.
At present, no report on the application of the steam explosion technology to the degradation of the shell membrane to prepare the functional hydrolysate is found.
Disclosure of Invention
The invention aims to overcome the difficulties and provide a method for preparing functional hydrolysate by degrading egg shell membrane by using steam explosion technology, which is to break disulfide bonds in the shell membrane protein under the conditions of high temperature, high pressure and high shearing force, effectively destroy disulfide bonds, hydrogen bonds and other chemical bonds in the shell membrane protein, degrade the shell membrane protein into polypeptide with lower molecular weight, improve the utilization rate of the polypeptide and prepare the functional hydrolysate. The method has the advantages of short treatment time, high efficiency and simple process, is suitable for industrial production, and is a shell membrane treatment technology with wide prospect.
The above purpose is realized by the following technical scheme:
a method for preparing functional hydrolysate by degrading egg shell membrane by steam explosion technology comprises the following steps:
1) steam explosion treatment
Placing the eggshell membrane in a steam explosion machine reactor, performing pressure maintaining treatment for 0.5-5 min under the condition that the pressure is 0.5-2.2 MPa, then instantly releasing the pressure, collecting a steam explosion sample, and storing for later use;
2) degradation of sample by dilute alkali method
And adding the steam explosion sample into a NaOH solution, carrying out oscillation reaction for 0.5-4 h at the temperature of 25-60 ℃, filtering to obtain a filtrate, and carrying out freeze drying to obtain the functional hydrolysate.
Further, in the step 1), the pressure is 1.6MPa, and the pressure-maintaining treatment time is 1 min.
Still further, in the step 1), the instant pressure relief time is 0.1 s.
Further, in the step 2), the alkali concentration of the NaOH solution is 0.2-0.6%; the material-liquid ratio of the steam explosion sample to the NaOH solution is 1: 10-25.
Further, the alkali concentration of the NaOH solution is 0.4%; and the feed-liquid ratio of the steam explosion sample to the NaOH solution is 1: 20.
Still further, in the step 2), the oscillation reaction conditions are as follows: the temperature is 25 ℃ and the reaction time is 1 h.
The theoretical basis of the research of the invention is as follows:
1. selection of steam explosion conditions
1.1 methods
a. Weighing 1000g of shell membrane, placing the shell membrane in a steam explosion machine reactor, respectively treating for 0.5-5 min under 0.5-2.2 MPa, and then instantly releasing pressure within 0.1 s; collecting steam explosion sample, and storing at-4 deg.C.
b. Weighing 1g of each shell membrane treated under each condition, adding 20mL of 0.4% NaOH solution into a sample, carrying out shaking reaction for 1h at the temperature of 25 ℃, centrifuging for 10min at 3000g, and pouring out the supernatant. The same solvent (10 mL) was added again to the precipitate and the procedure was repeated. Filtering the three groups of treated samples to obtain insoluble residues, washing the residues for three times, putting the residues into a 50 ℃ oven for drying, and weighing; the shell-membrane dissolution rate was calculated as follows:
1.2 conclusion
As shown in fig. 1: the dissolution rate of the eggshell membrane increases along with the increase of the steam explosion pressure, but the difference between 1.6MPa and 2.0MPa is not obvious; along with the increase of the pressure maintaining time, the dissolution rate of the shell membrane after steam explosion treatment is obviously increased, and when the treatment time is further prolonged to 2min, the dissolution rate is also increased, but the change is not obvious. Therefore, the optimal steam explosion pressure of the steam explosion treatment is 1.6MPa, and the pressure maintaining time is 1 min.
2. Selection of different solvents
2.1 method
Respectively taking 1g of a steam explosion sample and a shell membrane sample which is not subjected to steam explosion treatment, respectively setting the samples as an experimental group and a control group, adding 20mL of different solvents (0.05mol/L phosphate buffer solution (pH 8.0), 0.4% of NaOH solution with alkali concentration, 0.1mol/L HCl solution and 70% of ethanol), and placing the samples into a shaking water bath kettle for shaking reaction for 1 hour; centrifuging at 3000g for 10min, removing supernatant, adding 10mL of corresponding solvent into the precipitate, and repeating the above steps.
Filtering the treated experimental group and the control group to obtain insoluble residues, washing the residues for three times, putting the residues into a 50 ℃ oven for drying, weighing, and calculating the dissolution rate;
2.2 results
As shown in fig. 2: when 0.4% NaOH solution is used, the dissolution rate of the steam explosion sample is the highest, so the NaOH solution is used as the solvent.
3. Steam explosion assisted dilute alkali process optimization
3.1 method
Measuring the shell membrane dissolution rate in different alkali concentrations and material-liquid ratios, respectively taking 1g of steam explosion samples and shell membrane samples which are not subjected to steam explosion treatment as an experimental group and a control group, mixing the samples with NaOH solutions with different concentrations according to different material-water ratios, wherein the alkali concentration gradients are respectively 0, 0.2%, 0.4% and 0.6%, the material-liquid ratios are respectively 1:10, 1:15, 1:20 and 1:25, putting the samples into an oscillating water bath kettle at different temperatures (25 ℃, 40 ℃, 50 ℃ and 60 ℃) for carrying out oscillation reaction for 0.5h, 1h, 2h and 4h respectively, filtering to obtain insoluble residues, washing the residues for three times, putting the residues into a 50 ℃ oven for drying, weighing, and calculating the dissolution rate;
3.2 conclusion
a. When the alkali concentration of the NaOH solution is lower than 0.4%, the dissolution rate of the shell membrane is obviously increased along with the increase of the alkali concentration, and the change difference is not obvious when the concentration reaches 0.6%. This is probably because higher alkali concentrations are able to cleave the hydrogen bond between carbonyl and sulfate groups, thereby opening up the protein structure and exposing hydrophobic residues. The shell membrane is easy to dissolve under an extreme alkaline condition (such as 1mol/L NaOH), but the shell membrane can cause a great amount of protein to be degraded into low molecular polypeptide, so that the subsequent development and utilization of the shell membrane protein material are limited, and the alkali concentration of the NaOH solution is preferably 0.4 percent;
b. the shell membrane dissolution rate reaches the maximum when the feed-water ratio is 1:20, and the difference is not obvious when the feed-water ratio is larger, so the preferred value of the feed-water ratio of the steam explosion sample to the NaOH solution is 1:20
c. The dissolution rate of the shell film without steam explosion treatment is obviously increased along with the increase of degradation temperature, but the change amplitude of the steam explosion sample along with the increase of temperature is not obvious. With the increase of the reaction time, the dissolution rate of the shell membrane can be obviously improved in the first 1h, and the dissolution rate is not changed obviously by the longer reaction time; therefore, the reaction temperature was 25 ℃ and the reaction time was 1 hour (FIG. 3).
4. Effect of steam explosion treatment on disulfide bond content in Shell Membrane
4.1 method
(1) Synthesis of NTSB reagent:
a.0.1g of 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) dissolved in 10mL of 1mol/L Na2SO3Adjusting the pH of the solution to 7.5;
b. the reaction system was continuously oxygenated and reacted at a constant temperature of 38 ℃ under magnetic stirring until the solution changed from bright red to pale yellow (about 45min), and the reaction was terminated to obtain NTSB (the solution can be stored at-20 ℃ for one year).
(2) And (3) measuring the content of free sulfydryl:
weighing 15mg of a shell membrane sample, suspending the shell membrane sample in 0.8mL of buffer solution A (8mol/L urea, 3m mol/L EDTA, 1% SDS and 0.2mol/L Tris-HCl, pH 8.0), and carrying out vortex oscillation at room temperature at variable time; after 2.5h, 0.2mL buffer B (50mM DTNB reagent, 0.2mol/L Tris-HCl, pH 8.0) was added, vortexed again for 1h, centrifuged at 10000g at room temperature for 15min, the supernatant was taken and the absorbance A was measured at 412nm1(blank was a mixed solution of 0.8mL of buffer A and 0.2mL of buffer B), and the free thiol content was calculated according to the following formula.
(3) And (3) measuring the total thiol content:
15mg of a shell membrane sample (particle size 0.25mm) was weighed and suspended in 0.8m L buffer C (8mol/L urea, 3m mol/L EDTA, 1% SDS, 0.1mol/L Na)2SO3And 0.2mol/L Tris-HCl, pH 9.5), and performing vortex oscillation at room temperature at variable time; after 2.5h, 0.2mL of buffer D (50m mol/L NTSB assay) was addedAgent, 0.2mol/L Tris-HCl, pH 9.5); vortexing again for 1h, centrifuging at room temperature 10000g for 15min, collecting supernatant, and measuring absorbance A at 412nm2(blank control was a mixed solution of 0.8mL of buffer C and 0.2mL of buffer D), and the total thiol content was calculated according to the following formula;
(4) calculation of disulfide bond content
The disulfide bond content was calculated according to the following formula:
in the formula, n is the dilution multiple, and the molar extinction coefficient epsilon is 13600M-1cm-1。
4.2 conclusion
The steam explosion treatment can significantly reduce the content of disulfide bonds, which are major factors in the resistance of the shell membrane to degradation. With the increase of the steam explosion strength, the disulfide bond content is greatly reduced. Since 1 mole of disulfide bonds reacted to quantitatively generate 2 moles of cysteine residues, an increase in free thiol groups should be correlated with a decrease in disulfide crosslinking. Thus, compared with the control group, the experimental group has a lower disulfide bond content and a higher free thiol content. While other chemical bonds such as hydrogen bonds, salt bonds, peptide bonds and the like are destroyed by the alkali liquor, the disulfide bonds in the eggshell membrane can be destroyed to a certain extent, so that the content of the disulfide bonds is gradually reduced and the content of free sulfydryl is gradually increased along with the gradual increase of the alkali concentration from 0 to 0.6 percent, but the increase is not obvious (figure 4).
5. Effect of steam explosion treatment on solubility, emulsifiability, foaming characteristics and oxidation resistance of the shell membrane
5.1 methods
(1) And (3) determining the solubility:
a.
crushing and homogenizing 1g of eggshell membrane, dispersing in 100mL of deionized water, treating for 15min by using an ultrasonic instrument, and centrifuging for 15min by using a high-speed centrifuge at 4000 g;
b. respectively dissolving 1g of the shell membrane solution subjected to steam explosion treatment and the shell membrane solution not subjected to steam explosion treatment in 100mL of deionized water, stirring by adopting a homogenizer at 10000g for 1min, and centrifuging by adopting a high-speed centrifuge at 4000g for 15 min. Taking the supernatant as a liquid to be detected, determining the protein content in the supernatant by adopting a Coomassie brilliant blue method (referring to SN/T3926-:
(2) and (3) measuring the emulsification characteristic:
a. dissolving 1g of the shell membrane solution subjected to steam explosion treatment and the shell membrane solution not subjected to steam explosion treatment in 100mL of deionized water, and setting the solutions as an experimental group and a control group respectively;
b. respectively taking 45mL of shell membrane solution, adding 15mL of soybean oil, and dispersing for 1min by adopting 10000g of a high-speed disperser at room temperature to obtain emulsion.
c. Measurement of emulsification Activity
Absorbing 50 μ L of the above emulsion, adding into 5mL of 0.1% SDS solution, using 0.1% SDS solution as blank control, measuring absorbance at 500nm with ultraviolet spectrophotometer, and recording as A0Calculating the Emulsifying Activity (EA) of the shell membrane according to the following formula;
d. determination of emulsion stability
Standing the dispersed emulsion for 30min, transferring the middle part 5mL into a test tube, mixing, sucking 50 μ L, adding into 5mL 0.1% SDS solution, measuring absorbance at 500nm with spectrophotometer, and recording as A30The Emulsion Stability (ES) was calculated according to the following formula:
EA＝A0
(3) measurement of foaming Properties:
1g of the solution of the shell membrane subjected to steam explosion treatment and the solution of the shell membrane not subjected to steam explosion treatment are dissolved in 20mL of deionized water and respectively set as an experimental group and a control group. 20mL of the shell solution was poured into a 50mL centrifuge tube and the height of the solution was measured with a ruler and recorded as H010000g of stirring for 1min, and measuring the foam height H1Standing the centrifuge tube at room temperature for 10min, and measuring the foam height H again2. The Foaming Capacity (FC) and Foam Stability (FS) of the shell membrane samples were calculated using the following formulas:
(4) determination of DPPH radical scavenging Capacity:
and (3) putting 2mL of shell membrane solution into a colorimetric tube, adding 2mL of 1mmol/L DPPH ethanol solution, fully and uniformly mixing, reacting for 30min in a dark place at room temperature, and measuring the absorbance at 517nm after the reaction is finished. The control group is the absorbance of the mixed solution of 2mL of absolute ethyl alcohol solution and 2mL of sample solution, the blank group is the absorbance of the mixed solution of 2mL of DPPH solution and 2mL of distilled water, and absolute ethyl alcohol is used for zero setting. The formula for calculation of DPPH radical scavenging ability is as follows:
in the formula:
As- -absorbance of the experimental group
Ac- -absorbance of control group
AbAbsorbance of blank set.
5.2 conclusion
As can be seen from FIG. 5, the shell membrane dissolution rates of the experimental group and the control group both increase with the increase of the alkali concentration when the alkali concentration is 0-0.4%, the dissolution rate decreases when the alkali concentration reaches 0.6%, and an excessively high alkali concentration easily causes a large amount of degradation of protein peptide chains, so that the protein is degraded into low-molecular polypeptides, which is not beneficial to keeping the protein structure intact, and further influences the combination of the protein and Coomassie brilliant blue. However, in general, the shell membrane dissolution rate after the high-temperature and high-pressure treatment is significantly higher than that of the untreated group under each alkali concentration. The experimental result proves that the high-temperature high-pressure treatment is favorable for improving the dissolution rate of the shell membrane.
As can be seen from fig. 6A, the emulsifiability of the steam-exploded shell membrane significantly increased with the increase in alkali concentration before the alkali concentration was 0.4%, and slightly decreased with the alkali concentration of 0.6%, which was not significantly different from the former. The emulsibility of the shell membrane without steam explosion treatment is increased along with the increase of alkali concentration, but the increase is smaller than that of a steam explosion group. As can be seen from fig. 6B, the emulsion stability of the steam-exploded shell membrane was significantly increased with the increase in alkali concentration before the alkali concentration was 0.4%, and was slightly decreased at the alkali concentration of 0.6%. The emulsion stability of the shell membrane without steam explosion treatment is obviously lower than that of the shell membrane without steam explosion treatment, but still shows an ascending trend along with the increase of alkali concentration.
As can be seen from fig. 7A, the foaming properties of the envelope proteins in the experimental group increased significantly with increasing alkali concentration, and the foaming properties of the envelope proteins in the control group increased less, with insignificant variation. As can be seen from FIG. 7B, the foaming stability of the experimental group of shell membrane proteins is significantly increased with the increase of alkali concentration before the alkali concentration is 0.4%, while the foam stability is slightly reduced at the alkali concentration of 0.6%, which may be related to the occurrence of a large amount of hydrolysis of peptide bonds in the shell membrane fibers under the condition of higher alkali concentration, the decrease of the molecular weight of the proteins and the decrease of the strength of the interstitial fluid membrane. The control group shell membrane protein foaming stability was significantly lower than the experimental group, but still showed an increasing trend with increasing alkali concentration.
As can be seen from FIG. 8, the DPPH radical scavenging ability of the test and control shells increased with the increase of the concentration of the NaOH solution, indicating that the antioxidant activity of the shell sample increased with the increase of the concentration of the NaOH solution. The DPPH radical scavenging ability increases more slowly when the alkali concentration is 0-0.4%, and increases more rapidly when the alkali concentration increases to 0.6%. Compared with the control group of shell membranes which are not subjected to steam explosion treatment, the peptide bonds in the proteins of the experimental group of shell membranes under the high-temperature and high-pressure condition of steam explosion treatment are more strongly destroyed, so that part of the shell membrane proteins are degraded into discrete oligopeptides, short peptides with higher antioxidant activity are generated, and the antioxidant activity of the experimental group is higher than that of the control group.
6 Effect of different Condition treatments on the dissolution Rate of Eggshell membranes
6.1 method (1) four different treatments were performed on the shell membrane, respectively, the treatment methods were as follows:
a. steam explosion treatment group: weighing 1000g of shell membrane, placing the shell membrane in a steam explosion machine reactor, treating for 1min under 1.6MPa, then instantly releasing pressure within 0.1s, and collecting a steam explosion sample;
b. steam explosion alkali liquor treatment group: the steam explosion treatment method is the same as above. 1g of the steam-exploded shell membrane sample was added with 20mL of 0.4% NaOH solution and reacted for 1 hour at room temperature (25 ℃ C.) in a shaking water bath.
c. Alkali liquor treatment group: 1g of the shell membrane was weighed, 20mL of 0.4% NaOH solution was added, and the reaction was carried out in a shaking water bath at room temperature (25 ℃ C.) for 1 hour.
d. Blank group: 1g of the shell membrane was weighed, 20mL of distilled water was added, and the reaction was carried out for 1 hour at room temperature (25 ℃ C.) in a shaking water bath.
(2) The four groups of treated samples were taken out from the water bath, centrifuged at 3000g for 10min, and the supernatant was decanted. The same solvent (10 mL) was added again to the precipitates, and the above operation was repeated. Filtering the four groups of treated samples to obtain insoluble residues, washing the residues for three times, putting the residues into a 50 ℃ oven for drying, and weighing; the shell-membrane dissolution rate was calculated as follows:
6.2 conclusion
As can be seen from FIG. 10, the dissolution rates of the samples of the blank group, the steam explosion treatment group, the alkali solution treatment group and the steam explosion alkali solution treatment group were 5.07%, 32.4%, 9.2% and 91.6%, respectively. The dissolution rate of the blank group is obviously lower than that of the other three groups, and compared with steam explosion treatment and alkali liquor treatment, the dissolution rate of the shell membrane sample treated by steam explosion auxiliary alkali liquor is obviously increased. Therefore, the experimental result proves that the steam explosion assisted dilute alkali solution treatment is really helpful to improve the dissolution rate of the shell membrane.
7. Influence of different conditions on degree of hydrolysis of eggshell membrane
7.1 methods
(1) Steam explosion alkaline process
1000g of shell membrane is weighed and placed in a steam explosion reactor, treated for 1min under 1.6MPa, and then instantaneously decompressed within 0.1 s. And collecting a steam explosion sample. Adding 20mL of 0.4% NaOH solution into 1g of the steam-exploded shell membrane sample, carrying out oscillation reaction at 25 ℃ for 1h, taking out, centrifuging (3000g for 10min), and taking part of supernatant to determine the degree of hydrolysis.
(2) Method for enzymolysis of reducing agent
Weighing 5g eggshell membrane, adding 30mM sodium sulfite solution according to the feed-liquid ratio of 1:20 to prepare eggshell membrane suspension, adjusting the pH to the optimum pH (pH is 8.0) of alkaline protease by using 1M NaOH, adding the alkaline protease according to the enzyme-substrate eggshell membrane ratio of 9U/mg, continuously shaking for 6h at the optimum temperature (55 ℃) of the alkaline protease, taking out, centrifuging (3600g, 10min), and taking part of supernatant to determine the hydrolysis degree.
(3) And (3) complex enzymolysis:
weighing 5g of eggshell membrane, adding distilled water according to a material-liquid ratio of 1:20 to prepare an eggshell membrane suspension, adjusting the pH to the optimum pH of the enzyme by using 1M NaOH or 1M HCl, respectively and sequentially adding alkaline protease and papain according to an enzyme-substrate eggshell membrane ratio of 15U/mg of total enzyme addition, wherein the enzyme addition ratio is 1:1, carrying out enzymolysis for 2 hours for each enzyme, continuously shaking for 6 hours at the optimal temperature of each enzyme (the pH value of papain is 6.5, the enzymolysis temperature is 60 ℃, the pH value of alkaline protease is 8.0, and the enzymolysis temperature is 55 ℃), taking out, centrifuging (3600g, 10min), and taking part of supernatant to determine the hydrolysis degree.
(4) And (3) determining the degree of hydrolysis:
the content of free amino nitrogen is measured by adopting a national standard GB/T5009.39-2003 amino nitrogen formaldehyde value method, and the experiment is carried out for three times in parallel. 5mL of sample (supernatant from the three treatments described above) was aspirated, 60mL of distilled water was added, the magnetic stirrer was turned on, and the volume of the solution was 0.0Titrating 50mol/L NaOH standard solution until pH indicated by an acidimeter is 8.2, adding 10.0mL of neutral formaldehyde solution, and mixing uniformly. Then, the solution is titrated continuously to pH 9.2 with 0.050mol/L NaOH standard solution, and the volume of the consumed NaOH standard solution is recorded as V1. Simultaneously taking 65mL of distilled water, firstly using 0.050mol/L NaOH standard solution to adjust the pH value to 8.2, then adding 10.0mL of neutral formaldehyde solution, continuously using NaOH to titrate the solution to pH value 9.2, using the solution as a reagent blank test, recording the volume of NaOH consumed, and recording the volume as V2. The content of amino acid nitrogen in the sample is calculated by the following formula:
x represents the content of amino acid nitrogen in the sample, and the unit is g/mL; v1The volume of NaOH standard titration solution consumed after formaldehyde is added into a test sample is expressed, and the unit is mL; v2The volume of the NaOH standard titration solution consumed after formaldehyde is added in a reagent blank experiment is expressed in mL; c represents the concentration of NaOH standard titration solution, and the unit is mol/L; 0.014 indicates the mass of nitrogen in g equivalent to 1mL of a 1.000mol/L NaOH standard titration solution.
The total nitrogen content is determined by adopting a national standard GB 5009.5-2016 Kjeldahl method to determine the nitrogen content in the initial egg shell membrane.
The degree of shell membrane hydrolysis is calculated according to the following formula:
7.2 conclusion
As can be seen from FIG. 11, the degrees of hydrolysis by the steam explosion alkaline method, the reducing agent enzymolysis method and the complex enzymolysis method are 11.29%, 27.02% and 63%, respectively. Compared with a reducing agent enzymolysis method and a composite enzymolysis method, the degree of hydrolysis of the shell membrane sample treated by the steam explosion auxiliary alkali liquor is obviously increased. Therefore, the experimental result proves that the steam explosion assisted dilute alkali solution treatment is beneficial to improving the utilization rate of the shell membrane and preparing the functional hydrolysate.
The invention has the beneficial effects that:
(1) compared with the existing reduction method and enzymolysis method, the steam explosion assisted dilute alkali solution degradation method has the advantages of simple operation process, short treatment time, high efficiency and easy industrial production.
(2) The steam explosion technology is combined with dilute alkali solution treatment, so that the functional properties of shell membrane protein such as solubility, emulsifying property, foaming property and the like can be improved to different degrees, wherein the emulsifying property and the foaming property of the shell membrane protein are equivalent to those of the traditional alkali extraction and acid precipitation method, and the emulsifying stability and the foam stability are superior to those of the traditional alkali extraction and acid precipitation method.
Drawings
FIG. 1 is a graph of the dissolution rates of shell membrane samples in different solvents;
FIG. 2 is a graph showing the effect of steam explosion pressure (A) and dwell time (B) on the shell membrane dissolution rate;
FIG. 3 is the optimization of the material-liquid ratio (A), alkali concentration (B), temperature (C) and time (D) under the steam explosion shell membrane degradation process conditions;
FIG. 4 shows the change in disulfide bond and free thiol content;
FIG. 5 is a graph of the change in solubility of shell membrane samples at different base concentrations;
FIG. 6 shows the changes in emulsifiability (A) and emulsion stability (B) for the shell membrane samples;
FIG. 7 is a graph of the change in foaming (A) and bubble stability (B) for the shell membrane samples;
FIG. 8 is a graph of the change in oxidation resistance of the shell membrane sample;
FIG. 9 shows the changes before treatment (A) and after steam explosion degradation (B) of the shell membrane sample;
FIG. 10 is a graph of the difference in dissolution rates of samples under different treatment conditions;
FIG. 11 shows the difference in the degree of hydrolysis of the shell membrane in different treatments.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are intended to be only part of the present experiment and are not intended to be limiting, and the scope of the present invention should not be limited thereby.
Example 1
The method for preparing the functional hydrolysate 1 by degrading the eggshell membrane by utilizing the steam explosion technology comprises the following steps:
1) steam explosion treatment
Weighing 1000g of the mixture, placing the mixture in a steam explosion reactor, carrying out pressure maintaining treatment for 0.5min under the condition that the pressure is 1.4MPa, then instantly relieving the pressure for 0.1s, collecting a steam explosion sample, and storing the steam explosion sample at-4 ℃ for later use;
2) degradation of sample by dilute alkali method
Adding the steam explosion sample into NaOH solution with alkali concentration of 0.6% according to the feed-liquid ratio of 1:25, carrying out oscillation reaction for 0.5h at the temperature of 60 ℃, filtering to obtain filtrate, and carrying out freeze drying to obtain the functional hydrolysate 1.
Example 2
The method for preparing the functional hydrolysate 2 by degrading the eggshell membrane by utilizing the steam explosion technology comprises the following steps:
1) steam explosion treatment
Weighing 1000g of eggshell membrane, placing in a steam explosion machine reactor, performing pressure maintaining treatment for 1min under the condition that the pressure is 1.6MPa, then instantly releasing pressure for 0.1s, collecting a steam explosion sample, and storing at-4 ℃ for later use;
2) degradation of sample by dilute alkali method
Adding the steam explosion sample into NaOH solution with alkali concentration of 0.4% according to the feed-liquid ratio of 1:20, carrying out oscillation reaction for 1h at the temperature of 25 ℃, filtering to obtain filtrate, and carrying out freeze drying to obtain the functional hydrolysate 2.
Example 3
The method for preparing the functional hydrolysate 3 by degrading the eggshell membrane by utilizing the steam explosion technology comprises the following steps:
1) steam explosion treatment
Weighing 1000g of eggshell membrane, placing in a steam explosion machine reactor, performing pressure maintaining treatment for 2min under the condition that the pressure is 1.8MPa, then instantly releasing pressure for 0.1s, collecting a steam explosion sample, and storing at-4 ℃ for later use;
2) degradation of sample by dilute alkali method
Adding the steam explosion sample into NaOH solution with alkali concentration of 0.2% according to the feed-liquid ratio of 1:15, carrying out oscillation reaction for 2h at the temperature of 40 ℃, filtering to obtain filtrate, and carrying out freeze drying to obtain the functional hydrolysate 3.
Example 4
The method for preparing the functional hydrolysate 4 by degrading the eggshell membrane by utilizing the steam explosion technology comprises the following steps:
1) steam explosion treatment
Weighing 1000g of eggshell membrane, placing in a steam explosion machine reactor, performing pressure maintaining treatment for 3min under the condition that the pressure is 2MPa, then instantly releasing pressure for 0.1s, collecting a steam explosion sample, and storing at-4 ℃ for later use;
2) degradation of sample by dilute alkali method
Adding the steam explosion sample into NaOH solution with alkali concentration of 0 according to the feed-liquid ratio of 1:10, carrying out oscillation reaction for 4h at the temperature of 50 ℃, filtering to obtain filtrate, and carrying out freeze drying to obtain a functional hydrolysate 4.
As shown in fig. 2: the dissolution rate of the eggshell membrane increases along with the increase of the steam explosion pressure, but the difference between 1.6MPa and 2.0MPa is not obvious; along with the increase of the pressure maintaining time, the dissolution rate of the shell membrane after steam explosion treatment is obviously increased, and when the treatment time is further prolonged to 2min, the dissolution rate is also increased, but the change is not obvious. Therefore, the optimal steam explosion pressure of the steam explosion treatment is 1.6MPa, and the pressure maintaining time is 1 min.
When the alkali concentration of the NaOH solution is lower than 0.4%, the dissolution rate of the shell membrane is remarkably increased along with the increase of the alkali concentration, and the change difference is not remarkable when the concentration reaches 0.6%, so that the alkali concentration of the NaOH solution is preferably 0.4%; the shell membrane dissolution rate reaches the maximum when the feed-water ratio is 1:20, and the difference is not obvious when the feed-water ratio is larger, so the optimal value of the feed-water ratio of the steam explosion sample to the NaOH solution is 1: 20; the dissolution rate of the shell film without steam explosion treatment is obviously increased along with the increase of degradation temperature, but the change amplitude of the steam explosion sample along with the increase of temperature is not obvious. With the increase of the reaction time, the dissolution rate of the shell membrane can be obviously improved in the first 1h, and the dissolution rate is not changed obviously by the longer reaction time; therefore, the reaction temperature is 25 ℃ and the reaction time is 1 h.
Therefore, the best condition example is example 2.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (6)
1. A method for preparing functional hydrolysate by degrading egg shell membrane by steam explosion technology is characterized in that: the method comprises the following steps:
1) steam explosion treatment
Placing the eggshell membrane in a steam explosion machine reactor, performing pressure maintaining treatment for 0.5-5 min under the condition that the pressure is 0.5-2.2 MPa, then instantly releasing the pressure, collecting a steam explosion sample, and storing for later use;
2) degradation of sample by dilute alkali method
And adding the steam explosion sample into a NaOH solution, carrying out oscillation reaction for 0.5-4 h at the temperature of 25-60 ℃, filtering to obtain a filtrate, and carrying out freeze drying to obtain the functional hydrolysate.
2. The method for preparing functional hydrolysate by degrading eggshell membrane according to the steam explosion technology of claim 1, wherein the method comprises the following steps: in the step 1), the pressure is 1.6MPa, and the pressure maintaining time is 1 min.
3. The method for preparing functional hydrolysate by degrading eggshell membrane according to the steam explosion technology of claim 1, wherein the method comprises the following steps: in the step 1), the instant pressure relief time is 0.1 s.
4. The method for preparing functional hydrolysate by degrading eggshell membrane according to the steam explosion technology of claim 1, wherein the method comprises the following steps: in the step 2), the alkali concentration of the NaOH solution is 0.2-0.6%; the material-liquid ratio of the steam explosion sample to the NaOH solution is 1: 10-25.
5. The method for preparing functional hydrolysate by degrading eggshell membrane according to the steam explosion technology as claimed in claim 4, wherein the functional hydrolysate is prepared by the following steps: the alkali concentration of the NaOH solution is 0.4%; and the feed-liquid ratio of the steam explosion sample to the NaOH solution is 1: 20.
6. The method for preparing functional hydrolysate by degrading eggshell membrane according to the steam explosion technology as claimed in claim 4, wherein the functional hydrolysate is prepared by the following steps: in the step 2), the oscillation reaction conditions are as follows: the temperature is 25 ℃ and the reaction time is 1 h.
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