CN112375800A - Preparation method of sea cucumber visceral protein peptide - Google Patents
Preparation method of sea cucumber visceral protein peptide Download PDFInfo
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Abstract
The invention discloses a preparation method of sea cucumber visceral protein peptide, which comprises the following steps: the sea cucumber viscera protein peptide product with good antioxidant activity is prepared by taking sea cucumber viscera as a raw material, washing out impurities in intestines by clear water, soaking in electrolyzed water, performing primary enzymolysis, ultrafiltration, nanofiltration, secondary enzymolysis, decoloration and deodorization, performing ultrahigh-temperature instant sterilization, and performing spray drying. By adopting the technical scheme of the invention, the process of repeated heating sterilization in the production process can be reduced, the holothurian visceral protease can be fully utilized, the dosage of enzyme required by hydrolysis is reduced, the production efficiency and the product yield are improved, the production cost is reduced, the process is simple, and the method is suitable for industrial application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to the technical field of preparation of sea cucumber visceral protein peptides.
Background
The sea cucumber is rich in nutrient substances, is a traditional rare nourishing food in China, is rich in protein, trace elements and active substances, has low contents of fat and cholesterol, and has the effects of delaying senility, eliminating fatigue, improving immunity and the like. A large amount of internal organs are generated in the sea cucumber processing, and most of the internal organs of the sea cucumber are discarded except a small amount of salt-pickled sea cucumber processed into food, so that resources are wasted and the environment is polluted. The viscera of the sea cucumber has the same nutritive value as that of the sea cucumber, so that the precious sea cucumber resources can be fully utilized by utilizing the viscera of the sea cucumber to extract the protein peptide.
The protein peptide is usually composed of 2-3 amino acids, has biological activities of resisting oxidation, enhancing immunity, resisting hypertension, reducing cholesterol and the like, can inhibit the growth and the propagation of bacteria, viruses and tumor cells, has functions of improving the absorption and the transportation of trace elements and mineral substances of a human body, promoting growth and the like, and has important functions on the regulation of cell physiology and metabolic functions. The research shows that the protein taken by human body is absorbed not only in the form of amino acid, but also in the form of small peptide such as dipeptide or tripeptide after being acted by digestive enzyme, the small peptide is easier to be absorbed than free amino acid, and the biological value and the nutritional value of the protein peptide are higher than those of the free amino acid. Therefore, protein peptides with bioactive functions have wide application values in the fields of food and medical care, and the market demand for protein peptides is continuously increased.
In the preparation process of the protein peptide, because compound enzymolysis is needed to improve the enzymolysis degree of the protein, the optimum pH value of the enzymolysis needs to be continuously adjusted, and a large amount of inorganic salt is introduced as a result. Although part of the inorganic salts can eventually be removed by nanofiltration, a large amount of nutritive oligopeptides and free amino acids are also lost. Moreover, most of the protein peptide products from marine sources have deep color and heavy fishy smell, which affects the acceptance of consumers.
Therefore, it is an urgent technical problem to be solved by those skilled in the art to provide a preparation method which is simple in process, low in cost and capable of producing a sea cucumber visceral protein peptide product with excellent quality.
Disclosure of Invention
The invention aims to provide a preparation method of sea cucumber visceral protein peptide, which takes sea cucumber viscera as raw materials, and is prepared into the sea cucumber visceral protein peptide by using electrolyzed water for cleaning, and then carrying out processes such as enzymolysis, ceramic membrane filtration, nanofiltration membrane filtration, secondary enzymolysis, fishy smell removal, decoloration, drying and the like.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a preparation method of sea cucumber visceral protein peptide, which comprises the following steps:
s1, cleaning and soaking internal organs of sea cucumbers: cleaning viscera of Stichopus japonicus with tap water to remove impurities in intestine, draining, adding electrolytic water at a material-liquid ratio of 3: 1-6: 1(w/v), and soaking at 10-30 deg.C for 15-45 min;
s2, primary enzymolysis: adding a complex enzyme 1 solution into the sea cucumber viscera soaked in the electrolyzed water according to the feed-liquid ratio of 1: 1-2: 1(w/v), adjusting the pH of the solution to 8.0-9.0, and carrying out enzymolysis reaction at 45-55 ℃ for 1-3 h;
s3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis liquid to 6.0-7.0, filtering and separating by using a ceramic membrane, adding the retained protease and the incompletely-hydrolyzed sea cucumber visceral protein into a reconfigured complex enzyme 1 solution for continuous enzymolysis, and desalting and concentrating the obtained filtrate by using a nanofiltration membrane;
s4, secondary enzymolysis: adding complex enzyme 2 into the enzymolysis liquid intercepted by the nanofiltration membrane in the step S3, and carrying out enzymolysis reaction for 1-3h at the temperature of 45-55 ℃;
s5, decoloring and deodorizing: reacting the protease hydrolysate prepared in the step S4 for 10-15min at 45-55 ℃ by using activated carbon, and decoloring and deodorizing;
s6, sterilizing and drying: and (4) performing ultrahigh-temperature instant sterilization on the protease hydrolysate treated in the step S5 at the heating temperature of 140-.
Further, the electrolyzed water in the step S1 is prepared by electrolyzing 0.5-1% NaCl water solution for 30-60min under the conditions of current of 4-9A and flow rate of 3-5L/min by using a nano catalytic free radical generator, and the content of chloride ions in the electrolyzed water reaches 50-200 mg/kg.
Further, the compound enzyme 1 in the step S2 refers to the compound of collagenase, alkali protease and trypsin, the compound ratio is 2:2:1, and the mass concentration of the enzyme in the enzyme solution is 0.75-1.50 per mill.
Further, the molecular weight cut-off of the ceramic membrane in step S3 is 10000-20000Da, and the molecular weight cut-off of the nanofiltration membrane is 150-200 Da.
Further, the temperature range of ultrafiltration and nanofiltration in step S3 is 45-55 ℃.
Further, the complex enzyme 2 in the step S4 refers to the complex formulation of papain, neutral protease and flavourzyme, the complex formulation ratio is 2:2:1, and the mass percentage of the enzyme relative to the solid in the solution is 1.0-2.5%.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. when the aquatic product processing by-product is used for preparing protein peptide by enzymolysis, heating sterilization is usually adopted before and after enzymolysis due to worry about the influence of microorganisms in the enzymolysis process. Not only does this increase the cost of production, but heating can potentially destroy the bioactive function of the protein peptide, causing the protein peptide to darken in color. The invention adopts the electrolyzed water to clean the viscera of the sea cucumber, avoids repeated heating sterilization process, can shorten the production time and reduce the production cost. Meanwhile, as high-temperature sterilization is not adopted, the protease of the viscera of the sea cucumber can be fully utilized, and the dosage of the protease is reduced. Compared with acidic electrolyzed water, the electrolyzed water prepared by the sodium chloride solution through the nano catalytic electrolysis device contains more chlorine free radical ions, has the pH of 8.0-8.5, can be subjected to enzymolysis basically without adjusting the pH, has better sterilization effect and is more convenient to apply practically.
2. Because the temperature is kept at 45-55 ℃ all the time in the filtration of the ceramic membrane and the nanofiltration membrane, the membrane permeability can be improved, the enzymolysis can be continuously carried out, and the protease and the holothurian visceral protein with incomplete enzymolysis can be recycled, so that the preparation time of the protein peptide can be shortened, and the dosage of the protease can be reduced. The invention has mild treatment conditions, can effectively reduce the production cost and improve the product quality.
3. According to the invention, the composite enzymolysis of collagenase, alkaline protease and trypsin is selected in the primary enzymolysis, the molecular weight of polypeptide in the obtained protein enzymolysis liquid is mainly distributed in 3000-10000Da, after the enzymolysis liquid is subjected to the composite enzymolysis of papain, neutral protease and flavourzyme, the molecular weight of oligopeptide in the obtained secondary enzymolysis liquid is mainly distributed in 200-500Da, one part of the oligopeptide is enzymolyzed into oligopeptide or amino acid with the molecular weight lower than 200Da, and the molecular weight of the collagenase, the alkaline protease and the trypsin is larger than 20000Da, so that the protease and incompletely enzymolyzed sea cucumber visceral protein can be recovered by adopting ceramic membrane ultrafiltration before the secondary enzymolysis after the primary enzymolysis, and the loss of nutritional ingredients in the enzymolysis product can be reduced by using nanofiltration membrane desalination.
4. Through comprehensive measures such as washing off impurities in the sea cucumber visceral intestines by using clear water, soaking by using electrolyzed water, carrying out primary enzymolysis, ultrafiltration, nanofiltration, secondary enzymolysis, decoloration and deodorization, ultrahigh-temperature instant sterilization, drying and the like, the effects of shortening the production time, reducing the production cost, having a good sterilization effect, preparing a sea cucumber visceral protein peptide product with excellent quality and the like are achieved.
Drawings
The invention is further described with reference to the following figures and specific examples.
FIG. 1 shows the molecular weight distribution of sea cucumber visceral protein peptides.
FIG. 2 shows the total number of colonies of sea cucumber visceral protein peptides.
FIG. 3 shows the antioxidant activity of sea cucumber visceral protein peptide.
Detailed Description
Example 1
Experimental group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera is cleaned by tap water to remove impurities in intestines, and then 5L of electrolyzed water with the chloride ion concentration of 200mg/L (5L of NaCl solution with the mass concentration of 1% is electrolyzed by a nano catalytic free radical generator at the current of 9A and the flow rate of 3L/min for 60min to prepare the electrolyzed water, the chloride ion concentration in the electrolyzed water is about 200mg/L), and the electrolyzed water is statically soaked for 15min at the temperature of 30 ℃.
S2, primary enzymolysis: adding 30L of complex enzyme 1 solution (containing 18g collagenase, 18g alkaline protease and 9g trypsin) into the viscera of sea cucumber soaked in electrolyzed water, adjusting the pH of the solution to 8.0, and performing enzymolysis reaction at 45 deg.C for 3 h.
S3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis liquid to 6.0, filtering and separating at 45 ℃ by using a ceramic membrane (with the molecular weight cutoff of 10000Da), recovering the solution which cannot pass through an ultrafiltration membrane, adding the solution into the reconfigured complex enzyme 1 solution for continuous enzymolysis, desalting and concentrating the filtrate passing through the ceramic membrane by using a nanofiltration membrane (with the molecular weight cutoff of 200Da) at 45 ℃, intercepting by using the nanofiltration membrane to obtain about 6L of sea cucumber visceral protein enzymolysis liquid, and measuring by using a handheld refractometer, wherein the content of soluble solid matters in the enzymolysis liquid is 20%.
S4, secondary enzymolysis: adding 12g of papain, 12g of neutral protease and 6g of flavourzyme into the holothurian viscera protease hydrolysate, and carrying out enzymolysis reaction for 1h at 45 ℃.
S5, decoloring and deodorizing: adding 30g of active carbon into the sea cucumber visceral protein hydrolysate, decoloring and deodorizing at 45 ℃ for 15min, and removing the active carbon by using a disc centrifuge.
S6, sterilizing and drying: heating at 140 deg.C for 10 s for sterilization, and spray drying with spray dryer to obtain 1.21kg of milky white powder with water content of about 5% (w/w).
Control group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera is cleaned with tap water to remove intestinal impurities, drained, added with 5L of NaCl solution with the mass concentration of 1%, and soaked for 15min at 30 ℃.
S2, primary enzymolysis: adding 30L of complex enzyme 1 solution (containing 18g collagenase, 18g alkaline protease and 9g trypsin) into the viscera of sea cucumber soaked in electrolyzed water, adjusting the pH of the solution to 8.0, and performing enzymolysis reaction at 45 deg.C for 3 h.
S3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis liquid to 6.0, filtering and separating at 45 ℃ by using a ceramic membrane (with the molecular weight cutoff of 10000Da), recovering the solution which cannot pass through an ultrafiltration membrane, adding the solution into the reconfigured complex enzyme 1 solution for continuous enzymolysis, desalting and concentrating the filtrate passing through the ceramic membrane by using a nanofiltration membrane (with the molecular weight cutoff of 200Da) at 45 ℃, intercepting by using the nanofiltration membrane to obtain about 6L of sea cucumber visceral protein enzymolysis liquid, and measuring by using a handheld refractometer, wherein the content of soluble solid matters in the enzymolysis liquid is 20%.
S4, secondary enzymolysis: adding 12g of papain, 12g of neutral protease and 6g of flavourzyme into the holothurian viscera protease hydrolysate, and carrying out enzymolysis reaction for 1h at 45 ℃.
S5, decoloring and deodorizing: adding 30g of active carbon into the sea cucumber visceral protein hydrolysate, decoloring and deodorizing at 45 ℃ for 15min, and removing the active carbon by using a disc centrifuge.
S6, sterilizing and drying: heating at 140 deg.C for 10 s for sterilization, and spray drying with spray dryer to obtain 1.13kg of milky white powder with water content of about 5% (w/w).
Example 2
Experimental group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera is cleaned with tap water to remove intestinal impurities, drained, added with 7.5L of electrolyzed water with chloride ion concentration of 100mg/L (5L of NaCl solution with mass concentration of 1% is electrolyzed by a nano catalytic free radical generator at a current of 9A and a flow rate of 3L/min for 60min to prepare electrolyzed water, the chloride ion concentration in the electrolyzed water is about 200mg/L), and statically soaked at 20 ℃ for 30 min.
S2, primary enzymolysis: adding 25L of complex enzyme 1 solution (containing 27g collagenase, 27g alkaline protease and 13.5g trypsin) into the viscera of sea cucumber soaked in electrolyzed water, adjusting the pH of the solution to 8.5, and performing enzymolysis reaction at 50 deg.C for 2 h.
S3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis liquid to 6.5, filtering and separating at 50 ℃ by using a ceramic membrane (with the molecular weight cutoff of 15000Da), recovering the solution which cannot pass through an ultrafiltration membrane, adding the solution into the reconfigured complex enzyme 1 solution for continuous enzymolysis, desalting and concentrating the filtrate passing through the ceramic membrane at 50 ℃ by using a nanofiltration membrane (with the molecular weight cutoff of 200Da), and intercepting by using the nanofiltration membrane to obtain about 4.6L of holothurian visceral protease hydrolysate, wherein the content of soluble solids in the enzymolysis liquid is 25 percent by using a handheld refractometer.
S4, secondary enzymolysis: adding 7g of papain, 7g of neutral protease and 3.5g of flavourzyme into the holothurian viscera protease hydrolysate, and carrying out enzymolysis reaction for 2 hours at 50 ℃.
S5, adding 30g of activated carbon into the holothurian viscera protease hydrolysate, decoloring and deodorizing at 50 ℃ for 10min, and removing the activated carbon by using a disc centrifuge.
S6, sterilizing and drying: heating at 150 deg.C for 8 s for sterilization, and spray drying with spray dryer to obtain 1.14kg of milky white powder with water content of about 5% (w/w).
Control group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera is cleaned with tap water to remove intestinal impurities, drained, added with 7.5L of electrolyzed water with chloride ion concentration of 100mg/L (5L of NaCl solution with mass concentration of 1% is electrolyzed by a nano catalytic free radical generator at a current of 9A and a flow rate of 3L/min for 60min to prepare electrolyzed water, the chloride ion concentration in the electrolyzed water is about 200mg/L), and statically soaked at 20 ℃ for 30 min.
S2, primary enzymolysis: adding 25L of complex enzyme 1 solution (containing 27g collagenase, 27g alkaline protease and 13.5g trypsin) into the viscera of sea cucumber soaked in electrolyzed water, adjusting the pH of the solution to 8.5, and performing enzymolysis reaction at 50 deg.C for 2 h.
S3, secondary enzymolysis: after the pH of the enzymatic hydrolysate was adjusted to 6.5, 7g of papain, 7g of neutral protease and 3.5g of flavourzyme were added, and enzymatic reaction was carried out at 50 ℃ for 2 hours.
S4, ultrafiltration and nanofiltration: filtering and separating with ceramic membrane (with cut-off molecular weight of 15000Da) at 50 deg.C, desalting and concentrating the filtrate with nanofiltration membrane (with cut-off molecular weight of 200Da) at 50 deg.C, and cutting with nanofiltration membrane to obtain about 2.8L of sea cucumber viscera protease hydrolysate, wherein the content of soluble solid in the hydrolysate is 25% as determined by hand-held refractometer.
S5, decoloring and deodorizing: adding 30g of active carbon into the sea cucumber visceral protein hydrolysate, decoloring and deodorizing at 50 ℃ for 10min, and removing the active carbon by using a disk centrifuge.
S6, sterilizing and drying: heating at 150 deg.C for 8 s for sterilization, and spray drying with spray dryer to obtain 0.68kg of milky white powder with water content of about 5% (w/w).
Example 3
Experimental group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera is cleaned by tap water to remove impurities in intestines, and then 10L of electrolyzed water with the chloride ion concentration of 50mg/L (5L of NaCl solution with the mass concentration of 1% is electrolyzed by a nano catalytic free radical generator at the current of 9A and the flow rate of 3L/min for 60min to prepare the electrolyzed water, the chloride ion concentration in the electrolyzed water is about 200mg/L), and the electrolyzed water is statically soaked for 45min at the temperature of 10 ℃.
S2, primary enzymolysis: adding 15L of complex enzyme 1 solution (containing 36g collagenase, 36g alkaline protease and 18g trypsin) into the viscera of sea cucumber soaked in electrolyzed water, adjusting the pH of the solution to 9.0, and performing enzymolysis reaction at 55 deg.C for 1 h.
S3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis liquid to 7.0, filtering and separating at 55 ℃ by using a ceramic membrane (with the molecular weight cutoff of 20000Da), recovering the solution which cannot pass through an ultrafiltration membrane, adding the solution into the reconfigured complex enzyme 1 solution for continuous enzymolysis, desalting and concentrating the filtrate passing through the ceramic membrane by using a nanofiltration membrane (with the molecular weight cutoff of 150Da) at 55 ℃, intercepting by using the nanofiltration membrane to obtain about 4.10L of holothurian visceral protease hydrolysate, and measuring by using a handheld refractometer, wherein the content of soluble solids in the enzymolysis liquid is 30%.
S4, secondary enzymolysis: adding papain 5g, neutral protease 5g and flavourzyme 2.5g into the sea cucumber visceral protease hydrolysate, and carrying out enzymolysis reaction at 55 ℃ for 3 h.
S5, decoloring and deodorizing: adding 30g of active carbon into the sea cucumber viscera protease hydrolysate, decolorizing and deodorizing at 55 deg.C for 10min, and removing the active carbon by using a disk centrifuge.
S6, sterilizing and drying: heating at 160 deg.C for 5 s for sterilization, and spray drying with a spray dryer to obtain 1.21kg of milky white powder with water content of about 5% (w/w).
Control group
S1, cleaning and soaking internal organs of sea cucumbers: 30kg of sea cucumber viscera was washed with tap water to remove intestinal impurities, and 25L of 1% NaCl solution was added after draining.
S2, heating and sterilizing: heating the viscera of Stichopus japonicus soaked in electrolyzed water to 85 deg.C, maintaining the temperature for 20min, and sterilizing.
S3, primary enzymolysis: cooling to about 55 deg.C, adding 36g collagenase, 36g alkaline protease and 18g trypsin, adjusting pH of the solution to 9.0, and performing enzymolysis reaction at 55 deg.C for 1 h.
S4, secondary enzymolysis: adjusting the pH of the enzymolysis solution to 7.0, adding 5g of papain, 5g of neutral protease and 2.5g of flavourzyme, and carrying out enzymolysis reaction at 55 ℃ for 3 h.
S5, heating and sterilizing: heating to 85 deg.C, maintaining the temperature for 20min, sterilizing, and inactivating enzyme.
S6, ultrafiltration and nanofiltration: cooling to about 10 deg.C, filtering and separating with ceramic membrane (molecular weight cut-off is 20000 daltons) at 10 deg.C, desalting and concentrating the filtrate with nanofiltration membrane (molecular weight cut-off is 150 daltons) at 10 deg.C, and collecting 2.06L of holothurian viscera protease hydrolysate with Brix of 30%.
S7, decoloring and deodorizing: adding 30g of active carbon into the sea cucumber viscera protease hydrolysate, decolorizing and deodorizing at 55 deg.C for 10min, and removing the active carbon by using a disk centrifuge.
S8, spray drying: spray drying with a spray dryer to obtain 0.60kg of light yellow powder of sea cucumber visceral protein peptide with water content of about 5% (w/w).
Example 4
Molecular weight determination of sea cucumber visceral protein peptide product
And analyzing the molecular weight of the sea cucumber visceral protein peptide by using a high performance liquid chromatograph. The column was TSKgel G2000 SWXLL (300 mm. times.7.8 mm) and the mobile phase was acetonitrile/double distilled water/TFA (45:55:0.1, V/V/V). The results of measurement are shown in FIG. 1, using cytochrome C (12327Da), aprotinin (6533Da), oxidized glutathione (613Da), Gly-Gly-Gly (189Da) and Gly (75Da) as standards at a column temperature of 30 ℃ and a flow rate of 0.5mL/min and an ultraviolet detection wavelength of 214 nm.
As can be seen from FIG. 1, the molecular weight of the sea cucumber visceral protein peptide prepared by the invention is mainly distributed in 150-1000Da, and about 15% of oligopeptide/amino acid with the molecular weight less than 150Da is also contained. As can be seen from example 1, without pretreatment with electrolyzed water, the oligopeptide/amino acid ratio of the final product obtained, having a molecular weight of less than 150Da, is significantly increased, which may be caused by the action of microorganisms during the enzymatic hydrolysis. As can be seen from example 2 or example 3, when the electrolyzed water treatment or the conventional sterilization treatment is adopted, and the ultrafiltration and nanofiltration treatment is carried out after the secondary enzymolysis is finished, although the ratio of oligopeptide/amino acid with the molecular weight of less than 150Da is obviously reduced, the yield of the protein peptide is also obviously reduced.
Example 5
Colony count determination of sea cucumber visceral protein peptide product
5g of each of the sea cucumber visceral protein peptide products of the experimental group and the control group of examples 1 to 3 were mixed with 95ml of sterile physiological saline (0.85% NaCl solution), and then homogenized
100, Interscience, France) for 5min, diluting with sterile physiological saline 10 times, pouring 1ml of diluent with a proper concentration into a culture dish by a plate casting method, mixing with plate count agar, culturing at 30 ℃ for 48h, and calculating the total number of colonies, wherein the result is shown in fig. 2.
As can be seen from FIG. 2, in example 1, the sea cucumber viscera were treated with the electrolytic water prepared from NaCl solution instead of heating, and 1.21kg of milky sea cucumber viscera protein peptide powder with a water content of about 5% (w/w) was obtained, and the colony count was 2.5 CFU/g. In contrast, in example 1, the viscera of sea cucumber were treated by heating to obtain 1.21kg of milky white powder of sea cucumber viscera protein peptide with a water content of about 5% (w/w), and the colony count of the powder was 4.7 CFU/g. Therefore, the electrolytic water prepared by the NaCl solution can effectively inhibit the microbial production and reproduction in the enzymolysis process by replacing heating, and the product yield is obviously higher than that of the product prepared by the heating method.
Example 6
Determination of antioxidant function activity of sea cucumber protein peptide product
1. Measurement of hydroxyl radical scavenging activity: 0.3mL of 8mmol/L FeSO is sequentially added into 1.0mL of sea cucumber visceral protein peptide sample solution4，0.25mL 20mmol/L H2O21.0mL of 3mmol/L salicylic acid at 37 deg.CReacting in water bath for 30min, rapidly cooling with ice water, adding 0.45mL distilled water, shaking, centrifuging (5000r/min, 10min), and measuring absorbance at 510 nm. The OH radical clearance of the sample was calculated according to formula (1), wherein ASIs the absorbance value of the sample, ACAbsorbance values determined for the replacement of salicylic acid with distilled water, A0Absorbance values determined for the sample solution were replaced with distilled water. The measurement results are shown in FIG. 3.
2. Determination of DPPH radical scavenging Activity: 1.0mL of 0.2mmol/L DPPH ethanol solution is added into 1.0mL of the sea cucumber visceral protein peptide sample, and after the mixture is subjected to light-shielding reaction at room temperature for 30 minutes, the mixture is centrifuged at 10000r/min for 10 minutes. The absorbance was measured at a wavelength of 517nm using a UV-8000A spectrophotometer. DPPH radical clearance of the samples was calculated according to formula (1), wherein ASIs the absorbance of the sample, ACTo replace the absorbance of the ethanol DPPH solution with ethanol, A0The absorbance of the sample was replaced with distilled water. The measurement results are shown in FIG. 3.
3. Iron ion reducing ability: 1.0mL of phosphate buffer (PBS,0.2mol/L, pH 6.6) and 1.0mL of 1% potassium ferricyanide [ K ] were added to 1.0mL of the sea cucumber visceral protein peptide sample3Fe(CN)6]The solution was reacted in a water bath at 50 ℃ for 20 minutes. Thereafter, 1.0mL of 10% trichloroacetic acid was added to the mixed solution, and the mixture was centrifuged at 4000r/min for 10 minutes. 2mL of the supernatant was aspirated, and 2mL of distilled water and 0.4mL of a 0.1% ferric chloride solution (FeCl) were added3). After standing at room temperature for 10min, the absorbance value was measured at a wavelength of 700nm with a spectrophotometer. The measurement results are shown in FIG. 3.
As can be seen from FIG. 3, on one hand, the nanofiltration desalination is adjusted to be in front of the secondary enzymolysis, so that the antioxidant activity function of the product is not affected; on the other hand, the product yield was also significantly improved (example 2).
In conclusion, the sea cucumber visceral protein peptide with good antioxidant activity can be produced by washing out impurities in the sea cucumber visceral intestines by using clear water, soaking by using electrolyzed water, and carrying out the procedures of primary enzymolysis, ultrafiltration, nanofiltration, secondary enzymolysis, decoloration and deodorization, ultrahigh-temperature instant sterilization, drying and the like.
Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.
Claims (6)
1. A preparation method of sea cucumber visceral protein peptide is characterized by comprising the following steps: the method comprises the following steps:
s1, cleaning and soaking internal organs of sea cucumbers: cleaning viscera of Stichopus japonicus with tap water to remove impurities in intestine, draining, adding electrolytic water at a material-liquid ratio of 3 ׃ 1-6 ׃ 1(w/v), and soaking at 10-30 deg.C for 15-45 min;
s2, primary enzymolysis: adding a complex enzyme 1 solution into the sea cucumber viscera soaked in the electrolyzed water according to the feed-liquid ratio of 1 ׃ 1-2 ׃ 1(w/v), adjusting the pH of the solution to 8.0-9.0, and carrying out enzymolysis reaction at 45-55 ℃ for 1-3 h;
s3, ultrafiltration and nanofiltration: adjusting the pH of the enzymolysis solution to 6.0-7.0, performing ultrafiltration separation by using a ceramic membrane, adding the trapped protease and the incompletely-hydrolyzed sea cucumber visceral protein into the reconfigured compound enzyme 1 solution for continuous enzymolysis, and desalting and concentrating the obtained filtrate by using a nanofiltration membrane;
s4, secondary enzymolysis: adding a complex enzyme 2 solution into the enzymolysis liquid intercepted by the S3 nanofiltration membrane, and carrying out enzymolysis reaction for 1-3h at 45-55 ℃;
s5, decoloring and deodorizing: decoloring and deodorizing the protease hydrolysate prepared by the step S4 by using activated carbon;
s6, sterilizing and drying: and (3) carrying out ultrahigh-temperature instant sterilization on the protease hydrolysate treated by the S5 at the heating temperature of 140-160 ℃ for 5-10 seconds, and then carrying out spray drying to prepare the sea cucumber visceral protein peptide powder.
2. The method for producing a sea cucumber visceral protein peptide according to claim 1, wherein: in step S1, the electrolyzed water is prepared by electrolyzing 0.5-1% NaCl water solution for 30-60min under the conditions of 4-9A of current and 3-5L/min of flow rate by using a nano catalytic free radical generator, and the content of chloride ions in the electrolyzed water reaches 50-200 mg/kg.
3. The method for producing a sea cucumber visceral protein peptide according to claim 1, wherein: in step S2, the complex enzyme 1 refers to the complex formulation of collagenase, alkali protease and trypsin, the complex formulation ratio is 2:2:1, and the mass concentration of the enzyme in the enzyme solution is 0.75-1.50 ‰.
4. The method for producing a sea cucumber visceral protein peptide according to claim 1, wherein: in step S3, the molecular weight cut-off of the ceramic membrane is 10000-20000Da, and the molecular weight cut-off of the nanofiltration membrane is 150-200 Da.
5. The method for producing a sea cucumber visceral protein peptide according to claim 1, wherein: in step S3, the temperature range of ultrafiltration and nanofiltration is 45-55 ℃.
6. The method for producing a sea cucumber visceral protein peptide according to claim 1, wherein: in step S4, the complex enzyme 2 refers to the combination of papain, neutral protease and flavourzyme, the combination ratio is 2:2:1, and the mass percentage of the enzyme relative to the solid in the solution is 1.0-2.5%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151388A (en) * | 2021-04-19 | 2021-07-23 | 国肽生物工程（常德）有限公司 | Sea cucumber peptide with antioxidant and DPP-IV (dipeptidyl peptidase-IV) inhibiting functions and preparation method thereof |
| CN114807277A (en) * | 2022-04-12 | 2022-07-29 | 滨海宇美科技有限公司 | Method for preparing sea cucumber peptide by double enzymes step by step |
| CN114805625A (en) * | 2022-05-10 | 2022-07-29 | 滨海宇美科技有限公司 | Method for preparing sea cucumber polysaccharide from sea cucumber viscera |
| CN116440024A (en) * | 2023-06-13 | 2023-07-18 | 深圳阿尔法生物科技有限公司 | Sea collagen acne-removing repair cream from sea cucumber |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103734790A (en) * | 2014-01-24 | 2014-04-23 | 福建农林大学 | Method for assisting electrolyzed water soaking of sea cucumbers by ultrasonic waves |
| CN106259780A (en) * | 2016-08-17 | 2017-01-04 | 林春梅 | Sea cucumber intestine polypeptide cookies and preparation method thereof |
| CN111235205A (en) * | 2020-03-26 | 2020-06-05 | 琛蓝(美国)营养制品股份有限公司 | Preparation method and application of sea cucumber protein peptide |
-
2020
- 2020-11-10 CN CN202011246219.1A patent/CN112375800A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103734790A (en) * | 2014-01-24 | 2014-04-23 | 福建农林大学 | Method for assisting electrolyzed water soaking of sea cucumbers by ultrasonic waves |
| CN106259780A (en) * | 2016-08-17 | 2017-01-04 | 林春梅 | Sea cucumber intestine polypeptide cookies and preparation method thereof |
| CN111235205A (en) * | 2020-03-26 | 2020-06-05 | 琛蓝(美国)营养制品股份有限公司 | Preparation method and application of sea cucumber protein peptide |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151388A (en) * | 2021-04-19 | 2021-07-23 | 国肽生物工程（常德）有限公司 | Sea cucumber peptide with antioxidant and DPP-IV (dipeptidyl peptidase-IV) inhibiting functions and preparation method thereof |
| CN114807277A (en) * | 2022-04-12 | 2022-07-29 | 滨海宇美科技有限公司 | Method for preparing sea cucumber peptide by double enzymes step by step |
| CN114805625A (en) * | 2022-05-10 | 2022-07-29 | 滨海宇美科技有限公司 | Method for preparing sea cucumber polysaccharide from sea cucumber viscera |
| CN116440024A (en) * | 2023-06-13 | 2023-07-18 | 深圳阿尔法生物科技有限公司 | Sea collagen acne-removing repair cream from sea cucumber |
| CN116440024B (en) * | 2023-06-13 | 2023-10-17 | 深圳阿尔法生物科技有限公司 | Sea collagen acne-removing repair cream from sea cucumber |
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