CN112266944A - Industrial production method for obtaining chlorella protein peptide by acid-enzyme method - Google Patents

Industrial production method for obtaining chlorella protein peptide by acid-enzyme method Download PDF

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Publication number
CN112266944A
CN112266944A CN202011197302.4A CN202011197302A CN112266944A CN 112266944 A CN112266944 A CN 112266944A CN 202011197302 A CN202011197302 A CN 202011197302A CN 112266944 A CN112266944 A CN 112266944A
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chlorella
protein peptide
acid
feed liquid
enzyme
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Inventor
许团辉
李媛媛
陈璇
郑晓辉
吴奕武
陈蝶玲
王文谦
郑震堃
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Runke Bioengineering Fujian Co ltd
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Runke Bioengineering Fujian Co ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/06Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/145Extraction; Separation; Purification by extraction or solubilisation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/34Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Abstract

An industrial production method for obtaining chlorella protein peptide by an acid-enzyme method comprises the following steps: (1) swelling chlorella powder to obtain chlorella feed liquid; (2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1) for hydrolysis; (3) enzymolysis: adjusting pH of Chlorella feed liquid subjected to acid hydrolysis to 7-9, sequentially adding cellulase, alkaline protease, papain and neutral protease at 45-60 deg.C, performing enzymolysis for 4-8 hr, inactivating enzyme, and filtering to remove algae residue to obtain supernatant; (4) carrying out decoloration treatment on the supernatant to obtain decolored liquid; (5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide. The method combines acid hydrolysis and biological enzyme hydrolysis, has complementary advantages, and can improve the enzymolysis efficiency and the protein peptide extraction rate of the chlorella.

Description

Industrial production method for obtaining chlorella protein peptide by acid-enzyme method
Technical Field
The invention relates to the technical field of biology, in particular to an industrial production method for obtaining chlorella protein peptide by an acid-enzyme method.
Background
Chlorella (Chlorella spp.) is a unicellular green alga called "medicinal algae". The chlorella cell is oval or elliptical in shape, has a diameter of two to twelve micrometers, is easy to culture, wide in ecological distribution and high in growth speed, is a high-quality single-cell protein source, has a protein content of 50-65%, and can be used as a high-protein food to quickly supplement nutrition for human bodies and restore physical strength. Chlorella has an amino acid composition higher than the protein standard for human nutrition promulgated by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United nations, and is thus classified as a healthy food for humans in the 21 st century by the FAO. The Ministry of health in China approved the chlorella pyrenoidosa as a new resource food in 2012. Chlorella protein has antioxidant effect, and can scavenge free radicals in vivo; various proteins in chlorella have anti-tumor and immunoregulation effects; chlorella protein has the functions of detoxifying, protecting liver, reducing blood pressure and the like, has wide application prospect in health food and medicine, and is concerned by people.
The chlorella dry powder has poor solubility, strong algae fishy smell, thick and tough cell wall and poor protein digestion and absorption, and the small molecular peptides obtained by hydrolyzing the chlorella under proper conditions greatly improve the solubility and the taste, more importantly, the functional peptides such as antioxidant active peptides, antitumor active peptides, immunity-enhancing active peptides, activity-enhancing active peptides and the like are obtained, and meanwhile, the small molecular peptides can be directly and quickly absorbed, so that the digestion and absorption utilization rate is improved.
The current methods for obtaining protein peptide by hydrolyzing protein mainly comprise acid hydrolysis, alkali hydrolysis and biological enzyme hydrolysis.
The acid hydrolysis method is a method for obtaining protein peptide by hydrolyzing protein with acid under certain conditions. The acid hydrolysis has low cost, the product is hardly racemized, but excessive hydrolysis easily destroys sensitive amino acids such as tryptophan, asparagine and glutamine, and even destroys small-molecule peptides.
The alkaline hydrolysis method is a method for obtaining protein peptide by hydrolyzing protein with alkali under certain conditions. The hydrolysis of protein tryptophan by the alkali hydrolysis method is stable, but serine and threonine, particularly cystine and cysteine are easy to lose, and amino acid can be racemized after hydrolysis, so that L-type amino acid is changed into D-type amino acid, and the hydrolysis rate is low.
The enzyme hydrolysis method is a method for obtaining protein peptide by hydrolyzing protein by utilizing biological enzyme under a certain condition, has specific hydrolysis site, can selectively hydrolyze certain specific amino acid peptide bonds, has high safety, mild reaction condition, less side reaction, no damage to sensitive amino acid, and hydrolysis products superior to acid and alkali hydrolysis in the aspects of nutrition, flavor, process and the like.
Currently, many laboratory studies on chlorella active peptides are performed, but the industrial utilization of chlorella active peptides is low. Currently, industrial research on chlorella mainly focuses on single-enzyme or complex enzyme primary hydrolysis (for example, a preparation method of chlorella antitumor polypeptide disclosed in chinese patent application publication No. CN102851345A, a preparation method of chlorella antioxidant peptide disclosed in chinese patent application publication No. CN 104120160A), or hydrolyzing with an enzyme for a period of time, and adding a second enzyme (such as a preparation method of chlorella immunoactive peptide disclosed in the Chinese patent application publication No. CN 109402204A, and a preparation method of chlorella antioxidant polypeptide disclosed in the Chinese patent application publication No. CN 109207544A), but because the chlorella cell wall thickness is thick and rich in cellulose, the wall breaking rate of pretreatment such as swelling and homogenization is low, the biological enzymolysis effect is poor, and the extraction rate of protein peptide is low.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an industrial production method for obtaining chlorella protein peptide by an acid-enzyme method, and the method can improve the enzymolysis efficiency and the protein peptide extraction rate of chlorella. The technical scheme is as follows:
an industrial production method for obtaining chlorella protein peptide by an acid-enzyme method is characterized by comprising the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 0.5-2% of the dry weight of the chlorella feed liquid, and then hydrolyzing for 1-3 hours at the temperature of 35-45 ℃;
(3) enzymolysis: adjusting pH of Chlorella feed liquid to 7-9 (adjusted by sodium hydroxide), sequentially adding cellulase, alkaline protease, papain and neutral protease at 45-60 deg.C, performing enzymolysis for 4-8 hr, inactivating enzyme, and filtering to remove algae residue to obtain supernatant;
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
Preferably, in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 4-8 times of that of the chlorella powder, stirring at the speed of 30-50rpm, and swelling for 1-3 hours at the temperature of 35-45 ℃. Preferably, the stainless steel tank used in the step (1) is provided with a stirring system and a circulating water system, wherein the stirring system is used for stirring materials in the stainless steel tank, and the circulating water system is used for keeping the internal temperature of the stainless steel tank at 35-45 ℃.
Preferably, in step (2), the hydrochloric acid is added in a concentration of 30 to 38% by weight. More preferably, in step (2), the hydrochloric acid is added in a concentration of 36 to 38% by weight.
Preferably, in the step (3), the addition amount of the cellulase is 1-2% of the dry weight of the chlorella feed liquid, the addition amount of the alkaline protease is 1-3% of the dry weight of the chlorella feed liquid, the addition amount of the papain is 0.5-3% of the dry weight of the chlorella feed liquid, and the addition amount of the neutral protease is 1-3% of the dry weight of the chlorella feed liquid.
Preferably, in the step (3), the activity unit of the cellulase is 1-3 ten thousand U/g, the activity unit of the alkaline protease is 30-80 ten thousand U/g, the activity unit of the papain is 50-80 ten thousand U/g, and the activity unit of the neutral protease is 10-30 ten thousand U/g.
The cellulase is a compound of endo-beta-1, 4 glucan hydrolase, exo-beta-1, 4 glucan hydrolase and beta-1, 4 glucosidase, and the synergistic effect of the three can completely decompose cellulose, so that the wall of chlorella is damaged, and other proteases can be in full contact with chlorella protein to play a role. The alkaline protease is a serine type endoprotease, can hydrolyze peptide chains of protein molecules to generate polypeptide or amino acid, is stable at a pH value of 5-10, can only hydrolyze the peptide chains of the protein, has specificity on the carboxyl side of an opening point, and has strong capability of decomposing the protein. Papain belongs to thiol protease, can hydrolyze carboxyl terminals of arginine and lysine in protein and polypeptide, can preferentially hydrolyze peptide bonds of amino acid or aromatic L amino acid with 2 carboxyl groups at the N terminal of the peptide bonds, and has strong enzymolysis capability on plant protein. The neutral protease is bacterial protease, is endoprotease, has the characteristics of strong hydrolysis capacity, high speed, less bitter taste of hydrolysate and the like, can decompose peptide bonds in a protein peptide chain, and can further decompose the polypeptide decomposed by the alkaline protease into small peptides. The chlorella can be fully hydrolyzed and active peptides are reserved by selecting proper types of enzymes, proportion and adding sequence thereof and enzymolysis process conditions.
Preferably, in the step (3), the chlorella feed liquid is stirred at the rotating speed of 30-50rpm in the enzymolysis process.
Preferably, in the step (3), after the enzymolysis is finished, the enzymolysis liquid is heated to 80-95 ℃ to inactivate the enzyme for 10-20 minutes, then the enzymolysis liquid is cooled to 30-40 ℃, and then the algae residue in the enzymolysis liquid is filtered. Preferably, in the step (3), a plate-and-frame filter is adopted to filter algae residues in the enzymatic hydrolysate.
In a preferred embodiment, the decoloring in the step (4) is performed by: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 1-3% of that of the supernatant, and the weight of the added diatomite is 1-2% of that of the supernatant; then decolorizing at 45-60 deg.C for 40-60min under stirring (preferably at 30-50 rpm), and filtering with plate-and-frame filter. Preferably, the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 1-10 microns.
In a preferred embodiment, the filtering in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
(5-3) nanofiltration: and (5) adding purified water with the volume of 1-3 times of that of the small molecular chlorella protein peptide solution obtained in the step (5-2), and performing nanofiltration to obtain a chlorella protein peptide solution for spray drying.
In a more preferable mode, the ceramic membrane filter element used in the ceramic microfiltration equipment in the step (5-1) has a pore diameter of 30-50 nm.
More preferably, in step (5-2), the membrane filter cartridge used in the ultrafiltration apparatus allows substances having a molecular weight of less than 3000-10000Da (Dalton) to pass through.
In the more preferable step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150-500Da to pass through, and continuous three-stage nanofiltration is carried out, wherein the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration. The trapped fluid of the third-stage nanofiltration is the required chlorella protein peptide solution.
According to the invention, the microfiltration is carried out through the ceramic membrane, so that macromolecular substances are effectively intercepted, the ultrafiltration efficiency is favorably improved, the ultrafiltration membrane can be protected, and the service life of the ultrafiltration membrane is prolonged; ultrafiltration is carried out by an ultrafiltration membrane, so that the micromolecule peptide below 1000Da in the micromolecule chlorella protein peptide solution reaches more than 90 percent, and the content of the micromolecule is greatly improved; through nanofiltration, the chlorella protein peptide can be well desalted and debitterized, and can be effectively concentrated, so that the content of the chlorella protein peptide reaches more than 20-25%.
In a preferable scheme, in the step (5), a spray dryer is used for spray drying the chlorella protein peptide solution, the evaporation capacity of the spray drying is 2 t/h (ton/h), the air inlet temperature is 150-185 ℃, and the air outlet temperature is 60-85 ℃.
The invention has the following beneficial effects:
the invention combines acid hydrolysis and biological enzyme hydrolysis, has complementary advantages and improves the efficiency and the yield. The chlorella is firstly decomposed into smaller fragments by acid hydrolysis, the cell wall is destroyed, so that protein is exposed and can be fully contacted with enzyme, macromolecular protein can be hydrolyzed into smaller molecular protein, subsequent enzymolysis is facilitated, the enzymolysis efficiency and the protein peptide yield are improved, the dosage of the enzyme can be reduced, and meanwhile, part of amino acid is prevented from being destroyed due to excessive hydrolysis. Then, biological enzyme is utilized for further hydrolysis, the biological enzyme has strong specificity and mild reaction, and can carry out specific enzymolysis on the chlorella to obtain chlorella protein peptide.
The chlorella protein peptide obtained by the invention simultaneously contains a certain amount of active substances such as chlorella active polysaccharide, vitamins and the like, and has antioxidant activity through detection.
Detailed Description
Protein peptide extraction (%) = (total protein content in enzymatic supernatant/total protein content in chlorella vulgaris) × 100.
The molecular weight distribution of the protein peptide is determined by adopting high-efficiency size exclusion chromatography according to the national standard of GB 31645 and 2018 for food safety.
Determination of antioxidant Activity: ABTS free radical scavenging activity and DPPH free radical scavenging ability were measured by the method adopted in the Zengqiai doctor paper (Zengqiai, 2016).
Example 1
In this embodiment, the industrial production method for obtaining chlorella protein peptide by the acid-enzyme method comprises the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 5 times that of the chlorella powder, stirring at the speed of 50rpm, and swelling for 2 hours at the temperature of 45 ℃. The stainless steel tank used in the step (1) is provided with a stirring system and a circulating water system, wherein the stirring system is used for stirring materials in the stainless steel tank, and the circulating water system is used for keeping the internal temperature of the stainless steel tank at 45 ℃;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 1 percent of the dry weight of the chlorella feed liquid, and then hydrolyzing for 2 hours at the temperature of 45 ℃;
in the step (2), the weight percentage concentration of the added hydrochloric acid is 37 percent;
(3) enzymolysis: adjusting the pH value of chlorella feed liquid subjected to acid hydrolysis to 8.5 (adjusting with sodium hydroxide), sequentially adding cellulase, alkaline protease, papain and neutral protease at 52 ℃, performing enzymolysis for 6 hours (stirring the chlorella feed liquid at the rotation speed of 50rpm in the enzymolysis process), inactivating enzyme, and filtering algae residues in the enzymolysis liquid to obtain supernatant;
in the step (3), the addition amount of the cellulase is 2% of the dry weight of the chlorella feed liquid, the addition amount of the alkaline protease is 1% of the dry weight of the chlorella feed liquid, the addition amount of the papain is 2% of the dry weight of the chlorella feed liquid, and the addition amount of the neutral protease is 2% of the dry weight of the chlorella feed liquid;
the unit of the activity of the cellulase is 2 ten thousand U/g, the unit of the activity of the alkaline protease is 50 ten thousand U/g, the unit of the activity of the papain is 60 ten thousand U/g, and the unit of the activity of the neutral protease is 20 ten thousand U/g;
in the step (3), after the enzymolysis is finished, heating the enzymolysis liquid to 80 ℃ to inactivate the enzyme for 20 minutes, then cooling the enzymolysis liquid to 30 ℃, and then filtering algae residues in the enzymolysis liquid (filtering the algae residues in the enzymolysis liquid by using a plate and frame filter);
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
the process of the decoloring treatment in the step (4) is as follows: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 2% of that of the supernatant, and the weight of the added diatomite is 1% of that of the supernatant; then decoloring for 60min at 50 ℃ under the condition of stirring (the rotating speed of the stirrer is 40 rpm), and then carrying out plate-and-frame filtration;
the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 5 microns;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
The filtration in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
the aperture of the ceramic membrane filter element used by the ceramic microfiltration equipment in the step (5-1) is 50 nm;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
in the step (5-2), the membrane filter element used by the ultrafiltration device allows substances with molecular weight lower than 5000Da to pass through;
(5-3) nanofiltration: and (5) adding purified water with the volume being 1 time that of the small molecular chlorella protein peptide solution obtained in the step (5-2), and then performing nanofiltration to obtain the chlorella protein peptide solution for spray drying.
In the step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 300-500Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration.
And (5) performing spray drying on the chlorella protein peptide solution by using a spray dryer, wherein the evaporation capacity of the spray drying is 2 t/h (ton/hour), the air inlet temperature is 165 ℃, and the air outlet temperature is 78 ℃.
According to detection, the protein extraction rate of the embodiment is 56.7%, and the content of protein peptides below 1000Da is 92.1%.
Example 2
In this embodiment, the industrial production method for obtaining chlorella protein peptide by the acid-enzyme method comprises the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 4 times that of the chlorella powder, stirring at the speed of 40rpm, and swelling for 2 hours at the temperature of 40 ℃. The stainless steel tank used in the step (1) is provided with a stirring system and a circulating water system, wherein the stirring system is used for stirring materials in the stainless steel tank, and the circulating water system is used for keeping the internal temperature of the stainless steel tank at 40 ℃;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 1.5 percent of the dry weight of the chlorella feed liquid, and then hydrolyzing for 2 hours at the temperature of 45 ℃;
in the step (2), the weight percentage concentration of the added hydrochloric acid is 35 percent;
(3) enzymolysis: adjusting the pH value of chlorella feed liquid subjected to acid hydrolysis to 8.6 (adjusting by using sodium hydroxide), sequentially adding cellulase, alkaline protease, papain and neutral protease at 48 ℃, performing enzymolysis for 4 hours (stirring the chlorella feed liquid at the rotating speed of 50rpm in the enzymolysis process), and filtering algae residues in the enzymolysis liquid after enzyme deactivation to obtain a supernatant;
in the step (3), the adding amount of the cellulase is 1 percent of the dry weight of the chlorella feed liquid, the adding amount of the alkaline protease is 2 percent of the dry weight of the chlorella feed liquid, the adding amount of the papain is 1 percent of the dry weight of the chlorella feed liquid, and the adding amount of the neutral protease is 2 percent of the dry weight of the chlorella feed liquid;
the unit of the activity of the cellulase is 2 ten thousand U/g, the unit of the activity of the alkaline protease is 50 ten thousand U/g, the unit of the activity of the papain is 60 ten thousand U/g, and the unit of the activity of the neutral protease is 20 ten thousand U/g;
in the step (3), after the enzymolysis is finished, heating the enzymolysis liquid to 95 ℃ to inactivate the enzyme for 10 minutes, then cooling the enzymolysis liquid to 40 ℃, and then filtering algae residues in the enzymolysis liquid (filtering the algae residues in the enzymolysis liquid by using a plate and frame filter);
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
the process of the decoloring treatment in the step (4) is as follows: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 1% of that of the supernatant, and the weight of the added diatomite is 1% of that of the supernatant; then decoloring for 60min at the temperature of 45 ℃ under the condition of stirring (the rotating speed of a stirrer is 40 rpm), and then carrying out plate-and-frame filtration;
the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 10 microns;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
The filtration in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
the aperture of the ceramic membrane filter element used by the ceramic microfiltration equipment in the step (5-1) is 50 nm;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
in the step (5-2), the membrane filter element used by the ultrafiltration device allows substances with molecular weight lower than 5000Da to pass through;
(5-3) nanofiltration: and (5) adding purified water with the volume being 1 time that of the small molecular chlorella protein peptide solution obtained in the step (5-2), and then performing nanofiltration to obtain the chlorella protein peptide solution for spray drying.
In the step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150 and 300Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration.
And (5) performing spray drying on the chlorella protein peptide solution by using a spray dryer, wherein the evaporation capacity of the spray drying is 2 t/h (ton/hour), the air inlet temperature is 175 ℃, and the air outlet temperature is 81 ℃.
According to detection, the protein extraction rate of the embodiment is 57.3%, and the protein peptide content below 1000Da is 94.2%.
Example 3
In this embodiment, the industrial production method for obtaining chlorella protein peptide by the acid-enzyme method comprises the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 8 times that of the chlorella powder, stirring at the speed of 40rpm, and swelling for 1 hour at the temperature of 45 ℃. The stainless steel tank used in the step (1) is provided with a stirring system and a circulating water system, wherein the stirring system is used for stirring materials in the stainless steel tank, and the circulating water system is used for keeping the internal temperature of the stainless steel tank at 45 ℃;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 2% of the dry weight of the chlorella feed liquid, and then hydrolyzing for 2.5 hours at the temperature of 45 ℃;
in the step (2), the weight percentage concentration of the added hydrochloric acid is 35 percent;
(3) enzymolysis: adjusting the pH value of chlorella feed liquid subjected to acid hydrolysis to 7.5 (adjusting with sodium hydroxide), sequentially adding cellulase, alkaline protease, papain and neutral protease at 50 ℃, performing enzymolysis for 4.5 hours (stirring the chlorella feed liquid at the rotation speed of 50rpm in the enzymolysis process), and filtering algae residues in the enzymolysis liquid after enzyme deactivation to obtain supernatant;
in the step (3), the addition amount of the cellulase is 2% of the dry weight of the chlorella feed liquid, the addition amount of the alkaline protease is 1% of the dry weight of the chlorella feed liquid, the addition amount of the papain is 3% of the dry weight of the chlorella feed liquid, and the addition amount of the neutral protease is 1% of the dry weight of the chlorella feed liquid;
the unit of the activity of the cellulase is 2 ten thousand U/g, the unit of the activity of the alkaline protease is 50 ten thousand U/g, the unit of the activity of the papain is 60 ten thousand U/g, and the unit of the activity of the neutral protease is 20 ten thousand U/g;
in the step (3), after the enzymolysis is finished, heating the enzymolysis liquid to 85 ℃ to inactivate the enzyme for 15 minutes, then cooling the enzymolysis liquid to 35 ℃, and then filtering algae residues in the enzymolysis liquid (filtering the algae residues in the enzymolysis liquid by using a plate and frame filter);
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
the process of the decoloring treatment in the step (4) is as follows: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 1% of that of the supernatant, and the weight of the added diatomite is 1% of that of the supernatant; then decoloring for 40min at the temperature of 60 ℃ under the condition of stirring (the rotating speed of a stirrer is 40 rpm), and then carrying out plate-and-frame filtration;
the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 2 microns;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
The filtration in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
the aperture of the ceramic membrane filter element used by the ceramic microfiltration equipment in the step (5-1) is 30 nm;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
in the step (5-2), the membrane filter element used by the ultrafiltration device allows substances with the molecular weight of less than 3000Da to pass through;
(5-3) nanofiltration: and (5) adding purified water with the volume of 2.5 times that of the small molecular chlorella protein peptide solution obtained in the step (5-2), and performing nanofiltration to obtain a chlorella protein peptide solution for spray drying.
In the step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150 and 300Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration.
And (5) performing spray drying on the chlorella protein peptide solution by using a spray dryer, wherein the evaporation capacity of the spray drying is 2 t/h (ton/hour), the air inlet temperature is 172 ℃, and the air outlet temperature is 79 ℃.
According to detection, the protein extraction rate of the embodiment is 60.6%, and the protein peptide content below 1000Da is 94.5%.
Example 4
In this embodiment, the industrial production method for obtaining chlorella protein peptide by the acid-enzyme method comprises the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 6 times that of the chlorella powder, stirring at the speed of 40rpm, and swelling for 3 hours at the temperature of 40 ℃. The stainless steel tank used in the step (1) is provided with a stirring system and a circulating water system, wherein the stirring system is used for stirring materials in the stainless steel tank, and the circulating water system is used for keeping the internal temperature of the stainless steel tank at 40 ℃;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 2% of the dry weight of the chlorella feed liquid, and then hydrolyzing for 2 hours at the temperature of 45 ℃;
in the step (2), the weight percentage concentration of the added hydrochloric acid is 37 percent;
(3) enzymolysis: adjusting the pH value of chlorella feed liquid subjected to acid hydrolysis to 8 (adjusting by using sodium hydroxide), sequentially adding cellulase, alkaline protease, papain and neutral protease at the temperature of 55 ℃, performing enzymolysis for 5 hours (stirring the chlorella feed liquid at the rotating speed of 40rpm in the enzymolysis process), and filtering algae residues in the enzymolysis liquid after enzyme deactivation to obtain a supernatant;
in the step (3), the addition amount of the cellulase is 1.5 percent of the dry weight of the chlorella feed liquid, the addition amount of the alkaline protease is 3 percent of the dry weight of the chlorella feed liquid, the addition amount of the papain is 0.5 percent of the dry weight of the chlorella feed liquid, and the addition amount of the neutral protease is 3 percent of the dry weight of the chlorella feed liquid;
the unit of the activity of the cellulase is 2 ten thousand U/g, the unit of the activity of the alkaline protease is 50 ten thousand U/g, the unit of the activity of the papain is 60 ten thousand U/g, and the unit of the activity of the neutral protease is 20 ten thousand U/g;
in the step (3), after the enzymolysis is finished, heating the enzymolysis liquid to 90 ℃ to inactivate the enzyme for 20 minutes, then cooling the enzymolysis liquid to 35 ℃, and then filtering algae residues in the enzymolysis liquid (filtering the algae residues in the enzymolysis liquid by using a plate and frame filter);
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
the process of the decoloring treatment in the step (4) is as follows: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 1% of that of the supernatant, and the weight of the added diatomite is 1% of that of the supernatant; then decoloring for 60min at 50 ℃ under the condition of stirring (the rotating speed of the stirrer is 30 rpm), and then carrying out plate-and-frame filtration;
the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 5 microns;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
The filtration in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
the aperture of the ceramic membrane filter element used by the ceramic microfiltration equipment in the step (5-1) is 50 nm;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
in the step (5-2), the membrane filter element used by the ultrafiltration device allows substances with molecular weight lower than 10000Da to pass through;
(5-3) nanofiltration: and (5) adding 2 times of volume of purified water into the small-molecular chlorella protein peptide solution obtained in the step (5-2), and performing nanofiltration to obtain a chlorella protein peptide solution for spray drying.
In the step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150 and 300Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration.
And (5) performing spray drying on the chlorella protein peptide solution by using a spray dryer, wherein the evaporation capacity of the spray drying is 2 t/h (ton/hour), the air inlet temperature is 180 ℃, and the air outlet temperature is 75 ℃.
According to detection, the protein extraction rate of the embodiment is 61.4%, and the protein peptide content below 1000Da is 95.1%.
The antioxidant activity of the chlorella protein peptides of the above examples 1-4 was measured, and VC (vitamin C) was used as a positive control, and the measurement results showed that:
the half-clearing activity IC50 value of the positive control VC on ABTS free radicals is 4.42 mug/m L; the IC50 value of the positive control Vc on DPPH free radical was 4.58 μ g/m L.
The half-scavenging activity IC50 value of the chlorella protein peptide of example 1 on ABTS free radicals is 3.97 mug/m; the IC50 value for the Chlorella vulgaris peptide of example 1 on DPPH free radicals was 4.35 μ g/m L.
The half-scavenging activity IC50 value of the chlorella protein peptide of example 2 on ABTS free radicals is 3.92 mug/m; the IC50 value for the Chlorella vulgaris peptide of example 2 on DPPH free radicals was 4.47 μ g/m L.
The half-scavenging activity IC50 value of the chlorella protein peptide of example 3 on ABTS free radicals is 3.72 mug/m; the IC50 value for DPPH free radical for Chlorella proteinpeptide of example 3 was 4.77 μ g/m L.
The half-scavenging activity IC50 value of the chlorella protein peptide of example 4 on ABTS free radicals is 4.72 mug/m; the IC50 value for DPPH free radical for Chlorella proteinpeptide of example 4 was 4.40 μ g/m L.
From the above measurement results, it can be seen that the chlorella protein peptide obtained by the present invention has antioxidant activity close to that of VC (vitamin C).

Claims (10)

1. An industrial production method for obtaining chlorella protein peptide by an acid-enzyme method is characterized by comprising the following steps:
(1) swelling chlorella powder to obtain chlorella feed liquid;
(2) acid hydrolysis: adding hydrochloric acid into the chlorella feed liquid obtained after swelling in the step (1), wherein the addition amount of the hydrochloric acid is 0.5-2% of the dry weight of the chlorella feed liquid, and then hydrolyzing for 1-3 hours at the temperature of 35-45 ℃;
(3) enzymolysis: adjusting pH of Chlorella feed liquid subjected to acid hydrolysis to 7-9, sequentially adding cellulase, alkaline protease, papain and neutral protease at 45-60 deg.C, performing enzymolysis for 4-8 hr, inactivating enzyme, and filtering to remove algae residue to obtain supernatant;
(4) carrying out decoloration treatment on the supernatant to obtain decolored liquid;
(5) filtering the decolorized solution obtained in the step (4) to obtain chlorella protein peptide solution, and spray drying the chlorella protein peptide solution to obtain chlorella protein peptide.
2. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: in the step (1), the method for swelling the chlorella powder comprises the following steps: pumping the chlorella powder into a stainless steel tank by using a vacuum pump, adding purified water with the weight 4-8 times of that of the chlorella powder, stirring at the speed of 30-50rpm, and swelling for 1-3 hours at the temperature of 35-45 ℃.
3. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: in the step (2), the weight percentage concentration of the added hydrochloric acid is 30-38%.
4. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: in the step (3), the addition amount of the cellulase is 1-2% of the dry weight of the chlorella feed liquid, the addition amount of the alkaline protease is 1-3% of the dry weight of the chlorella feed liquid, the addition amount of the papain is 0.5-3% of the dry weight of the chlorella feed liquid, and the addition amount of the neutral protease is 1-3% of the dry weight of the chlorella feed liquid.
5. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1 or 4, wherein the method comprises the steps of: in the step (3), the unit of the activity of the cellulase is 1-3 ten thousand U/g, the unit of the activity of the alkaline protease is 30-80 ten thousand U/g, the unit of the activity of the papain is 50-80 ten thousand U/g, and the unit of the activity of the neutral protease is 10-30 ten thousand U/g.
6. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: in the step (3), in the enzymolysis process, the chlorella feed liquid is stirred at the rotating speed of 30-50 rpm.
7. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: in the step (3), after the enzymolysis is finished, the enzymolysis liquid is heated to 80-95 ℃ to inactivate the enzyme for 10-20 minutes, then the enzymolysis liquid is cooled to 30-40 ℃, and then the algae residues in the enzymolysis liquid are filtered.
8. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: the process of the decoloring treatment in the step (4) is as follows: pumping the supernatant obtained in the step (3) into a decoloring tank, and adding activated carbon and diatomite, wherein the weight of the added activated carbon is 1-3% of that of the supernatant, and the weight of the added diatomite is 1-2% of that of the supernatant; then decoloring for 40-60min at 45-60 ℃ under the condition of stirring, and then performing plate-and-frame filtration;
the activated carbon is plant activated carbon, wherein the proportion of the activated carbon with the particle size of 300-800 meshes is more than or equal to 90 percent; the filter cloth used for plate-frame filtration is made of nylon material, and the aperture is 1-10 microns.
9. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: the filtration in the step (5) comprises the following steps:
(5-1) microfiltration: adding the decolorized solution obtained in the step (4) into a stainless steel storage tank, and filtering by using tubular ceramic microfiltration equipment;
(5-2) ultrafiltration: ultrafiltering the permeate obtained by the filtering treatment in the step (5-1) by using an ultrafiltration device to obtain a micromolecule chlorella protein peptide solution;
(5-3) nanofiltration: adding purified water with the volume of 1-3 times that of the small molecular chlorella protein peptide solution obtained in the step (5-2), and performing nanofiltration to obtain a chlorella protein peptide solution for spray drying;
the aperture of the ceramic membrane filter element used by the ceramic microfiltration equipment in the step (5-1) is 30-50 nm;
in the step (5-2), the membrane filter element used by the ultrafiltration device allows substances with molecular weight lower than 3000-10000Da to pass through;
in the step (5-3), the membrane filtration filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150-.
10. The industrial production method for obtaining chlorella protein peptide by the acid-enzyme method according to claim 1, wherein the method comprises the steps of: and (5) performing spray drying on the chlorella protein peptide solution by using a spray dryer, wherein the evaporation capacity of the spray drying is 2 tons/h, the air inlet temperature is 150-185 ℃, and the air outlet temperature is 60-85 ℃.
CN202011197302.4A 2020-10-31 2020-10-31 Industrial production method for obtaining chlorella protein peptide by acid-enzyme method Pending CN112266944A (en)

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