CN111154824A - Industrial production method for obtaining spirulina antioxidant oligopeptide through high-concentration two-step enzymolysis - Google Patents
Industrial production method for obtaining spirulina antioxidant oligopeptide through high-concentration two-step enzymolysis Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
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Abstract
The invention relates to the technical field of biotechnology, in particular to an industrial production method for obtaining spirulina antioxidant oligopeptide by high-concentration two-step enzymolysis, and the produced product is easy to digest and absorb, has good water solubility and stability and antioxidant activity, can expand the application range of spirulina in health food and medicine industries and improve the functional efficacy of spirulina; the method comprises the following steps: s1, swelling spirulina powder; s2, hydraulic cavitation wall breaking; s3, enzymolysis; s4, centrifuging; s5, decoloring; s6, plate-frame filtration, the spirulina protein peptide solution obtained by the invention has the characteristics of easy digestion and absorption, oligopeptide as a main component, high activity, good instant solubility, no fishy smell, good taste, easy dissolution and easy absorption, has stronger antioxidant activity, is suitable for industrial mass production, is beneficial to the development of the spirulina industry, and expands the application range of the spirulina in the health food and medicine industry.
Description
Technical Field
The invention relates to the technical field of biotechnology, in particular to an industrial production method for obtaining spirulina antioxidant oligopeptide through high-concentration two-step enzymolysis.
Background
Spirulina (Spirulina) belongs to the phylum of Cyanophyta, the family Oscillatoriaceae, and is a kind of spiral prokaryotic algae composed of single cells or multiple cells, and has 35 million years of ancient biology on the earth. The spirulina contains 60-70% of protein, and also contains abundant mineral substances such as VA, VB, selenium, zinc and the like. Spirulina was processed into a blue biscuit by inhabitants in africa, america, mexico, etc. since the 15 th century. In the 60 s of the 20 th century, the international biologists paid attention to the fact that the nutrient has high nutritional value; in the early 70 s of the 20 th century, spirulina was recognized as the "best food resource in tomorrow" by the food conference of the united nations; and also recommended by the Food and Agriculture Organization (FAO) of the United nations in turn: "21 st century optimal food"; recommended by the United nations scientific and educational organization: "tomorrow optimal and perfect food"; reputed by the world food association of the united nations: "the most ideal nutrient source in the 21 st century"; determined by the World Health Organization (WHO): "best health products for human 21 st century"; the second international conference on microbial proteins identified in 1972: "future super nutritious food"; in 1974, the method is accepted by food conference in the united nations: super nutrient food. In the early 70 s, the Ministry of agriculture and the national Commission of science in China put spirulina into the items of 'seven five', 'eight five' attack and popularization. Because of its high safety, spirulina is not affected by eating so far, and can be popularized and eaten in large quantities for a long time without any toxic and side effect in human body, and the spirulina is listed as common food management in 8 months of 2004 (No. 17 bulletin of 2004 by ministry of health) by ministry of health in China.
A large number of researches show that the spirulina has the effects of scavenging free radicals, resisting oxidation and resisting inflammation; supplementing high-quality protein, vitamins and minerals, relieving fatigue, promoting differentiation and proliferation of hematopoietic progenitor cells, and promoting protein synthesis; enhancing immunity, and promoting immune system function; has anti-tumor effect, and can inhibit growth of tumor cells and induce apoptosis of tumor cells; nerve tissue and DNA can be protected from being damaged; can improve the radiation resistance of organisms and the like, and is widely applied to the development of health-care food and medicines. At present, spirulina powder, tablets and capsules are mainly used in the market, and due to the poor solubility, strong fishy smell and poor protein stability of dry powder, precipitation and denaturation are easy to occur, so that the loss of biological activity is caused, and meanwhile, the further development of spirulina in the fields of food health care and medicine is limited due to the fact that the protein has large molecular weight and is not easy to digest and absorb by human bodies. The spirulina selects proper enzyme to hydrolyze to obtain small molecular peptide, which not only greatly improves the solubility and improves the taste, but also obtains the functional peptides such as antioxidant active peptide, immunoregulation active peptide, antitumor active peptide, antihypertensive active peptide, anti-radiation active peptide and the like, and simultaneously the small molecular active peptide can be directly and rapidly absorbed, thereby improving the digestion, absorption and utilization rate. Currently, the utilization of spirulina is mainly focused on spirulina powder or tablets, but research and application of spirulina active peptides are less, and industrial utilization of spirulina active peptides is less. At present, the research and application of spirulina protein peptide are mainly as follows: a double enzymolysis preparation method of spirulina antitumor polypeptide with publication number of CN 104561207A; a preparation method of novel spirulina powder with publication number of CN108753874A for small molecule active peptide; the invention researches and develops an industrial production method for obtaining spirulina antioxidant oligopeptide by high-concentration two-step enzymolysis.
Disclosure of Invention
In order to solve the technical problems, the invention provides an industrial production method for obtaining the spirulina antioxidant oligopeptide by high-concentration two-step enzymolysis, which is easy to digest and absorb, good in water solubility and stability, has antioxidant activity, expands the application range of spirulina in health food and medicine industries and improves the functional efficacy of the spirulina.
The invention relates to an industrial production method for obtaining spirulina antioxidant oligopeptide by high-concentration two-step enzymolysis, which comprises the following steps:
s1, swelling spirulina powder: pumping the spirulina powder into a stainless steel tank by using a vacuum pump, adding 80-65% (w/v) of purified water, stirring at the speed of 30-50 rpm/min, and swelling for 6-9 hours at the temperature of 20-25 ℃;
s2, hydraulic cavitation wall breaking: carrying out cavitation wall breaking on the swollen spirulina liquid by using a hydraulic cavitator, wherein the rotating speed of the hydraulic cavitator is 1200-600 rad/min, the inlet pressure is 0.4-0.6 MPa, and the outlet pressure is 0.1MPa, and treating for 2-3 times;
s3, enzymolysis: adding the spirulina liquid subjected to hydrodynamic cavitation wall breaking into an enzyme reaction kettle, adjusting the pH to 9.2-9.5 by using sodium hydroxide, adding 2-4% of alkaline protease by weight of the spirulina, and performing enzymolysis for 9-12 hours at the temperature of 47-52 ℃; adjusting the pH value to 6.4-6.8, respectively adding papain accounting for 1-3% of the weight of the spirulina and flavourzyme accounting for 1-3% of the weight of the spirulina, carrying out enzymolysis for 9-12 h at the temperature of 55-60 ℃, and finally heating the enzymolysis liquid to 80 ℃ for carrying out enzyme deactivation treatment for 20-30 min;
s4, centrifugation: centrifuging the enzymolysis liquid by using a disc centrifuge at 4500-6000 rpm to obtain a supernatant;
s5, decoloring treatment: pumping the supernatant into a decoloring tank, adding 1-3% of decoloring active carbon and 2% of diatomite, stirring at a rotating speed of 30-50 rpm, and decoloring at 50 ℃ for 40-60 min;
s6, plate and frame filtration: filtering the decolorized solution with plate frame to obtain golden yellow or orange red bright spirulina protein peptide solution.
Further, the stainless steel tank used in S1 is provided with a stirring system and a circulating water system, and the circulating water system controls the temperature of the feed liquid to be 20-28 ℃.
Further, the hydrodynamic cavitation device in the S2 includes a cavitator, an air supply device, and a hydrodynamic cavitation apparatus connected to the cavitator, the cavitator is a core component of the system, and includes a rotating shaft, a box, a motor, and a sealing ring, and the rotating shaft includes a central cavity, a venturi channel, and a wake vortex cavitation flange.
Further, in the step S3, the activity unit of the alkaline protease is 30-80 ten thousand units, the activity unit of the papain is 50-80 ten thousand units, and the activity unit of the neutral protease is 10-30 ten thousand units.
Further, the rotation speed of the butterfly centrifuge used in the S4 is 4000-8000 rpm, and the power is 16 kw.
Further, the decolorizing active carbon in S5 is plant active carbon, and the content of particles with the particle size of 300-800 meshes is more than or equal to 90%.
Furthermore, the filter cloth used for filtering the slab frame in S6 is made of nylon material, and the aperture of the filter cloth is 1-10 μm.
Compared with the prior art, the invention has the beneficial effects that: the spirulina protein peptide solution is obtained by the operations of swelling, hydrodynamic cavitation, enzymolysis, centrifugation, decoloration, plate-frame filtration and the like on spirulina powder. At present, methods such as an acid-base method, high-pressure homogenization, repeated freeze thawing and the like are mostly adopted for wall breaking treatment of spirulina, the acid-base method is high in efficiency, but acid and base have destructive effects on small peptides and amino acids and influence the quality of spirulina protein peptides; the technology applies the hydrodynamic cavitation technology for sewage treatment to the wall breaking of the spirulina, has high efficiency and low energy consumption, avoids the wall breaking of the spirulina by acid and alkali treatment, reduces the damage of acid and alkali to the spirulina peptide amino acid, does not generate harmful substances, retains nutrient substances, improves the functional efficacy of the spirulina, generates less bitter substances, and simultaneously, the taste is greatly improved by flavor protease and activated carbon decoloration. The substrate concentration adopted by the existing spirulina enzymolysis technology is more than 3-8%, the technology adopted by the invention can enable the spirulina concentration to reach 20-35%, the enzymolysis with high substrate concentration is favorable for improving the protein peptide content of the spirulina, improving the utilization rate of enzyme, being favorable for concentrating protein peptide, saving energy, improving efficiency and simultaneously being favorable for obtaining antioxidant peptide with higher activity; the technology adopts alkaline protease for enzymolysis, and adds papain and flavourzyme after reaching the proper pH value of the papain and the flavourzyme, thereby avoiding the use of a large amount of acid and alkali and reducing the introduction of foreign substances.
The spirulina protein peptide solution obtained by the invention has the characteristics of easy digestion and absorption, oligopeptide main component, high activity, good instant solubility, no fishy smell, good taste, easy dissolution and easy absorption, has stronger antioxidant activity, is suitable for industrial mass production, is beneficial to the development of the spirulina industry, and expands the application range of the spirulina in the health food and medicine industry.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Protein peptide extraction rate (%) (total protein content in the enzymatic supernatant/total protein content in the spirulina) × 100;
the method for measuring the molecular weight distribution of the protein peptide is characterized in that the high-efficiency size exclusion chromatography is adopted to measure the collagen peptide according to the national standard of GB 31645-2018 food safety;
determination of antioxidant Activity: ABTS free radical scavenging activity and DPPH free radical scavenging ability were measured by the method of Zengqiao Ph thesis (Zengqiao 2016).
Example 1
Swelling spirulina powder: adding 20kg of spirulina powder into a stainless steel temporary storage tank, adding 80L of purified water, stirring at 30rpm/min, and swelling at 20 deg.C for 8 h;
hydraulic cavitation wall breaking: carrying out air-talk wall breaking on the swollen spirulina liquid by using a hydraulic cavitator, and treating for 3 times at the rotating speed of the cavitator of 1200-1600 rad/min, the inlet pressure of 0.6MPa and the outlet pressure of 0.1 MPa;
enzymolysis: pumping the spirulina liquid subjected to hydrodynamic cavitation wall breaking into an enzyme reaction kettle, adjusting the pH to 9.3 by using sodium hydroxide, adding 0.75kg of alkaline protease in weight of the spirulina, carrying out enzymolysis for 12h at 48 ℃, and treating by using alkaline protease to obtain the spirulina liquid with the pH of 6.4; adding papain 0.5kg and flavourzyme 0.5kg respectively, performing enzymolysis at 58 deg.C for 12 hr, heating the enzymolysis solution to 80 deg.C, and inactivating enzyme for 20 min;
centrifuging: centrifuging the enzymolysis solution by using a disc centrifuge at the rotating speed of 6000rpm to obtain supernatant;
and (3) decoloring treatment: pumping the supernatant into a decolorizing tank, adding 1.6kg of activated carbon and 1.6kg of diatomite, and decolorizing at 50 deg.C for 40min under stirring at 50 rpm;
plate-frame filtration: filtering the decolorized solution with plate frame to obtain golden yellow clear spirulina protein peptide solution.
The detection shows that the protein extraction rate is 58.2 percent, and the content of protein peptide below 3000Da is 84.7 percent. The result of taking VC as a positive control shows that: the positive control Vc and the half-clearing activity IC50 value of the spirulina protein peptide on ABTS free radical is 4.36 mu g/mL and 3.89 mu g/m; the positive controls Vc and Spirulina protein peptide had IC50 values of 4.46. mu.g/mL and 4.27. mu.g/mL for DPPH free radical.
Example 2
Swelling spirulina powder: pumping 250kg of spirulina powder into a stainless steel tank by using a vacuum pump, adding 800L of purified water, stirring at the speed of 30rpm/min, and swelling for 7h at 20 ℃;
hydraulic cavitation wall breaking: performing air-talk wall breaking on the swollen spirulina liquid by using a hydraulic cavitator, and treating for 3 times at the rotating speed of the cavitator of 1600rad/min, the inlet pressure of 0.6MPa and the outlet pressure of 0.1 MPa;
enzymolysis: pumping the spirulina liquid subjected to hydrodynamic cavitation wall breaking into an enzyme reaction kettle, adjusting the pH to 9.4 by using sodium hydroxide, adding 7.5kg of alkaline protease in weight of the spirulina, performing enzymolysis for 10h at 48 ℃, and treating by using alkaline protease to obtain the spirulina liquid with the pH of 6.5; adding papain 7.5kg and flavourzyme 7.5kg respectively, performing enzymolysis at 58 deg.C for 12h, heating the enzymolysis solution to 80 deg.C, and inactivating enzyme for 30 min;
centrifuging: centrifuging the enzymolysis solution at 6000rpm by using a disc centrifuge to obtain supernatant;
and (3) decoloring treatment: pumping the supernatant into a decolorizing tank, adding 16kg of activated carbon and 16kg of diatomite, and decolorizing at 50 ℃ for 60min under stirring at 30 rpm;
plate-frame filtration: filtering the decolorized solution with plate frame to obtain golden yellow clear spirulina protein peptide solution.
The detection shows that the protein extraction rate is 56.7 percent, and the content of protein peptide below 3000Da is 83.6 percent. The result of taking VC as a positive control shows that: the positive control Vc and the half-clearing activity IC50 value of the spirulina protein peptide on ABTS free radical is 4.32 mug/mL and 3.93 mug/m; the positive controls Vc and Spirulina protein peptide had IC50 values of 4.55. mu.g/mL and 4.16. mu.g/mL for DPPH free radical.
Example 3
Swelling spirulina powder: sucking 300kg of spirulina powder into a stainless steel tank by using a vacuum pump, adding 8000L of purified water, stirring at the speed of 30rpm/min, and swelling for 8h at 20 ℃;
hydraulic cavitation wall breaking: performing air-talk wall breaking on the swollen spirulina liquid by using a hydraulic cavitator, and treating for 3 times at the rotating speed of the cavitator of 1600rad/min, the inlet pressure of 0.6MPa and the outlet pressure of 0.1 MPa;
enzymolysis: pumping the spirulina liquid subjected to hydrodynamic cavitation wall breaking into an enzyme reaction kettle, adjusting the pH to 9.4 by using sodium hydroxide, adding 90kg of alkaline protease, performing enzymolysis at 50 ℃ for 12h, and treating by using alkaline protease to obtain the pH of 6.5; adding papain 90kg and flavourzyme 90kg respectively, performing enzymolysis at 58 deg.C for 12h, heating the enzymolysis solution to 80 deg.C, and inactivating enzyme for 30 min;
centrifuging: centrifuging the enzymolysis solution at 6000rpm by using a disc centrifuge to obtain supernatant;
and (3) decoloring treatment: pumping the supernatant into a decolorizing tank, adding 160kg of activated carbon and 160kg of diatomite, and decolorizing at 50 ℃ for 60min under stirring at 30 rpm;
plate-frame filtration: filtering the decolorized solution with plate-frame filter to obtain orange red bright spirulina protein peptide solution.
The detection shows that the protein extraction rate is 57.3%, the protein peptide content below 3000Da is 86.6%, and VC is used as a positive control result to show that: the positive control Vc and the half-clearing activity IC50 value of the spirulina protein peptide on ABTS free radical is 4.57 mu g/mL and 3.83 mu g/m; the positive controls Vc and Spirulina protein peptide had IC50 values of 4.69. mu.g/mL and 4.35. mu.g/mL for DPPH free radical.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. An industrial production method for obtaining spirulina antioxidant oligopeptide by high-concentration two-step enzymolysis is characterized by comprising the following steps:
s1, swelling spirulina powder: pumping the spirulina powder into a stainless steel tank by using a vacuum pump, adding 80-65% (w/v) of purified water, stirring at the speed of 30-50 rpm/min, and swelling for 6-9 hours at the temperature of 20-25 ℃;
s2, hydraulic cavitation wall breaking: carrying out cavitation wall breaking on the swollen spirulina liquid by using a hydraulic cavitator, wherein the rotating speed of the hydraulic cavitator is 1200-600 rad/min, the inlet pressure is 0.4-0.6 MPa, and the outlet pressure is 0.1MPa, and treating for 2-3 times;
s3, enzymolysis: adding the spirulina liquid subjected to hydrodynamic cavitation wall breaking into an enzyme reaction kettle, adjusting the pH to 9.2-9.5 by using sodium hydroxide, adding 2-4% of alkaline protease by weight of the spirulina, and performing enzymolysis for 9-12 hours at the temperature of 47-52 ℃; adjusting the pH value to 6.4-6.8, respectively adding papain accounting for 1-3% of the weight of the spirulina and neutral protease accounting for 1-3% of the weight of the spirulina, carrying out enzymolysis for 9-12 h at the temperature of 55-60 ℃, and finally heating the enzymolysis liquid to 80 ℃ for enzyme deactivation treatment for 20-30 min;
s4, centrifugation: centrifuging the enzymolysis liquid by using a disc centrifuge at 4500-6000 rpm to obtain a supernatant;
s5, decoloring treatment: pumping the supernatant into a decoloring tank, adding 1-3% of decoloring active carbon and 2% of diatomite, stirring at a rotating speed of 30-50 rpm, and decoloring at 50 ℃ for 40-60 min;
s6, plate and frame filtration: filtering the decolorized solution with plate frame to obtain golden yellow or orange red bright spirulina protein peptide solution.
2. The industrial production method of spirulina oxidation resistant oligopeptide with high concentration by two-step enzymolysis according to claim 1, wherein the stainless steel tank used in S1 is provided with a stirring system and a circulating water system, and the circulating water system controls the temperature of the feed liquid to be 20-28 ℃.
3. The industrial production method of spirulina oxidation resistant oligopeptide by high concentration two-step enzymolysis as claimed in claim 1, wherein the hydrodynamic cavitator in S2 comprises a cavitator, an air supply device and a hydrodynamic cavitation device connected with the cavitator, the cavitator is a core component of the system and comprises a rotating shaft, a box body, a motor and a sealing ring, and the rotating shaft comprises a central cavity, a venturi tube channel and a wake vortex cavitation flange.
4. The industrial production method of spirulina oxidation resistant oligopeptide by high concentration two-step enzymolysis according to claim 1, wherein the activity unit of alkaline protease in S3 is 30-80 ten thousand units, the activity unit of papain is 50-80 ten thousand units, and the activity unit of neutral protease is 10-30 ten thousand units.
5. The industrial production method of spirulina oxidation resistant oligopeptide with high concentration by two-step enzymolysis according to claim 1, wherein the rotation speed of a butterfly centrifuge used in S4 is 4000-8000 rpm, and the power is 16 kw.
6. The industrial production method of spirulina antioxidant oligopeptide with high concentration through two-step enzymolysis according to claim 1, wherein the decolorizing active carbon in S5 is plant active carbon, and the content of particles with the particle size of 300-800 meshes is more than or equal to 90%.
7. The industrial production method of spirulina oxidation resistant oligopeptide with high concentration by two-step enzymolysis according to claim 1, wherein the filter cloth used for filtering the plate frame in S6 is made of nylon material and has a pore size of 1-10 μm.
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