CN109207544B - Preparation method of chlorella antioxidant polypeptide - Google Patents
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Abstract
The invention provides a preparation method of chlorella antioxidant polypeptide, which is characterized in that a high-pressure homogenization method is combined with cellulase/pectinase for enzymolysis and extraction of chlorella protein, and after enzymolysis of specific protease hydrolysate, the chlorella protein is separated by an ultrafiltration membrane to obtain chlorella enzymolysis peptide with different molecular weight regions. The chlorella polypeptide with the molecular weight range of 0-3.5KDa after being subjected to enzymolysis by the specific protease enzymolysis liquid has better ability of clearing DPPH and hydroxyl free radicals in vitro and clearing active oxygen clusters of liver cancer cells in vitro than other protease enzymolysis products and chlorella polypeptides in other molecular weight regions, and more effective inhibiting ability of the formation of a peroxidation product malondialdehyde in animal bodies and glutathione peroxidase activity promoting ability. The invention depends on the specific protease enzymolysis liquid to prepare the chlorella bioactive peptide with better antioxidant function, which is beneficial to the development and utilization of health-care food and medical products with antioxidant function.
Description
Technical Field
The invention provides a preparation method of chlorella antioxidant polypeptide, belonging to the field of biotechnology.
Background
Chlorella (Chlorella) is a universal unicellular green algae, is widely distributed in nature, can be grown and propagated by using organic carbon sources under autotrophic and heterotrophic conditions, has large biomass, and is the only organism on the earth which can grow 4 times in 20 hours. The chlorella is extremely rich in nutrition, contains more than 50% of crude protein, basically balanced essential amino acid composition, contains unsaturated fatty acid, bioactive polysaccharide, nucleic acid, vitamins, trace elements, mineral substances, chlorophyll and the like, has extremely high nutritional value, and has various biological pharmacological activities of preventing and treating peptic ulcer, resisting tumors, enhancing immunity, resisting radiation, resisting pathogenic microorganisms, preventing and treating anemia, reducing blood fat, resisting atherosclerosis and the like. Therefore, besides being widely applied to aquaculture, chlorella has been developed into foods, beverages, medical health products and the like in many countries and regions.
At present, the production of chlorella health products takes wall-broken dry powder as a raw material. The chlorella dry powder obtained by wall breaking, concentration and extraction is a mixture composed of nucleoprotein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), a plurality of vitamins, amino acids, polysaccharide, complex protein bodies, ferment, glycoprotein, a plurality of plant hormones and the like. Since Chlorella has such an effective health-care function, the dry powder mixture is also called Chlorella Growth Factor (CGF). Meanwhile, many researches suggest that the dry powder also contains 'Moming factor' with unique biological activity and physiological function. However, no relevant research has been available to demonstrate what components constitute the main substances exerting high biological activity in chlorella, and what respective biological activities and functions different components have. Obviously, the shortages of the research seriously restrict the further development of the medical value of the chlorella, in particular the development of high-valued chlorella health care products.
Proteins are the main players of life activities, and are closely related to life and various forms of life activities, and the movement of life cannot be separated from proteins. It is believed that nucleic acids, vitamins, trace elements, minerals, chlorophyll, etc. in chlorella do not differ substantially from similar nutrients from other sources. The content of protein in the chlorella reaches 50% of dry weight, the health care function of the chlorella is necessarily closely related to the protein and polypeptide contained in the chlorella, and a large part of the activity function of the chlorella is derived from the protein and polypeptide components contained in the chlorella.
The bioactive peptide is an important active substance with the functions of resisting oxidation, resisting fatigue, assisting in reducing blood pressure, regulating immunity, resisting tumors, resisting bacteria, reducing blood sugar and the like, and usually contains 3 to 20 amino acid residues. Short peptide chains of these amino acids are inactive within the sequence of the parent protein, but are released by gastrointestinal digestion, food processing or fermentation. The bioactive peptide obtained by hydrolyzing food protein has high safety and easy absorption. The biological activity of the bioactive peptide is closely related to the structural characteristics, amino acid sequence, relative molecular mass, amino acid side chain group and the like of the self peptide chain. After the food protein from the same source is subjected to enzymolysis by different proteases, polypeptides with different peptide chain structures, amino acid sequences, molecular weights and side chain groups can be obtained due to different protease recognition sites. Many domestic and foreign studies have confirmed that bioactive peptides obtained by subjecting proteins from the same source to enzymolysis by different proteases often have different biological activities, and proteins from different sources often have the same biological activities after being subjected to enzymolysis by the same protease, such as: different polypeptides obtained by carrying out enzymolysis on the same milk protein by using different microbial enzymes or trypsin, pepsin and the like respectively have different biological activities of reducing blood pressure, enhancing immunity, resisting oxidation and the like; the polypeptides with different molecular weight regions and amino acid sequences obtained by enzymolysis of proteins from different foods (milk, soybean, fish, microalgae and the like) by pepsin usually have obvious health-care effect of enhancing immunity. Therefore, it is necessary to screen the most suitable kind of protease for digesting specific food proteins according to the different biological activities we need to obtain.
The antioxidant peptide is a peptide with strong functions of inhibiting biological macromolecule peroxidation and eliminating free radicals in vivo. The action mechanism of the antioxidant peptide is directly acting on free radicals or indirectly consuming substances which are easy to generate the free radicals, so that further reaction is prevented. At present, many studies report that various animal and plant-derived protein hydrolysates have antioxidant activity. Although related researches have been carried out on chlorella protein serving as a raw material, antioxidant polypeptides are obtained after enzymolysis by using different proteases, such as: wang is equal to 2017, extracting chlorella protein by using an alkaline method, and carrying out enzymolysis by using flavourzyme to obtain enzymolysis polypeptide with antioxidant activity; han et al isolated a tripeptide with antioxidant activity from Chlorella vulgaris in 2015; the Wangchao Run is equal to 2014, chlorella protein is used as a raw material, and different antioxidant polypeptides are obtained through acid protease enzymolysis, separation and purification; sheih, et al 2009, used pepsin to enzymolyze chlorella and separated and purified to obtain 11 peptide with antioxidant activity. However, the antioxidant effect of the antioxidant polypeptides obtained by the research is mostly based on in vitro and cell level detection, the research rarely supports the in vivo animal experiment results, and meanwhile, it is difficult to determine which enzymolysis products among different enzymolysis polypeptides have the optimal antioxidant polypeptides.
The research further proves that polypeptide products obtained by enzymolysis of different proteases in microalgae source protein have antioxidant activity of different degrees. Alzahrani is equal to 2017, 3 microalgal proteins are extracted by an ultrasonic method, and further subjected to independent enzymolysis by alkaline protease, flavourzyme and trypsin respectively, and the antioxidant effects of enzymolysis products are compared, so that the enzymolysis polypeptides have certain antioxidant activity, but only the polypeptides hydrolyzed by the alkaline protease have the optimal antioxidant activity. In view of this, modern biotechnology is utilized to carry out high-value development on chlorella, which is a high-quality microalgae protein resource, and by selecting the optimal protease combination and optimizing the formula of enzymolysis liquid and the enzymolysis process, chlorella antioxidant polypeptide with good antioxidant effect can be expected to be obtained and used for further developing food, health care and medical products related to the antioxidant polypeptide.
Disclosure of Invention
The invention provides a preparation method of chlorella antioxidant polypeptide, which can obtain chlorella antioxidant polypeptide with good antioxidant effect in vitro, cells and animal bodies, thereby better expanding the application of chlorella in the fields of food, health care and medicine.
The invention adopts the following technical scheme:
1. preparation of chlorella cell wall enzymolysis liquid and specific protein enzymolysis liquid
1) The chlorella cell wall enzymolysis liquid comprises the following components in percentage by weight:
after the chlorella cell wall enzymolysis liquid is prepared, concentrated hydrochloric acid with the mass fraction of 36% is used for adjusting the pH value to 3.0-5.0;
2) the specific protein enzymolysis liquid comprises the following components in percentage by weight:
after the specific protease hydrolysate is prepared, adjusting the pH to 7.5-9.5 by using 1mol/L NaOH solution;
2. the chlorella antioxidant polypeptide is prepared by the following steps:
1) adding chlorella powder into 0.2-0.3 mol/L NaOH dilute alkali solution according to the mass ratio of the chlorella powder to the NaOH dilute alkali solution of 1: 7-10 by taking protein core chlorella powder as a raw material, and soaking for 60min at the temperature of 60 ℃ to obtain pretreated chlorella liquid;
2) homogenizing the pretreated chlorella solution under high pressure for 2-4 times under the pressure of 30-60 MPa, centrifuging for 20min at 5000rpm, temporarily storing the obtained supernatant A at 4 ℃, adding the obtained precipitate into chlorella cell wall enzymolysis liquid for enzymolysis, wherein the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 3-7 h, the stirring speed is 150rpm, and centrifuging for 20min at 5000rpm after enzymolysis to obtain a supernatant B;
3) mixing the supernatant A and the supernatant B, performing single-effect energy-saving concentration under the normal pressure condition until the volume ratio before and after concentration reaches 3-6: 1, and performing vacuum freeze drying on the concentrated solution to obtain chlorella protein powder;
4) adding chlorella protein powder into specific protein enzymolysis liquid for enzymolysis, carrying out enzymolysis at 40-70 ℃, carrying out enzyme deactivation treatment in 90 ℃ water for 30min after carrying out enzymolysis for 30-60 min, rapidly cooling to room temperature, and centrifuging at 4000rpm for 20min to obtain supernatant C;
5) and (3) performing ultrafiltration separation on the supernatant C through ultrafiltration membranes with molecular weight cut-offs of 10KDa, 5KDa and 3.5KDa respectively under the conditions of membrane inlet pressure of 1MPa and dialysis flow of 40mL/min to obtain chlorella antioxidant polypeptide liquids with molecular weight ranges of more than 10KDa, 5-10kDa, 3.5-5kDa and 0-3.5kDa respectively, collecting chlorella antioxidant polypeptide liquids with different molecular weights, and performing vacuum freeze drying respectively to obtain powdered chlorella antioxidant polypeptides with different molecular weights. The antioxidant activity of the chlorella antioxidant polypeptide liquid with 0-3.5kDa is strongest.
The vacuum freeze drying is carried out under the conditions as follows: freezing at-40 deg.C for 30min and holding time for 120 min; setting the drying temperature of-20 ℃, the drying time of 30min, the holding time of 120min and the vacuum of 0.25 in the first stage of drying; in stage 2, drying temperature is-15 deg.C, drying time is 30min, holding time is 120min, and vacuum is 0.25; in stage 3, the drying temperature is-5 ℃, the drying time is 30min, the holding time is 500min, and the vacuum is 0.25; in the 4 th stage, the drying temperature is 0 ℃, the drying time is 30min, the holding time is 1500min, and the vacuum is 0.3; in the 5 th stage, the drying temperature is 5 ℃, the drying time is 30min, the holding time is 120min, and the vacuum is 0.3; in the 6 th stage, the drying temperature is 10 ℃, the drying time is 30min, the holding time is 120min, and the vacuum is 0.3; setting the temperature of the analysis and drying at 20 ℃, the time of 30min, the holding time of 120min and the vacuum of 0.3.
Compared with the prior art, the invention has the following obvious advantages and better functional effects: the chlorella protein is taken as a starting point, and the extraction rate of the chlorella protein is effectively improved through the cellulase/pectinase cell wall enzymolysis liquid obtained through high-pressure homogenization combined optimization; the obtained chlorella protein is subjected to specific protein enzymolysis liquid, enzymolysis process control and ultrafiltration separation to obtain a chlorella polypeptide product with the optimal antioxidant activity and a specific molecular weight range (0-3.5 KDa). The chlorella antioxidant polypeptide obtained by the invention has the best antioxidant activity: (1) has better in vitro total reducing capability than chlorella enzymolysis products of other protease enzymolysis; (2) has better ability of eliminating DPPH and hydroxyl free radical in vitro, ability of eliminating active oxygen cluster (ROS) of liver cancer cells in vitro, and more effective ability of inhibiting formation of peroxidation product Malondialdehyde (MDA) and promoting activity of glutathione peroxidase (GSH-PX) in animal bodies than other polypeptide products with large molecular weight after enzymolysis of the same specific protease hydrolysate. Therefore, the chlorella antioxidant polypeptide obtained by the invention is more beneficial to further developing food, health care and medical products related to the antioxidant polypeptide.
Drawings
FIG. 1 is a graph showing the comparison of DPPH and hydroxyl radical scavenging capacity in vitro of polypeptides with different molecular weights obtained by hydrolyzing chlorella protein prepared in example 1 with a specific protease hydrolysate used in the present invention.
FIG. 2 is a diagram showing the effect of the chlorella antioxidant polypeptide with molecular weight of 0-3.5KDa in reducing active oxygen in liver cancer cells.
FIG. 3 is a graph showing the comparison of the MDA content in serum and the GSH-PX activity of old mice after gavage of Chlorella polypeptides with different molecular weights obtained by the present invention.
Detailed Description
The following embodiments and drawings are provided to further illustrate the embodiments of the present invention, and all equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the present invention.
Example 1
1. Preparation of chlorella cell wall enzymolysis liquid and specific protein enzymolysis liquid
The cellulase, pectinase and various proteases are purchased from Beijing Solebao scientific Co.
1) The chlorella cell wall enzymolysis liquid comprises the following components in percentage by weight:
the pH value of the chlorella cell wall enzymolysis liquid is adjusted to 4.0 by concentrated hydrochloric acid with the mass fraction of 36 percent after being prepared.
2) The specific protein enzymolysis liquid comprises the following components in percentage by weight:
after the specific protease hydrolysate is prepared, regulating the pH to 9.5 by using 1mol/L NaOH solution;
2. the chlorella antioxidant polypeptide is prepared by the following steps:
1) weighing 500g of chlorella powder, adding the chlorella powder into 4.5L of 0.3mol/L NaOH dilute alkali solution, and soaking for 60min at the temperature of 60 ℃ to obtain pretreated chlorella solution;
2) homogenizing the pretreated chlorella solution obtained in the step 1) under high pressure for 3 times under the pressure of 45MPa, centrifuging the homogenized chlorella solution at 5000rpm for 20min, temporarily storing the obtained supernatant A at 4 ℃, adding the obtained precipitate into chlorella cell wall enzymolysis solution, performing enzymolysis for 6h at 45 ℃ and the stirring speed of 150rpm, and centrifuging the treated chlorella solution at 5000rpm for 20min after enzymolysis to obtain a supernatant B;
3) mixing the supernatant A and the supernatant B obtained in the step 2), performing single-effect energy-saving concentration under normal pressure until the volume ratio before and after concentration reaches 5:1, and performing vacuum freeze drying on the concentrated solution to obtain chlorella protein powder;
4) adding the chlorella protein powder obtained in the step 3) into a specific protein enzymolysis liquid for enzymolysis, carrying out enzymolysis for 60min at 50 ℃, then carrying out enzyme deactivation treatment for 30min in water at 90 ℃, rapidly cooling to room temperature, and centrifuging for 20min at 4000rpm to obtain a supernatant;
5) and 4) performing ultrafiltration separation on the supernatant fluid under the conditions of 1MPa membrane-entering pressure and 40mL/min dialysis flow through ultrafiltration membranes with molecular weight cut-off of 10KDa, 5KDa and 3.5KDa respectively to obtain chlorella antioxidant polypeptide liquids with molecular weight ranges of more than 10KDa, 5-10kDa, 3.5-5kDa and 0-3.5kDa respectively, collecting chlorella antioxidant polypeptide liquids in different molecular weight regions, and performing vacuum freeze drying to obtain powdery chlorella antioxidant polypeptide.
The vacuum freeze-drying conditions in the above steps are as follows: freezing at-40 deg.C for 30min and holding time for 120 min; setting the temperature of-20 ℃ in the 1 st stage, the time of 30min, the holding time of 120min, the vacuum of 0.25, the temperature of-15 ℃ in the 2 nd stage, the time of 30min, the holding time of 120min, the vacuum of 0.25, the temperature of-5 ℃ in the 3 rd stage, the time of 30min, the holding time of 500min, the vacuum of 0.25, the temperature of 0 ℃ in the 4 th stage, the time of 30min, the holding time of 1500min, the vacuum of 0.3, the temperature of 5 ℃ in the 5 th stage, the time of 30min, the holding time of 120min, the vacuum of 0.3, the temperature of 10 ℃ in the 6 th stage, the time of 30min, the holding time of 120min and the vacuum of 0.3 in the 1 st stage; setting the temperature of the analysis and drying at 20 ℃, the time of 30min, the holding time of 120min and the vacuum of 0.3.
3. Determination of in vitro antioxidant Activity
1) Determination of Total reducing Capacity
2.5mL of the Chlorella antioxidant polypeptide sample solution prepared in this example was added with 2.5mL of phosphate buffer (0.2mol/L, pH 6.6) and 2.5mL of 0.01g/mL K3Fe(CN)6Reacting the solution in water bath at 50 ℃ for 20min, rapidly cooling, adding 2.5mL of 10% trichloroacetic acid solution, taking 5mL of reaction solution, adding 5mL of ddH2O and 1mg/mL FeCl31mL of the solution is uniformly mixed, the absorbance value of the solution is measured at 700nm after 10min, water is used as a blank, and the higher the absorbance value is, the stronger the reduction capability is.
2) Determination of hydroxyl radical scavenging Capacity
2mL of the chlorella antioxidant polypeptide sample solution prepared in the example was added with 2mL of 9mM FeSO42mL of 9mM salicylic acid (in absolute ethanol), 2mL of 8.8mM H2O2And 2mL ddH2O, reaction at 37 ℃ for 30min with ddH2O is used as reference, and the absorbance at the wavelength of 510nm is measured to calculate the hydroxyl radical scavenging capacity. And evaluating the capability of the chlorella enzymolysis polypeptide for removing hydroxyl free radicals by using ascorbic acid as a positive control.
Hydroxyl radical scavenging rate (%) ﹦ [ 1- (A1-A2)/A0] x 100%
Wherein A1 is the absorbance of the sample set; a2 is no color-developing agent H2O2The light absorption value of the solution set; a0 is the absorbance of distilled water instead of the sample blank.
3) Determination of DPPH radical scavenging Capacity
2mL of the chlorella antioxidant polypeptide sample solution prepared in the embodiment is taken, 2mL of 0.04g/mL DPPH solution (prepared by absolute ethyl alcohol) is added, the mixture is mixed uniformly and then stands for 30min in the dark, the light absorption value is measured at the wavelength of 517nm, and the DPPH free radical scavenging capacity is calculated. Ascorbic acid is used as a positive control to evaluate the capability of the chlorella enzymolysis polypeptide to eliminate DPPH free radicals.
DPPH clearance (%) [ 1- (a1-a2)/a0] x 100%
Wherein A1 is the absorbance of the sample when reacted with DPPH; a2 is the light absorption value of ethanol instead of the sample; a0 is the absorbance of distilled water instead of the sample.
4. Assay of cellular level antioxidant Activity
The method comprises the following steps of (1) inoculating Bel 7402 liver cancer cells which are in good growth state and in logarithmic growth phase into a six-hole plate, respectively adding 100 mu g/mL, 200 mu g/mL, 400 mu g/mL and 800 mu g/mL chlorella antioxidant polypeptides with molecular weight of 0-3.5kDa after the cells adhere to the wall for 24 hours, and detecting the ROS level in the cells according to the following method after 24 hours of treatment: preparing 10 mu M ROS fluorescent probe DCFH-DA by using serum-free 1640 culture solution, sucking out the culture solution in a six-well plate, adding 1mL DCFH-DA (except for blank control hole) with the concentration of 10 mu M into each hole, and placing the mixture in CO2Incubating for 20min in constant temperature incubator in dark place. The DCFH-DA solution was aspirated away from the sun, and the cells were washed 3 times with a serum-free 1640 medium, and then the fluorescence intensity was observed by a fluorescence microscope. A higher fluorescence intensity indicates a higher intracellular ROS level.
5. Determination of antioxidant Activity in animals
After being adaptively fed for 7 days, 12-month-old aged mice were randomly divided into 5 groups by weight, 10 mice per group: the control group is a model group, a total polypeptide gavage group, a 5-10kDa polypeptide gavage group, a 3.5-5kDa polypeptide gavage group and a 0-3.5kDa polypeptide gavage group. The gavage dosage of 50mg/kg per day is 0.2 mL/per control group, and the control group is administered with double distilled water with equal amount, and the gavage is continued for 5 weeks. During the experiment, the feeding dose was adjusted according to the change in body weight, and all mice were not restricted from eating and drinking water.
After the last gastric lavage, all mice are fasted for 12 hours without water prohibition, eyeball blood is taken into an anticoagulation tube under the anesthesia state, the anticoagulation tube is kept stand for 30min at room temperature, and the anticoagulation tube is centrifuged for 10min at 4 ℃ and 3000rpm, and the obtained supernatant is the mouse serum. The MDA content, SOD and GSH-PX activity level in animal serum are respectively detected according to the instruction of a kit (purchased from Nanjing institute of bioengineering).
The results of in vitro, cell and in vivo animal antioxidant detection show that: the chlorella vulgaris antioxidant polypeptide with the molecular weight of 0-3.5KDa has the optimal antioxidant activity, has better in-vitro total reduction capacity than chlorella vulgaris enzymolysis products subjected to enzymolysis by other proteases, can better eliminate DPPH and hydroxyl free radical in vitro than other polypeptide products with the molecular weight of the same protein enzymolysis liquid subjected to enzymolysis, can effectively reduce the ROS level in liver cancer cells, and can more effectively inhibit the generation of a peroxidation product MDA in an animal body and remarkably improve the GSH-PX activity as can be seen from a graph (1) and a graph (2).
Example 2
1. Preparation of chlorella cell wall enzymolysis liquid and specific protein enzymolysis liquid
The cellulase, pectinase and various proteases are purchased from Beijing Solebao scientific Co.
1) The chlorella cell wall enzymolysis liquid comprises the following components in parts by weight:
adjusting the pH value to 4.0 by using concentrated hydrochloric acid with the mass fraction of 36%;
2) the specific protein enzymolysis liquid comprises the following components in parts by weight:
adjusting the pH value to 7.5-9.5 by using 1mol/L NaOH solution;
2. prepared by the following steps:
1) weighing 750g of chlorella powder, adding the chlorella powder into 4.25L of 0.3mol/L NaOH dilute alkali solution, and soaking for 60min at the temperature of 60 ℃ to obtain pretreated chlorella solution;
2) homogenizing the pretreated chlorella solution obtained in the step 1) under high pressure for 2 times under 55MPa, centrifuging at 5000rpm for 20min, temporarily storing the obtained supernatant A at 4 ℃, adding the obtained precipitate into chlorella cell wall enzymolysis liquid, performing enzymolysis for 5h at 50 ℃ and at the stirring speed of 150rpm, and centrifuging at 5000rpm for 20min after enzymolysis to obtain a supernatant B;
3) mixing the supernatant A and the supernatant B obtained in the step 2), performing single-effect energy-saving concentration under the normal pressure condition until the concentration volume ratio reaches 1:4, and performing vacuum freeze drying on the concentrated solution to obtain chlorella protein powder;
4) adding the chlorella protein powder obtained in the step 3) into a specific protein enzymolysis liquid for enzymolysis, carrying out enzymolysis for 50min at 45 ℃, then carrying out enzyme deactivation treatment for 30min in water at 90 ℃, rapidly cooling to room temperature, and centrifuging for 20min at 4000rpm to obtain a supernatant;
5) and 4) performing ultrafiltration separation on the supernatant fluid under the conditions of 1MPa membrane-entering pressure and 40mL/min dialysis flow through ultrafiltration membranes with molecular weight cut-off of 10KDa, 5KDa and 3.5KDa respectively to obtain chlorella enzymolysis polypeptide liquid with molecular weight ranges of more than 10KDa, 5-10kDa, 3.5-5kDa and 0-3.5kDa respectively, collecting chlorella enzymolysis polypeptide liquid in different molecular weight regions, and performing vacuum freeze drying to obtain chlorella polypeptide powder. The in vitro, cell and in vivo animal antioxidant assay and results are the same as in example 1.
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| CN112451651A (en) * | 2020-11-30 | 2021-03-09 | 湖北瑞邦生物科技有限公司 | Application of chlorella pyrenoidosa peptide |
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