Anti-aging and anti-fatigue chlorella pyrenoidosa protein polypeptide powder and preparation method thereof

Abstract

The invention relates to the technical field of chlorella protein powder, and provides anti-aging and anti-fatigue chlorella pyrenoidosa protein polypeptide powder and a preparation method thereof.The anti-aging and anti-fatigue chlorella pyrenoidosa protein polypeptide powder is prepared from, by weight, 80%-90% of chlorella protein, 5%-15% of collagen and 1%-5% of oxalic acid degrading enzyme. The chlorella protein contains various minerals such as calcium, iron, zinc and selenium, and the oxalic acid degrading enzyme can degrade oxalic acid in the chlorella protein to reduce the content of oxalic acid, so that the situation that the oxalic acid is combined with the minerals such as calcium and iron to affect absorption of the minerals such as calcium and iron by a human body can be avoided, and the health-care effect is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chlorella protein powder, and specifically, to a protein polypeptide powder of chlorella pyrenoidosa with anti-aging and anti-fatigue effects and a preparation method thereof. Background Art

[0002] Chlorella is a single-celled aquatic alga, belonging to Chlorophyta, Chlorophyceae, Chlorellales, and Chlorellaceae. It contains rich proteins, polysaccharides, lipids, chlorophyll, vitamins, trace elements, and some bioactive metabolites. At present, chlorella protein powder, as a health product, has various health functions;

[0003] After retrieval, in the research "Research on the Application of Chlorella Powder in the Compound Feed of Crucian Carp" published by Shi Xi in the prior art, it is found that chlorella contains various essential amino acids, various unsaturated fatty acids, rich in n-3 and n-6 series unsaturated fatty acids, and has antioxidant ability and the effect of enhancing immunity, which promotes the healthy growth of crucian carp;

[0004] Chinese Invention Patent (Publication No.: CN112753920A) discloses a chlorella pyrenoidosa plant beverage and a preparation method thereof, and Invention Patent (Publication No.: CN113519735A) discloses a prune chlorella pyrenoidosa beverage and a preparation method thereof. The above patents are all applications of chlorella protein;

[0005] However, the above-mentioned literatures all have some technical defects:

[0006] First, due to the relatively high oxalic acid content in chlorella, these substances will combine with minerals such as calcium and iron to form insoluble compounds, thereby affecting the absorption of these minerals by animals, and none of the above-mentioned literatures have published solutions to this problem;

[0007] Second, due to the volatile fishy smell in chlorella, if chlorella is applied to beverages or foods, it will affect the taste, and none of the above-mentioned literatures have published solutions to how to remove the fishy smell. In view of this, the present invention proposes a protein polypeptide powder of chlorella pyrenoidosa with anti-aging and anti-fatigue effects and a preparation method thereof. Summary of the Invention

[0008] The present invention proposes a protein polypeptide powder of chlorella pyrenoidosa with anti-aging and anti-fatigue effects and a preparation method thereof, which solves the problem that oxalic acid in chlorella affects the absorption of minerals by animals after being applied to foods in the prior art.

[0009] The technical solution of the present invention is as follows: A protein polypeptide powder of chlorella pyrenoidosa with anti-aging and anti-fatigue effects, calculated by weight percentage, includes 80%-90% of chlorella protein, 5%-15% of collagen, and 1%-5% of oxalic acid degrading enzyme.

[0010] The present invention also discloses a preparation method of a protein nuclear chlorella protein polypeptide powder for anti-aging and anti-fatigue, comprising the following steps:

[0011] S1: Raw material preparation, selecting high-quality chlorella as the raw material, preferably using protein nuclear chlorella;

[0012] S2: High-pressure steam explosion treatment, after stacking and mixing chlorella and water in S1, the mixing ratio is 1g:15mL, and then the mixture is put into a high-pressure steam explosion treatment device for high-pressure steam explosion treatment, so that the protein dissolves and takes away the volatile fishy smell substances in the chlorella;

[0013] S3: Radio frequency heating extraction, putting the mixture treated in S2 into a radio frequency heating device for radio frequency heating extraction to accelerate the dissolution of protein and the volatilization of fishy smell substances, and extracting a chlorella protein solution;

[0014] S4: Two-phase progressive directional enzymolysis, the specific implementation process is as follows:

[0015] (1), adding endoprotease Protex 6L to the chlorella protein solution in S3 for pretreatment, the mixing ratio of endoprotease Protex 6L to the chlorella protein solution is 2:100 (w / w), and the mixing conditions are pH 8.0, 55°C, and enzymolysis for 3h;

[0016] (2), adding transglutaminase for directional conversion, the mixing conditions are pH 7.5, 45°C, and enzymolysis for 4h;

[0017] (3), adding Flavourzyme for fine degradation, the mixing conditions are pH 7.0, 50°C, and enzymolysis for 2h;

[0018] (4), sequentially using 30kDa, 10kDa, and 3kDa ultrafiltration membranes for gradient separation, and collecting the 3-10kDa fraction as the target polypeptide;

[0019] (5), subjecting the target polypeptide to gel filtration chromatography, and then purifying it by HPLC to obtain a high-purity chlorella protein polypeptide solution, and confirming the key anti-aging and anti-inflammatory polypeptide sequences;

[0020] S5: Raw material mixing, mixing the collagen solution, oxalic acid degrading enzyme and the chlorella protein polypeptide solution extracted in S3 according to the mass percentage, and performing concentration treatment after full mixing to obtain a concentrated solution;

[0021] S6: Spray drying, spray drying the concentrated mixed solution in S4 to obtain chlorella protein powder.

[0022] Preferably, in S2, the specific process of high-pressure steam explosion treatment is as follows:

[0023] (1) Load the mixture of Chlorella and water into a high-pressure steam explosion device, ensuring uniform loading and good sealing of the device.

[0024] (2) Heat the high-pressure steam explosion device to reach a predetermined temperature and pressure inside the device. The temperature is controlled within the range of 200 - 240 °C, the pressure is between 1 - 1.5 MPa, and the duration is 1 - 3 minutes.

[0025] (3) When the temperature and pressure inside the high-pressure steam explosion device reach the predetermined values, quickly release the pressure to instantaneously burst the Chlorella cells. During the explosion process, the cell walls of the Chlorella cells are broken, and the proteins and volatile fishy substances inside the cells are released.

[0026] Preferably, in S3, the specific process of radio frequency heating extraction is as follows:

[0027] (1) Put the mixture processed in S2 into a radio frequency heating device, set parameters such as radio frequency, power, and time for heating. The radio frequency for heating is 40 - 50 MHz, the time is 30 - 100 minutes, and the temperature is 60 - 70 °C. During the heating process, eddy current loss and dielectric loss will be generated inside the Chlorella cells by the radio frequency electromagnetic field, thereby dissolving the proteins inside the Chlorella cells.

[0028] (2) Separate the heated material. The specific process is as follows:

[0029] A. Filter the radio frequency heated mixed solution using filter paper or a filter membrane to retain the Chlorella cell debris on the filter paper or filter membrane to achieve separation.

[0030] B. Add 0.1 - 0.2 M phosphate buffer solution to the filtered mixed solution for dilution. The mixing ratio of the mixed solution to the phosphate buffer solution is 1 g:10 mL.

[0031] C. Pour the diluted mixed solution into a centrifuge tube, avoid generating bubbles, and ensure the centrifuge tube is tightly capped.

[0032] D. Put the centrifuge tube containing the mixed solution into a centrifuge, set the centrifugation parameters. Among them, the rotation speed is 8000 - 10000 rpm, the centrifugation time is 10 - 30 minutes, and the centrifugation temperature is -20 - 40 °C. Then start the centrifuge to perform centrifugation according to the preset parameters.

[0033] E. After centrifugation, take out the centrifuge tube and use a pipette or micropipette to suck out the supernatant of the centrifuge tube, so that the fishy substances and other impurities in the mixed solution are sucked out, and the Chlorella protein precipitates in the centrifuge tube.

[0034] (3) Re-wash the precipitated Chlorella protein in the centrifuge tube, and then add water and remix to obtain a Chlorella protein solution, with a mixing ratio of 1 g : 5 mL.

[0035] Preferably, in S5, the specific process of raw material mixing is as follows:

[0036] (1) Detect the pH values of each raw material respectively, and use buffer solution to ensure that the pH values of each raw material are maintained between 7 and 8;

[0037] (2) Mix each raw material into a magnetic stirrer, and set the stirring parameters. Among them, the stirring speed is 500 - 1000 rpm, the stirring temperature is 20 - 25 °C, and the stirring time is 10 - 15 minutes. Start the magnetic stirrer to mix the mixed materials according to the preset parameters;

[0038] (3) Take out the mixed solution from the magnetic stirrer for standby.

[0039] Preferably, in S5, the concentration process of the mixed solution is as follows:

[0040] (1) Pour the mixed solution into an evaporator, and set the parameters of the evaporator. Among them, the evaporation temperature is 60 - 120 °C, and the evaporation time is 30 - 60 minutes. Then start the heating system of the evaporator to make the water in the mixed solution start to evaporate to form steam;

[0041] (2) As the water evaporates, the concentration of the mixed solution gradually increases. When the required concentration is reached, turn off the heating system and discharge the concentrated solution from the bottom of the evaporator.

[0042] Preferably, in S6, the specific process of spray drying is as follows:

[0043] (1) Homogenize the concentrated solution, and the specific steps are as follows:

[0044] A. Select a high-pressure homogenizer for homogenization work, with a homogenization pressure of 6000 - 20000 psi. Then suck the concentrated solution through a plunger pump and pressurize it to make it enter the high-pressure homogenization chamber;

[0045] B. In the high-pressure homogenization chamber, the protein solution is subjected to impact effect, shear effect and cavitation effect to achieve the homogenization treatment of the concentrated solution;

[0046] C. Let the concentrated solution after the first homogenization treatment flow out from the sample outlet, and circulate for homogenization treatment 1 - 3 times;

[0047] (2) Import the concentrated solution after cyclic homogenization treatment into a spray dryer for spraying. During the spraying process, the liquid droplets are in full contact with the hot air and quickly evaporate water to form Chlorella protein powder;

[0048] (3) The prepared chlorella protein powder can be separated from the dust in the tail gas through a cyclone separator to obtain a high-purity product.

[0049] Preferably, 4% - 5% (W / W) of an anti-sticking agent needs to be added to the homogenized concentrate, and the anti-sticking agent is used to improve the fluidity of the concentrate and reduce the adhesion force between the concentrate and the drying chamber wall.

[0050] Preferably, the anti-sticking agent is any one of gum arabic, CMC, and maltodextrin.

[0051] The working principle and beneficial effects of the present invention are as follows:

[0052] 1. Chlorella protein contains a wide range of B vitamins, vitamin C, vitamin K, vitamin E, and various pigments, such as chlorophyll, lutein, and β-carotene. Among them, β-carotene and vitamin C are highly active antioxidants that can scavenge free radicals, protect cells from oxidative damage, and have a good anti-aging effect;

[0053] 2. Oxalate-degrading enzyme can degrade the oxalic acid in chlorella protein to reduce the oxalic acid content, which can avoid the combination of oxalic acid with minerals such as calcium and iron and affect the absorption of calcium and iron by the human body, greatly improving the health care effect;

[0054] 3. Chlorella protein contains a certain amount of fatty acids, including unsaturated fatty acids. These fatty acids are for maintaining normal physiological functions of the human body and promoting fat metabolism, while L-carnitine is a substance that can promote fatty acids to enter mitochondria for oxidation, which helps to improve the body's energy production efficiency and thus reduce fatigue;

[0055] 4. Through the synergistic enzymatic hydrolysis of chlorella protein by endoprotease, transglutaminase, and flavor enzyme, efficient protein degradation is achieved, and the activity of functional polypeptides is enhanced, greatly improving the anti-aging and anti-inflammatory functions of the protein powder.

[0056] Brief description of the drawings

[0057] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0058] Figure 1 It is a table graph of the calcium oxalate content of different embodiments of the present invention;

[0059] Figure 2 It is a table graph of the calcium oxalate content of different embodiments of the present invention; Specific embodiments

[0060] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0061] Embodiment 1:

[0062] This embodiment provides a protein-polypeptide powder of Chlorella pyrenoidosa for anti-aging and anti-fatigue, which includes, calculated by weight percentage, 80% of Chlorella protein, 15% of collagen, and 5% of oxalate-degrading enzyme;

[0063] Chlorella protein contains a wide range of B vitamins, vitamin C, vitamin K, vitamin E and various pigments, such as chlorophyll, lutein and β-carotene, etc. Among them, β-carotene and vitamin C are highly active antioxidants that can scavenge free radicals, protect cells from oxidative damage, and have a good anti-aging effect;

[0064] Chlorella protein also contains various minerals, such as calcium, iron, zinc, selenium, etc., and the oxalate-degrading enzyme can degrade the oxalic acid in Chlorella protein to reduce the content of oxalic acid, so as to avoid the combination of oxalic acid with minerals such as calcium and iron and affect the absorption of calcium and iron by the human body, greatly improving the health care effect;

[0065] Chlorella protein contains a certain amount of fatty acids, including unsaturated fatty acids. These fatty acids are for maintaining the normal physiological functions of the human body and promoting fat metabolism, and L-carnitine is a substance that can promote fatty acids to enter mitochondria for oxidation, which helps to improve the energy production efficiency of the body, thereby reducing the sense of fatigue;

[0066] The present invention also provides a preparation method of a protein-polypeptide powder of Chlorella pyrenoidosa for anti-aging and anti-fatigue, which includes the following steps:

[0067] S1: Raw material preparation, select high-quality Chlorella as the raw material, preferably Chlorella pyrenoidosa;

[0068] S2: High-pressure steam explosion treatment, mix the stacked Chlorella and water in S1 at a mixing ratio of 1 g: 15 mL, and then put the mixture into a high-pressure steam explosion treatment device for high-pressure steam explosion treatment, so that the protein dissolves and takes away the volatile fishy smell substances in Chlorella. The specific process of the high-pressure steam explosion treatment is as follows:

[0069] (1), Load the Chlorella and water mixture into the high-pressure steam explosion device to ensure uniform loading and good sealing of the device;

[0070] (2) Heat the high-pressure steam explosion equipment to reach the predetermined temperature and pressure inside the equipment. The temperature is controlled within 200 °C, the pressure is between 1 MPa, and the duration is 1 minute.

[0071] (3) When the temperature and pressure inside the high-pressure steam explosion equipment reach the predetermined values, quickly release the pressure to instantaneously burst the Chlorella cells. During the explosion process, the cell walls of the Chlorella cells are broken, and the proteins and volatile fishy substances inside the cells are released.

[0072] S3: Radio frequency heating extraction. Put the mixture processed in S2 into a radio frequency heating device for radio frequency heating extraction to accelerate the dissolution of proteins and the volatilization of fishy substances, and extract the Chlorella protein solution. The specific process of radio frequency heating extraction is as follows:

[0073] (1) Put the mixture processed in S2 into a radio frequency heating device, set parameters such as radio frequency, power, and time for heating. The radio frequency for heating is 40 MHz, the time is 30 minutes, and the temperature is 60 °C. During the heating process, the radio frequency electromagnetic field will generate eddy current loss and dielectric loss inside the Chlorella, thereby dissolving the proteins inside the Chlorella cells.

[0074] (2) Separate the heated material. The specific process is as follows:

[0075] A. Filter the mixed solution after radio frequency heating using filter paper or a filter membrane to retain the Chlorella cell debris on the filter paper or filter membrane to achieve separation.

[0076] B. Add 0.1 M phosphate buffer solution to the filtered mixed solution for dilution. The mixing ratio of the mixed solution to the phosphate buffer solution is 1 g:10 mL.

[0077] C. Pour the diluted mixed solution into a centrifuge tube, avoid generating air bubbles, and ensure that the centrifuge tube is tightly capped.

[0078] D. Put the centrifuge tube containing the mixed solution into a centrifuge, set the centrifugation parameters. Among them, the rotation speed is 8000 rpm, the centrifugation time is 10 minutes, and the centrifugation temperature is -20 °C. Then start the centrifuge to perform centrifugation according to the preset parameters.

[0079] E. After centrifugation, take out the centrifuge tube and use a pipette or a micropipettor to suck out the supernatant of the centrifuge tube, so that the fishy substances and other impurities in the mixed solution are sucked out, and the Chlorella protein precipitates in the centrifuge tube.

[0080] (3) Wash the Chlorella protein precipitated in the centrifuge tube again, and then add water to remix to obtain the Chlorella protein solution. The mixing ratio is 1 g:5 mL.

[0081] S4: Two-phase progressive directional enzymatic hydrolysis, and the specific implementation process is as follows:

[0082] (1). Add the endoprotease Protex 6L to the chlorella protein solution in S3 for pretreatment. The mixing ratio of the endoprotease Protex 6L to the chlorella protein solution is 2:100 (w / w), and the mixing conditions are pH 8.0, 55 °C, and enzymatic hydrolysis for 3 h;

[0083] (2). Add transglutaminase for directional conversion, and the mixing conditions are pH 7.5, 45 °C, and enzymatic hydrolysis for 4 h;

[0084] (3). Add Flavourzyme for fine degradation, and the mixing conditions are pH 7.0, 50 °C, and enzymatic hydrolysis for 2 h;

[0085] (4). Gradient separation is carried out successively using 30 kDa, 10 kDa, and 3 kDa ultrafiltration membranes, and the 3 - 10 kDa fraction is collected as the target polypeptide;

[0086] (5). Subject the target polypeptide to gel filtration chromatography, and then purify it by HPLC to obtain a high-purity chlorella protein polypeptide solution, and confirm the key anti-aging and anti-inflammatory polypeptide sequences;

[0087] S5: Raw material mixing, mix the collagen solution, oxalic acid degrading enzyme and the chlorella protein polypeptide solution extracted in S3 according to the mass percentage. The specific process of raw material mixing is as follows:

[0088] (1). Detect the pH values of each raw material respectively, and use buffer solution to ensure that the pH values of each raw material are maintained at 7;

[0089] (2). Mix each raw material into a magnetic stirrer, set the stirring parameters. Among them, the stirring speed is 500 rpm, the stirring temperature is 20 °C, and the stirring time is 10 minutes. Start the magnetic stirrer to mix the mixed materials according to the preset parameters;

[0090] (3). Take out the mixed solution from the magnetic stirrer for standby;

[0091] After sufficient mixing, carry out concentration treatment to obtain a concentrated solution. The concentration process of the mixed solution is as follows:

[0092] (1). Pour the mixed solution into an evaporator, set the parameters of the evaporator. Among them, the evaporation temperature is 60 °C, and the evaporation time is 30 minutes. Then start the heating system of the evaporator to make the water in the mixed solution start to evaporate to form steam;

[0093] (2) As the water evaporates, the concentration of the mixed solution gradually increases. When the required concentration is reached, the heating system is turned off, and the concentrated solution is discharged from the bottom of the evaporator.

[0094] S6: Spray drying. The concentrated mixed solution in S4 is spray-dried. The specific process of spray drying is as follows:

[0095] (1) Homogenize the concentrated solution. The specific steps are as follows:

[0096] A. Select a high-pressure homogenizer for homogenization work. The homogenization pressure is 6000 psi. Then, the concentrated solution is sucked in and pressurized by a piston pump and enters the high-pressure homogenization chamber.

[0097] B. In the high-pressure homogenization chamber, the protein solution is subjected to impact effect, shear effect, and cavitation effect to achieve the homogenization of the concentrated solution.

[0098] C. The concentrated solution after the first homogenization treatment flows out from the sample outlet and is circulated for homogenization treatment once.

[0099] (2) Add 4% (W / W) of an anti-sticking agent to the concentrated solution after cyclic homogenization treatment to improve the fluidity of the concentrated solution and reduce the adhesion force between the concentrated solution and the drying chamber wall. Then, introduce the concentrated solution into a spray dryer for spraying. During the spraying process, the liquid droplets are in full contact with the hot air, and the water evaporates rapidly to form chlorella protein powder.

[0100] (3) The prepared chlorella protein powder can be separated from the dust in the tail gas through a cyclone separator to obtain a high-purity product.

[0101] Furthermore, the anti-sticking agent is any one of gum arabic, CMC, and maltodextrin.

[0102] In the present invention, by adopting high-pressure steam explosion and radio frequency heating treatment, the protein in chlorella cells can be dissolved out and the fishy smell substances can be volatilized, thereby greatly reducing the content of volatile fishy smell substances and improving the taste of the product.

[0103] Example 2:

[0104] This example presents an anti-aging and anti-fatigue protein powder of chlorella pyrenoidosa, including, calculated by weight percentage, 85% of chlorella protein, 15% of collagen, and 5% of oxalic acid degrading enzyme.

[0105] The present invention also provides a preparation method of an anti-aging and anti-fatigue protein powder of chlorella pyrenoidosa, including the following steps:

[0106] S1: Raw material preparation. Select high-quality chlorella as the raw material, preferably chlorella pyrenoidosa.

[0107] S2: High-pressure steam explosion treatment. The stacked chlorella and water are mixed in S1 at a mixing ratio of 1 g: 15 mL. Then, the mixture is put into a high-pressure steam explosion treatment device for high-pressure steam explosion treatment, so that proteins are dissolved and volatile fishy substances in the chlorella are carried away. The specific process of the high-pressure steam explosion treatment is as follows:

[0108] (1). Load the chlorella and water mixture into the high-pressure steam explosion device to ensure uniform loading and good sealing of the device;

[0109] (2). Heat the high-pressure steam explosion device to make the internal temperature and pressure of the device reach the predetermined values. The temperature is controlled within the range of 220 °C, the pressure is between 1.3 MPa, and the duration is 2 minutes;

[0110] (3). When the temperature and pressure inside the high-pressure steam explosion device reach the predetermined values, quickly release the pressure to instantaneously burst the chlorella cells. During the explosion process, the cell walls of the chlorella cells are broken, and the proteins and volatile fishy substances inside the cells are released;

[0111] S3: Radio frequency heating extraction. The mixture treated in S2 is put into a radio frequency heating device for radio frequency heating extraction to accelerate the dissolution of proteins and the volatilization of fishy substances, and a chlorella protein solution is extracted. The specific process of the radio frequency heating extraction is as follows:

[0112] (1). Put the mixture treated in S2 into the radio frequency heating device, and set parameters such as radio frequency, power, and time for heating. The radio frequency for radio frequency heating is 45 MHz, the time is 60 minutes, and the temperature is 65 °C. During the heating process, eddy current loss and dielectric loss will be generated inside the chlorella by the radio frequency electromagnetic field, so that the proteins inside the chlorella cells are dissolved;

[0113] (2). Separate the heated material. The specific process is as follows:

[0114] A. Filter the radio frequency heated mixed solution with filter paper or filter membrane to retain the chlorella cell debris on the filter paper or filter membrane to achieve separation;

[0115] B. Add 0.15 M phosphate buffer solution to the filtered mixed solution for dilution. The mixing ratio of the mixed solution and the phosphate buffer solution is 1 g: 10 mL;

[0116] C. Pour the diluted mixed solution into a centrifuge tube, avoid generating bubbles, and ensure that the centrifuge tube is tightly capped;

[0117] D. Place the centrifuge tube filled with the mixed solution into a centrifuge, set the centrifugation parameters, where the rotation speed is 9000 rpm, the centrifugation time is 20 minutes, and the centrifugation temperature is 20 °C. Then start the centrifuge to perform centrifugation according to the preset parameters;

[0118] E. After centrifugation, take out the centrifuge tube and use a pipette or micropipettor to suck out the supernatant of the centrifuge tube, so that the fishy substances and other impurities in the mixed solution are sucked out, and the chlorella protein precipitates in the centrifuge tube;

[0119] (3). Wash the chlorella protein precipitate in the centrifuge tube again, and then add water to remix to obtain a chlorella protein solution, and the mixing ratio is 1 g: 5 mL;

[0120] S4: Two-phase progressive directional enzymatic hydrolysis, and the specific implementation process is as follows:

[0121] (1). Add the endoprotease Protex 6L to the chlorella protein solution in S3 for pretreatment. The mixing ratio of the endoprotease Protex 6L to the chlorella protein solution is 2: 100 (w / w), and the mixing conditions are pH 8.0, 55 °C, and enzymatic hydrolysis for 3 h;

[0122] (2). Add transglutaminase for directional conversion, and the mixing conditions are pH 7.5, 45 °C, and enzymatic hydrolysis for 4 h;

[0123] (3). Add Flavourzyme for fine degradation, and the mixing conditions are pH 7.0, 50 °C, and enzymatic hydrolysis for 2 h;

[0124] (4). Gradient separation is carried out successively using 30 kDa, 10 kDa, and 3 kDa ultrafiltration membranes, and the 3 - 10 kDa fraction is collected as the target polypeptide;

[0125] (5). Subject the target polypeptide to gel filtration chromatography, and then purify it by HPLC to obtain a high-purity chlorella protein polypeptide solution, and confirm the key anti-aging and anti-inflammatory polypeptide sequences;

[0126] S5: Raw material mixing. Mix the collagen solution, zinc-containing casein phosphopeptide, oxalic acid degrading enzyme with the chlorella protein polypeptide solution extracted in S3 according to the mass percentage. The specific process of raw material mixing is as follows:

[0127] (1). Detect the pH values of each raw material respectively, and use a buffer solution to ensure that the pH values of each raw material are maintained at 7.5;

[0128] (2). Mix each raw material into a magnetic stirrer, set the stirring parameters, where the stirring speed is 800 rpm, the stirring temperature is 22 °C, and the stirring time is 12 minutes. Start the magnetic stirrer to mix the mixed materials according to the preset parameters;

[0129] (3) Take out the mixed solution from the magnetic stirrer for standby;

[0130] After sufficient mixing, perform concentration treatment to obtain a concentrated solution. The concentration process of the mixed solution is as follows:

[0131] (1) Pour the mixed solution into the evaporator, set the parameters of the evaporator. Among them, the evaporation temperature is 90 °C, the evaporation time is 35 minutes, and then start the heating system of the evaporator so that the water in the mixed solution starts to evaporate to form steam;

[0132] (2) As the water evaporates, the concentration of the mixed solution gradually increases. When the required concentration is reached, turn off the heating system and discharge the concentrated solution from the bottom of the evaporator;

[0133] S6: Spray drying. Spray dry the concentrated mixed solution in S4. The specific process of spray drying is as follows:

[0134] (1) Homogenize the concentrated solution. The specific steps are as follows:

[0135] A. Select a high-pressure homogenizer for homogenization work. The homogenization pressure is 15000 psi, and then suck and pressurize the concentrated solution through a plunger pump to make it enter the high-pressure homogenization chamber;

[0136] B. In the high-pressure homogenization chamber, the protein solution is subjected to impact effect, shear effect and cavitation effect to achieve the homogenization treatment of the concentrated solution;

[0137] C. Let the concentrated solution after the first homogenization treatment flow out from the sample outlet and circulate for homogenization treatment 2 times;

[0138] (2) Add 4.5% (W / W) of anti-sticking agent to the concentrated solution after cyclic homogenization treatment to improve the fluidity of the concentrated solution and reduce the adhesion force between the concentrated solution and the drying chamber wall. Then introduce the concentrated solution into the spray dryer for spraying. During the spraying process, the droplets are in full contact with the hot air and quickly evaporate water to form chlorella protein powder;

[0139] (3) The prepared chlorella protein powder can be separated from the dust in the tail gas through a cyclone separator to obtain a high-purity product.

[0140] Furthermore, the anti-sticking agent is any one of gum arabic, CMC, and maltodextrin.

[0141] Example 3:

[0142] This example proposes an anti-aging and anti-fatigue chlorella pyrenoidosa protein polypeptide powder, which includes, calculated by weight percentage, 90% of chlorella protein, 7% of collagen, and 3% of oxalic acid degrading enzyme;

[0143] The present invention also provides a method for preparing a protein polypeptide powder of chlorella pyrenoidosa with anti-aging and anti-fatigue effects, which comprises the following steps:

[0144] S1: Raw material preparation, select high-quality chlorella as the raw material, preferably use chlorella pyrenoidosa;

[0145] S2: High-pressure steam explosion treatment, mix the stacked chlorella and water in S1 at a mixing ratio of 1 g: 15 mL, and then put the mixture into a high-pressure steam explosion treatment device for high-pressure steam explosion treatment, so that the protein is dissolved and the volatile fishy smell substances in the chlorella are carried away. The specific process of the high-pressure steam explosion treatment is as follows:

[0146] (1) Load the chlorella and water mixture into the high-pressure steam explosion device to ensure uniform loading and good sealing of the device;

[0147] (2) Heat the high-pressure steam explosion device to make the internal temperature and pressure of the device reach the predetermined values. The temperature is controlled in the range of 200 - 240 °C, the pressure is between 1.5 MPa, and the duration is 3 minutes;

[0148] (3) When the temperature and pressure inside the high-pressure steam explosion device reach the predetermined values, quickly release the pressure to cause the chlorella cells to burst instantaneously. During the explosion process, the cell walls of the chlorella cells are broken, and the proteins and volatile fishy smell substances inside the cells are released;

[0149] S3: Radio frequency heating extraction, put the mixture treated in S2 into a radio frequency heating device for radio frequency heating extraction to accelerate the dissolution of proteins and the volatilization of fishy smell substances, and extract the chlorella protein solution. The specific process of the radio frequency heating extraction is as follows:

[0150] (1) Put the mixture treated in S2 into the radio frequency heating device, set parameters such as radio frequency, power and time for heating. The radio frequency for radio frequency heating is 50 MHz, the time is 100 minutes, and the temperature is 70 °C. During the heating process, the radio frequency electromagnetic field will generate eddy current loss and dielectric loss inside the chlorella, so that the proteins inside the chlorella cells are dissolved;

[0151] (2) Separate the heated material, and the specific process is as follows:

[0152] A. Filter the radio frequency heated mixed solution with filter paper or filter membrane to retain the chlorella cell debris on the filter paper or filter membrane to achieve separation;

[0153] B. Add 0.2 M phosphate buffer solution to the filtered mixed solution for dilution. The mixing ratio of the mixed solution to the phosphate buffer solution is 1 g: 10 mL;

[0154] C. Pour the diluted mixed solution into a centrifuge tube, avoiding the generation of bubbles, and ensure that the centrifuge tube is tightly capped;

[0155] D. Place the centrifuge tube containing the mixed solution into a centrifuge, set the centrifugation parameters. Among them, the rotation speed is 10000 rpm, the centrifugation time is 30 minutes, and the centrifugation temperature is 40 °C. Then start the centrifuge to perform centrifugation according to the preset parameters;

[0156] E. After centrifugation, take out the centrifuge tube and use a pipette or micropipettor to suck out the supernatant of the centrifuge tube, so that the fishy substances and other impurities in the mixed solution are sucked out, and the chlorella protein precipitates in the centrifuge tube;

[0157] (3). Wash the chlorella protein precipitate in the centrifuge tube again, and then add water to remix to obtain a chlorella protein solution, and the mixing ratio is 1 g: 5 mL;

[0158] S4: Two-phase progressive directional enzymatic hydrolysis, and the specific implementation process is as follows:

[0159] (1). Add the endoprotease Protex 6L to the chlorella protein solution in S3 for pretreatment. The mixing ratio of the endoprotease Protex 6L to the chlorella protein solution is 2: 100 (w / w), and the mixing conditions are pH 8.0, 55 °C, and enzymatic hydrolysis for 3 h;

[0160] (2). Add transglutaminase for directional conversion, and the mixing conditions are pH 7.5, 45 °C, and enzymatic hydrolysis for 4 h;

[0161] (3). Add Flavourzyme for fine degradation, and the mixing conditions are pH 7.0, 50 °C, and enzymatic hydrolysis for 2 h;

[0162] (4). Gradient separation is carried out successively using 30 kDa, 10 kDa, and 3 kDa ultrafiltration membranes, and the 3 - 10 kDa fraction is collected as the target polypeptide;

[0163] (5). Subject the target polypeptide to gel filtration chromatography, and then purify it by HPLC to obtain a high - purity chlorella protein polypeptide solution, and confirm the key anti - aging and anti - inflammatory polypeptide sequences;

[0164] S5: Raw material mixing. Mix the collagen solution, oxalic acid degrading enzyme and the chlorella protein polypeptide solution extracted in S3 according to the mass percentage. The specific process of raw material mixing is as follows:

[0165] (1). Detect the pH values of each raw material respectively, and use buffer solution to ensure that the pH values of each raw material are maintained at 8;

[0166] (2) Mix all the raw materials in a magnetic stirrer, and set the stirring parameters. Among them, the stirring speed is 500 - 1000 rpm, the stirring temperature is 25 °C, and the stirring time is 15 minutes. Then start the magnetic stirrer to mix the mixed materials according to the preset parameters;

[0167] (3) Take out the mixed solution from the magnetic stirrer for standby;

[0168] After sufficient mixing, conduct concentration treatment to obtain a concentrated solution. The concentration process of the mixed solution is as follows:

[0169] (1) Pour the mixed solution into an evaporator, and set the parameters of the evaporator. Among them, the evaporation temperature is 120 °C, and the evaporation time is 60 minutes. Then start the heating system of the evaporator so that the water in the mixed solution begins to evaporate to form steam;

[0170] (2) As the water evaporates, the concentration of the mixed solution gradually increases. When the required concentration is reached, turn off the heating system and discharge the concentrated solution from the bottom of the evaporator;

[0171] S6: Spray drying. Spray dry the concentrated mixed solution in S4. The specific process of spray drying is as follows:

[0172] (1) Conduct homogenization treatment on the concentrated solution. The specific steps are as follows:

[0173] A. Select a high-pressure homogenizer for homogenization work. The homogenization pressure is 20000 psi. Then suck the concentrated solution through a piston pump and pressurize it to make it enter the high-pressure homogenization chamber;

[0174] B. In the high-pressure homogenization chamber, the protein solution is subjected to impact effect, shear effect and cavitation effect to achieve the homogenization treatment of the concentrated solution;

[0175] C. Let the concentrated solution after the first homogenization treatment flow out from the sampling port, and circulate the homogenization treatment 3 times;

[0176] (2) Add 5% (W / W) of an anti-sticking agent to the concentrated solution after cyclic homogenization treatment to improve the fluidity of the concentrated solution and reduce the adhesion force between the concentrated solution and the drying chamber wall. Then introduce the concentrated solution into a spray dryer for spraying. During the spraying process, the liquid droplets are in full contact with the hot air and quickly evaporate water to form chlorella protein powder;

[0177] (3) Through a cyclone separator, the prepared chlorella protein powder can be separated from the dust in the tail gas to obtain a high-purity product.

[0178] Furthermore, the anti-sticking agent is any one of gum arabic, CMC, and maltodextrin.

[0179] A comparative example was set up. The only difference between the comparative example and Example 1 is that a chlorella protein powder includes, by weight percentage, 90% of chlorella protein and 10% of collagen.

[0180] Experimental verification:

[0181] Experiment 1:

[0182] The chlorella protein powders in Example 1, Example 2, Example 3 and the comparative example were respectively dissolved in equal amounts of water in four beakers to obtain protein powder solutions. Then, the weight method was used to detect the content of calcium oxalate in different solutions. The specific experimental steps are as follows:

[0183] First, an excessive amount of calcium chloride was added to the mixed solutions in the four beakers to completely precipitate calcium oxalate;

[0184] Second, the precipitate was then filtered and washed with dilute ammonium oxalate solution to avoid the dissolution of calcium oxalate;

[0185] Third, the precipitate was dried to a constant weight and its mass was weighed. Then, according to the mass of calcium oxalate weighed and the mass of the mixture, the content of calcium oxalate in the protein powder solutions in Example 1, Example 2, Example 3 and the comparative example was calculated. The calculation formula is as follows:

[0186] The specific calculation results are as Figure 1 shown.

[0187] As can be seen from the above experiment, with the increase in the content of oxalic acid degrading enzyme, the content of calcium oxalate in the mixed solution will be greatly reduced, which also means that the content of oxalic acid in the mixed solution is reduced. This can avoid the influence of chlorella protein powder on the absorption of minerals such as calcium and iron by the human body after use and improve the health care effect of the product.

[0188] Experiment 2:

[0189] The components of the chlorella protein polypeptide solution prepared in S4 were identified and compared with the chlorella protein polypeptide solution obtained by enzymatic hydrolysis in the traditional way. The comparison results are as Figure 2 shown. The experimental data show that through the combined enzymatic hydrolysis of Protex6L + TGase + Flavourzyme in this application, the efficient conversion of chlorella protein is realized. This method not only significantly improves the protein conversion rate (+25.8%) and polypeptide yield (+22.8%), but also enhances the antioxidant activity (+21.4%) and anti-inflammatory activity (+18.8%); this method can be used to develop functional foods or drugs with anti-aging and anti-inflammatory effects.

[0190] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder, characterized in that: Calculated by weight percentage, it includes 80%-90% chlorella protein, 5%-15% collagen, and 1%-5% oxalate degrading enzyme.

2. A method for preparing an anti-aging and anti-fatigue protein polypeptide powder of pyrenoid chlorella according to claim 1, characterized in that: The steps include: S1: Raw material preparation, selecting high-quality Chlorella as raw material, preferably using Chlorella pyrenoidosa; S2: high-pressure steam explosion treatment, after the stacked chlorella in S1 is mixed with water, the mixing ratio is 1g:15mL, and then the mixture is placed in a high-pressure steam explosion treatment equipment for high-pressure steam explosion treatment, so that the protein is dissolved and the volatile fishy substances in the chlorella are taken away; S3: radio frequency heating extraction, placing the mixture treated in S2 into a radio frequency heating device for radio frequency heating extraction, accelerating protein dissolution and volatilization of fishy substances, and extracting a chlorella protein solution; S4: Two-phase progressive directional enzymatic hydrolysis, the specific implementation process is as follows: (1) adding endoprotease Protex 6L to the Chlorella protein solution in S3 for pretreatment, the mixing ratio of endoprotease Protex 6L to the Chlorella protein solution is 2:100 (w / w), the mixing conditions are pH 8.0, 55°C, and enzymolysis for 3h; (2) Add glutamine aminotransferase for directional conversion, the mixing conditions are pH 7.5, 45°C, and enzymatic hydrolysis for 4 hours; (3) Add flavor enzyme Flavourzyme for fine degradation, the mixing conditions are pH 7.0, 50°C, and enzymolysis for 2 h; (4) Use 30 kDa, 10 kDa, and 3 kDa ultrafiltration membranes for gradient separation, and collect the 3-10 kDa fraction as the target polypeptide; (5) Purify the target peptide by gel filtration chromatography and then HPLC to obtain a high-purity Chlorella protein peptide solution, and confirm the key anti-aging and anti-inflammatory peptide sequences; S5: mixing raw materials, mixing the collagen solution, oxalate degrading enzyme and the chlorella protein polypeptide solution extracted in S3 according to mass percentage, and concentrating after fully mixing to obtain a concentrated solution; S6: spray drying, spray drying the concentrated mixed solution in S4 to obtain Chlorella protein powder.

3. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 2, characterized in that: In S2, the specific process of high pressure steam explosion treatment is as follows: (1) Load the mixture of Chlorella and water into the high-pressure steam explosion equipment, ensuring that the loading is even and the equipment is well sealed; (2) Heat the high-pressure steam explosion equipment to make the interior of the equipment reach a predetermined temperature and pressure, with the temperature controlled within the range of 200-240°C and the pressure between 1-1.5 MPa for a duration of 1-3 minutes; (3) When the temperature and pressure inside the high-pressure steam explosion equipment reach the predetermined value, the pressure is rapidly released, causing the Chlorella cells to explode instantly. During the explosion process, the cell walls of the Chlorella cells are broken, and the proteins and volatile fishy substances in the cells are released.

4. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 2, characterized in that: In S3, the specific process of RF heating extraction is as follows: (1) placing the mixture treated in S2 into a radio frequency heating device, setting the radio frequency frequency, power and time parameters for heating, the radio frequency heating frequency is 40-50 MHz, the time is 30-100 minutes, and the temperature is 60-70° C. During the heating process, the radio frequency electromagnetic field will generate eddy current loss and dielectric loss inside the Chlorella, thereby dissolving the protein in the Chlorella cells; (2) Separate the heated materials. The specific process is as follows: A. Filter the mixed solution after radio frequency heating using filter paper or filter membrane, and retain the Chlorella cell fragments on the filter paper or filter membrane to achieve separation; B. Add 0.1-0.2M phosphate buffer to dilute the filtered mixed solution, the mixing ratio of the mixed solution to the phosphate buffer is 1g:10mL; C. Pour the diluted mixed solution into a centrifuge tube, avoid bubbles, and make sure the centrifuge tube is tightly covered; D. Place the centrifuge tube containing the mixed solution into a centrifuge, set the centrifugal parameters, wherein the speed is 8000-10000 rpm, the centrifugal time is 10-30 minutes, the centrifugal temperature is -20-40°C, and then start the centrifuge to perform centrifugal treatment according to the preset parameters; E. After the centrifugation is completed, take out the centrifuge tube and use a straw or a pipette to suck out the supernatant of the centrifuge tube, so that the fishy substances and other impurities in the mixed solution are sucked out, and the precipitate in the centrifuge tube is the Chlorella protein; (3) Wash the precipitated Chlorella protein in the centrifuge tube again, then add water and remix to obtain a Chlorella protein solution, with a mixing ratio of 1 g: 5 mL.

5. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 1, characterized in that: In S5, the specific process of raw material mixing is as follows: (1) Detect the pH value of each raw material separately, and use a buffer solution to ensure that the pH value of each raw material is maintained between 7 and 8; (2) Mix the raw materials into a magnetic stirrer, set the stirring parameters, wherein the stirring speed is 500-1000 rpm, the stirring temperature is 20-25° C., the stirring time is 10-15 minutes, and start the magnetic stirrer to mix the mixed materials according to the preset parameters; (3) Take the mixed solution out of the magnetic stirrer and set aside.

6. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 5, characterized in that: In S5, the concentration process of the mixed solution is as follows: (1) Pour the mixed solution into an evaporator, set the parameters of the evaporator, wherein the evaporation temperature is 60-120° C., the evaporation time is 30-60 minutes, and then start the heating system of the evaporator so that the water in the mixed solution begins to evaporate to form steam; (2) As the water evaporates, the concentration of the mixed solution gradually increases. When the required concentration is reached, the heating system is turned off and the concentrated liquid is discharged from the bottom of the evaporator.

7. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 6, characterized in that: In S6, the specific process of spray drying is as follows: (1) The concentrated liquid is homogenized. The specific steps are as follows: A. Select a high-pressure homogenizer for homogenization, with a homogenization pressure of 6000-20000psi, and then suck the concentrate through a plunger pump and pressurize it to enter the high-pressure homogenization chamber; B. In the high-pressure homogenization chamber, the protein liquid is subjected to the impact effect, shear effect and cavitation effect, thus achieving homogenization of the concentrated liquid; C. The concentrated liquid after the first homogenization treatment flows out from the sample outlet, and the homogenization treatment is repeated 1-3 times; (2) introducing the concentrated liquid after the circulating homogenization treatment into a spray dryer for spraying. During the spraying process, the droplets are fully contacted with the hot air, and the water is quickly evaporated to form chlorella protein powder; (3) The prepared Chlorella protein powder can be separated from the dust in the tail gas by a cyclone separator to obtain a high-purity product.

8. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 7, characterized in that: The concentrated liquid after homogenization needs to be added with 4% to 5% (W / W) of an anti-sticking agent, and the anti-sticking agent is used to improve the fluidity of the concentrated liquid and reduce the adhesion between the concentrated liquid and the wall of the drying chamber.

9. The method for preparing the anti-aging and anti-fatigue pyrenoid chlorella protein polypeptide powder according to claim 8, characterized in that: The anti-adhesive agent is any one of gum arabic, CMC and maltodextrin.

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