Preparation method of chlorella pyrenoidosa peptide

Abstract

The invention belongs to the field of preparation of chlorella pyrenoidosa peptides, and particularly discloses a preparation method of a chlorella pyrenoidosa peptide. The chlorella pyrenoidosa peptide effectively solves the problem that the chlorella pyrenoidosa peptide is dark in color and cannot be used for light-color matrix products such as transparent beverages and medical dressings Comprising the following steps: (1) adding chlorella pyrenoidosa powder into water, and performing high-pressure homogenization for multiple times to obtain wall-broken chlorella pyrenoidosa feed liquid; (2) heating the feed liquid, keeping the temperature, adding alkaline protease and papain for enzymolysis, and performing enzyme deactivation; (3) centrifuging, collecting supernate, and enabling the supernate to pass through a ceramic membrane, so as to obtain chlorella enzymatic hydrolysate; and (4) heating the chlorella enzymatic hydrolysate to 60 DEG C, adding activated carbon, activated clay and a hydrogen peroxide solution, carrying out heat preservation stirring, heating to 90-95 DEG C, carrying out heat preservation stirring, carrying out filter pressing and refined filtration to obtain a chlorella peptide decoloring solution, and sterilizing and concentrating the chlorella peptide decoloring solution to obtain a chlorella peptide concentrated solution. The chlorella peptide produced by the method is almost white, has no peculiar fishy smell of raw materials, and is wide in application range.

Description

Technical Field

[0001] This invention belongs to the field of preparation of protein-nucleated Chlorella peptides, and particularly relates to a method for preparing protein-nucleated Chlorella peptides. Background Technology

[0002] Chlorella proteoglycans contains unique and diverse functional macroscopic and microscopic nutrients, including proteins, omega-3 polyunsaturated fatty acids, polysaccharides, vitamins, and minerals. The protein it contains includes 17 amino acids, among which the essential amino acids are similar to the RDA (Recommended Daily Allowance) values ​​recommended by the Food and Agriculture Organization of the United Nations and the World Health Organization. Furthermore, Chlorella proteoglycans possesses health benefits such as antioxidant activity, immune enhancement, metabolic regulation, lipid-lowering, blood pressure-lowering, and anti-tumor effects. Currently, increasing research shows that antioxidation is a crucial step in preventing aging and death; eliminating excessive free radicals can prevent many age-related diseases caused by free radicals. Compared to synthetic antioxidants, which may have cumulative carcinogenic effects, Chlorella proteoglycan peptides are naturally safe and have the effect of stimulating the activity of intracellular oxidases, particularly catalase activity. Its antioxidant activity also has a certain correlation with the prevention and treatment of diabetes. Chlorella peptides reduce the burden on the liver by chelating heavy metals (such as lead and mercury) and removing environmental toxins such as BPA.

[0003] With people's dietary concepts trending towards green and healthy options, Chlorella proteoglycans, a natural product with health benefits, is undoubtedly a hot topic in future food research. Chlorella not only perfectly aligns with the current demand for healthy eating but also conforms to my country's development and utilization of new resources. Currently, the application of existing green or yellow Chlorella proteoglycan peptides in the food, cosmetics, and pharmaceutical fields is limited due to their color. Natural chlorophyll and carotenoids give Chlorella proteoglycan peptides their dark green or yellow color, making them unsuitable for use in light-colored matrix products such as transparent beverages and medical dressings.

[0004] Chinese invention patent CN119685168A, titled "White Mutant Chlorella proteoglycans FZU65-W1 and Its Application," describes the induction of Chlorella proteoglycans into a chlorophyll-deficient strain. While this strain eliminates the need for decolorization and can be used to prepare colorless Chlorella proteoglycan peptides, it suffers from lower protein content compared to conventional Chlorella proteoglycans. Chlorella growth factor (CGF), which is related to chlorophyll metabolism, may have reduced CGF content in the mutant strain, potentially affecting core functions such as immune regulation and cell repair. Chinese invention patent CN115894619A, titled "A Short Peptide, a Chlorella Proteoglycans Polypeptide Extract, Its Preparation Method and Application," primarily describes a Chlorella peptide that inhibits pancreatic lipase activity. However, this invention focuses solely on its function, neglecting the application requirements related to its color. Therefore, it is necessary to develop a process for preparing nearly white Chlorella proteoglycan peptides using conventional green Chlorella proteoglycans as raw material. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing Chlorella protein-nucleated peptides, which effectively solves the problem that Chlorella protein-nucleated peptides are dark green or yellow and cannot be used in light-colored matrix products such as transparent beverages and medical dressings.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing Chlorella protein-core peptides includes the following steps: S1, adding dried Chlorella protein-core powder in small amounts to water at a ratio of 10 to 20 times the mass of the Chlorella protein-core powder, stirring and dissolving evenly, and homogenizing under high pressure at 30 to 60 MPa for 3 to 5 times to obtain a broken Chlorella protein-core peptide solution.

[0008] S2. Heat the high-pressure homogenized Chlorella vulgaris protein-core liquid to 45℃~55℃, keep it at this temperature, add alkaline protease and papain for 3h~6h to inactivate the enzymes.

[0009] S3. Centrifuge the cell wall-broken Chlorella protein hydrolysate at 8000 r / min for 15 min, collect the supernatant, pass the supernatant through a ceramic membrane, collect the permeate, and obtain a clear Chlorella protein hydrolysate.

[0010] S4. Heat the clarified Chlorella proteolytic solution to 60°C, then add activated carbon, activated clay and hydrogen peroxide solution and keep it warm and stir for 20 min to 40 min. Then heat it to 90°C to 95°C and keep it warm and stir for 20 min to 60 min. After plate and frame filtration and titanium rod activated carbon filter, the clarified Chlorella proteolytic peptide decolorizing solution is obtained.

[0011] S5. After ultra-high temperature instantaneous sterilization, the decolorized solution of Chlorella peptide is concentrated by reverse osmosis technology to obtain Chlorella peptide concentrate.

[0012] Furthermore, in step S2, the alkaline protease activity is 350,000 U / g to 400,000 U / g, and the amount of alkaline protease added is 0.5% to 4% of the mass of Chlorella pulveratum powder; the papain activity is 100,000 U / g to 600,000 U / g, and the amount of papain added is 0.2% to 2% of the mass of Chlorella pulveratum powder.

[0013] Furthermore, in step S4, the activated carbon is in powder form, and the amount of activated carbon added is 2% to 10% of the mass of Chlorella pulveratum powder.

[0014] Furthermore, in step S4, the decolorization rate of the activated clay is ≥95%, and the amount of activated clay added is 1% to 5% of the mass of Chlorella pyrenoidosa powder.

[0015] Furthermore, in step S4, the concentration of the hydrogen peroxide solution is 30%, and the amount of hydrogen peroxide solution added is 0.2% to 2% of the mass of Chlorella pyrenoidosa powder.

[0016] Compared with the prior art, the beneficial technical effects of the present invention are:

[0017] (1) The present invention uses ceramic membrane to retain activated carbon, activated clay and hydrogen peroxide for decolorization, covering a wider decolorization range and greatly improving the decolorization effect. Moreover, no organic solvents are used, which preserves the activity of protein core chlorella peptide as much as possible and reduces environmental pollution.

[0018] (2) The present invention uses activated carbon and activated clay in combination for decolorization, which is low in cost and can be regenerated at high temperature, reducing solid waste generation and is suitable for large-scale industrial use.

[0019] (3) The protein-nucleated chlorella peptide produced by this invention is nearly white, the aqueous solution is nearly colorless, and it has no fishy smell characteristic of the raw materials, thus having a wider range of applications. Detailed Implementation

[0020] Example 1: The preparation method of Chlorella protein-core peptide in this example includes the following steps: S1, adding dried Chlorella protein-core powder to water in small amounts and multiple times in a volume equal to 10 times the mass of Chlorella protein-core powder, stirring and dissolving evenly, and homogenizing under high pressure at 60 MPa for 3 times to obtain Chlorella protein-core peptide solution with broken cell walls.

[0021] S2. Heat the high-pressure homogenized Chlorella vulgaris protein-core liquid to 55°C, keep it warm, add alkaline protease and papain for 3 hours to inactivate the enzymes.

[0022] The alkaline protease has an enzyme activity of 400,000 U / g, and the amount of alkaline protease added is 4% of the mass of Chlorella pulveratum powder; the papain has an enzyme activity of 100,000 U / g, and the amount of papain added is 1% of the mass of Chlorella pulveratum powder.

[0023] S3. Centrifuge the cell wall-broken Chlorella protein hydrolysate at 8000 r / min for 15 min, collect the supernatant, pass the supernatant through a ceramic membrane, collect the permeate, and obtain a clear Chlorella protein hydrolysate.

[0024] S4. Heat the clarified Chlorella proteinensis enzymatic hydrolysate to 60°C, then add powdered activated carbon, activated clay and hydrogen peroxide solution and keep warm and stir for 20 minutes. Then heat to 90°C~95°C and keep warm and stir for 20 minutes. After plate and frame filtration and titanium rod activated carbon filter, obtain the clarified Chlorella proteinensis peptide decolorizing solution.

[0025] The activated carbon is added at 7% of the mass of the Chlorella pyrenoidosa powder. The activated clay has a decolorization rate of ≥95%, and the amount of activated clay added is 5% of the mass of the Chlorella pyrenoidosa powder. The concentration of the hydrogen peroxide solution is 30%, and the amount of hydrogen peroxide solution added is 1% of the mass of the Chlorella pyrenoidosa powder.

[0026] Activated carbon and activated clay are low in cost and can be regenerated at high temperatures, reducing solid waste generation and making them suitable for large-scale industrial use.

[0027] S5. After ultra-high temperature instantaneous sterilization, the decolorized solution of Chlorella peptide is concentrated by reverse osmosis technology to obtain Chlorella peptide concentrate.

[0028] Example 2: The preparation method of Chlorella protein-core peptide in this example includes the following steps: S1, adding dried Chlorella protein-core powder to water in small amounts and multiple times in a volume equal to 15 times the mass of Chlorella protein-core powder, stirring and dissolving evenly, and homogenizing under high pressure at 30 MPa for 5 times to obtain Chlorella protein-core peptide solution with broken cell walls.

[0029] S2. Heat the high-pressure homogenized Chlorella vulgaris protein-core liquid to 45°C, keep it warm, add alkaline protease and papain for 4 hours to inactivate the enzymes.

[0030] The alkaline protease has an enzyme activity of 400,000 U / g, and the amount of alkaline protease added is 2% of the mass of Chlorella pulveratum powder; the papain has an enzyme activity of 600,000 U / g, and the amount of papain added is 0.2% of the mass of Chlorella pulveratum powder.

[0031] S3. Centrifuge the cell wall-broken Chlorella protein hydrolysate at 8000 r / min for 15 min, collect the supernatant, pass the supernatant through a ceramic membrane, collect the permeate, and obtain a clear Chlorella protein hydrolysate.

[0032] S4. Heat the clarified Chlorella proteinensis enzymatic hydrolysate to 60°C, then add powdered activated carbon, activated clay and hydrogen peroxide solution and keep warm and stir for 20 minutes. Then heat to 90°C~95°C and keep warm and stir for 60 minutes. After plate and frame filtration and titanium rod activated carbon filter, obtain the clarified Chlorella proteinensis peptide decolorizing solution.

[0033] The activated carbon is added at 10% of the mass of Chlorella pyrenoidosa powder. The activated clay has a decolorization rate of ≥95%, and the amount of activated clay added is 1% of the mass of Chlorella pyrenoidosa powder. The concentration of the hydrogen peroxide solution is 30%, and the amount of hydrogen peroxide solution added is 2% of the mass of Chlorella pyrenoidosa powder.

[0034] S5. After ultra-high temperature instantaneous sterilization, the decolorized solution of Chlorella peptide is concentrated by reverse osmosis technology to obtain Chlorella peptide concentrate.

[0035] Example 3: The preparation method of Chlorella protein-core peptide in this example includes the following steps: S1, adding dried Chlorella protein-core powder to water in small amounts and multiple times in a volume equal to 20 times the mass of Chlorella protein-core powder, stirring and dissolving evenly, and homogenizing under high pressure at 60 MPa for 3 times to obtain Chlorella protein-core peptide solution with broken cell walls.

[0036] S2. Heat the high-pressure homogenized Chlorella vulgaris protein-core liquid to 45℃~55℃, keep it at this temperature, add alkaline protease and papain for 6 hours to inactivate the enzymes.

[0037] The alkaline protease has an enzyme activity of 350,000 U / g, and the amount of alkaline protease added is 0.5% of the mass of Chlorella pulveratum powder; the papain has an enzyme activity of 600,000 U / g, and the amount of papain added is 2% of the mass of Chlorella pulveratum powder.

[0038] S3. Centrifuge the cell wall-broken Chlorella protein hydrolysate at 8000 r / min for 15 min, collect the supernatant, pass the supernatant through a ceramic membrane, collect the permeate, and obtain a clear Chlorella protein hydrolysate.

[0039] S4. Heat the clarified Chlorella proteinensis enzymatic hydrolysate to 60°C, then add powdered activated carbon, activated clay and hydrogen peroxide solution and keep warm and stir for 40 min. Then heat to 90°C~95°C and keep warm and stir for 20 min. After plate and frame filtration and titanium rod activated carbon filter, obtain the clarified Chlorella proteinensis peptide decolorizing solution.

[0040] The activated carbon is added at 2% of the mass of the Chlorella pyrenoidosa powder. The activated clay has a decolorization rate of ≥95%, and the amount of activated clay added is 3% of the mass of the Chlorella pyrenoidosa powder. The concentration of the hydrogen peroxide solution is 30%, and the amount of hydrogen peroxide solution added is 0.2% of the mass of the Chlorella pyrenoidosa powder.

[0041] S5. After ultra-high temperature instantaneous sterilization, the decolorized solution of Chlorella peptide is concentrated by reverse osmosis technology to obtain Chlorella peptide concentrate.

[0042] Comparative Example 1: Referring to Example 1, the difference between this comparative example and Example 1 is that: in step S4, this comparative example only uses activated carbon for decolorization and does not use activated clay and hydrogen peroxide solution.

[0043] Comparative Example 2: Referring to Example 2, the difference between this comparative example and Example 2 is that in step S4, this comparative example only uses activated clay for decolorization and does not use activated carbon and hydrogen peroxide solution.

[0044] Comparative Example 3: Referring to Example 3, the difference between this comparative example and Example 3 is that in step S4, this comparative example only uses hydrogen peroxide solution for decolorization and does not use activated carbon and activated clay.

[0045] The flavor of the chlorella peptides prepared in the examples and comparative examples was determined by sensory evaluation. The sensory evaluation panel consisted of 20 sensory evaluators (10 men and 10 women) who evaluated the flavor of the chlorella peptides prepared in Examples 1-3 and Comparative Examples 1-3, respectively. The sensory scoring criteria were as follows: a score of 0 to 5 was assigned to indicate the severity of any algal odor or other unpleasant off-odors, from mild to severe. 0 points indicated no algal odor or other unpleasant off-odors, and 5 points indicated extremely severe algal odor or other unpleasant off-odors. Scoring was accurate to one decimal place, and the final score was the average, retained to two decimal places.

[0046] The sensory evaluation results are shown in Table 1. The flavor of the Chlorella peptides in Examples 1-3 is better than that in Comparative Examples 1-3. Moreover, the Chlorella peptide prepared in Example 1 has the best flavor, with virtually no algae-like smell or other unacceptable off-odors.

[0047] Table 1 Flavor scores of Chlorella peptides extracted using different processes

[0048] Flavor rating Example 1 0.23 Example 2 0.45 Example 3 0.38 Comparative Example 1 2.21 Comparative Example 2 1.87 Comparative Example 3 1.56

[0049] The whiteness of the chlorella peptides prepared in each example and comparative example was determined. The whiteness measurement method was as follows: the wavelength of the whiteness meter was adjusted to 457 nm and calibrated with a standard white plate; the chlorella peptide powder was passed through a 150-mesh sieve and mixed evenly, then compacted with a constant pressure powder compactor until the surface was smooth and texture-free before measurement. Three measurements were taken from the same batch of samples, and the average value of the three measurements was taken, retaining two decimal places. The measurement results are shown in Table 2. The higher the whiteness value, the whiter the color; a whiteness value above 50 indicates that the color is close to white.

[0050] Table 2. Whiteness of Chlorella peptides extracted using different processes

[0051] Whiteness value Example 1 71.23 Example 2 61.40 Example 3 64.63 Comparative Example 1 15.57 Comparative Example 2 14.36 Comparative Example 3 16.82

[0052] As shown in Table 2, the Chlorella peptide powder prepared in Examples 1-3 is white in color, exhibiting good whiteness and decolorization effect, thus having a wider range of applications. In contrast, the whiteness value of the Chlorella peptide prepared in Comparative Examples 1-3 is only around 15, far below 50.

[0053] The molecular weights of Examples 1-3 were determined according to the method specified in GB / T 22492 (Soybean Peptide Powder). The results are shown in Table 3.

[0054] Table 3. Molecular weights of Chlorella peptides extracted using different processes

[0055]

[0056]

[0057] The protein hydrolysates of the chlorella peptides prepared in Examples 1-3 all had a molecular weight of less than 1000u, and the proportion of these hydrolysates was greater than 85%, which is more conducive to human digestion and absorption compared to large molecular weight protein peptides.

[0058] In summary, this invention utilizes a ceramic membrane for retention combined with activated carbon, activated clay, and hydrogen peroxide for decolorization, covering a wider decolorization range and significantly improving the decolorization effect. Furthermore, it eliminates the use of organic solvents, preserving the activity of the chlorella peptide nucleus as much as possible and reducing environmental pollution. The chlorella peptide nucleus produced by this invention is nearly white, its aqueous solution is nearly colorless, and it has no characteristic fishy odor of the raw materials, thus possessing a wider range of applications.

[0059] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a protein-nucleated Chlorella peptide, characterized in that, Includes the following steps: S1. Add the dried Chlorella pulveratum powder in small amounts to water at a ratio of 10 to 20 times the weight of the Chlorella pulveratum powder, stir until dissolved, and homogenize under high pressure at 30 to 60 MPa for 3 to 5 times to obtain Chlorella pulveratum slurry with broken cell walls. S2. Heat the high-pressure homogenized Chlorella vulgaris protein-core liquid to 45℃~55℃, keep it at this temperature, add alkaline protease and papain for 3h~6h to inactivate the enzymes. S3. Centrifuge the cell wall broken Chlorella protein hydrolysate at 8000 r / min for 15 min, collect the supernatant, pass the supernatant through a ceramic membrane, collect the permeate, and obtain a clear Chlorella protein hydrolysate. S4. Heat the clarified Chlorella proteoglycans enzymatic hydrolysate to 60°C, then add activated carbon, activated clay and hydrogen peroxide solution and keep warm and stir for 20 min to 40 min. Then heat to 90°C to 95°C and keep warm and stir for 20 min to 60 min. After pressure filtration and fine filtration, a clear Chlorella proteoglycans peptide decolorizing solution is obtained. S5. Sterilize and concentrate the decolorizing solution of Chlorella protein nucleus peptide to obtain concentrated Chlorella protein nucleus peptide solution.

2. The method for preparing a protein-nucleated chlorella peptide according to claim 1, characterized in that, In step S2, the alkaline protease activity is 350,000 U / g to 400,000 U / g, and the amount of alkaline protease added is 0.5% to 4% of the mass of Chlorella pyrenoidosa powder. The papain enzyme activity is 100,000 U / g to 600,000 U / g, and the amount of papain added is 0.2% to 2% of the mass of Chlorella vulgaris powder.

3. The method for preparing a protein-nucleated Chlorella peptide according to claim 1, characterized in that, In step S4, the activated carbon is in powder form, and the amount of activated carbon added is 2% to 10% of the mass of Chlorella pyrenoidosa powder.

4. The method for preparing a protein-nucleated Chlorella peptide according to claim 3, characterized in that, In step S4, the decolorization rate of activated clay is ≥95%, and the amount of activated clay added is 1% to 5% of the mass of Chlorella pyrenoidosa powder.

5. The method for preparing a protein-nucleated Chlorella peptide according to claim 4, characterized in that, In step S4, the concentration of hydrogen peroxide solution is 30%, and the amount of hydrogen peroxide solution added is 0.2% to 2% of the mass of Chlorella pyrenoidosa powder.

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