Chlorella pyrenoidosa high-F-value oligopeptide as well as preparation method and application thereof

Abstract

According to the chlorella pyrenoidosa high-F-value peptide and the preparation method thereof, parameters in the enzymolysis and adsorption processes are strictly controlled, and the enzymolysis of the incision enzyme and the excision enzyme not only can efficiently hydrolyze chlorella protein and generate more peptide fragments with biological activity, but also can be used for preparing the chlorella pyrenoidosa high-F-value peptide. And the characteristics of high industrial production cost and poor stability caused by wide enzyme cutting sites are solved. The prepared chlorella pyrenoidosa high-F-value peptide reaches 36.90 + / -8.18, the F value of the chlorella pyrenoidosa high-F-value peptide is increased by 12.72 times compared with the chlorella pyrenoidosa raw material (2.90 + / -0.07), and in addition, the molecular weight of the high-F-value oligopeptide is 1t; peptides with the molecular weight of 1000 Da account for 95-99.40%, the characteristics of oligopeptides are met, the molecular weight is small, and digestion and absorption are easy. The prepared high-F-value oligopeptide can accelerate metabolism of ethyl alcohol in the liver and protect the liver from being damaged by the ethyl alcohol, and has the effects of dispelling the effects of alcohol and protecting the liver.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide preparation, and specifically relates to a pyrenoidosa chlorella high F value oligopeptide and a preparation method and application thereof. Background Art

[0002] High F value oligopeptides are oligopeptide systems composed of 3-9 amino acids, with a ratio of branched-chain amino acid content to aromatic amino acid content exceeding 20. High F value oligopeptides have multiple functional activities due to their unique amino acid composition, such as antioxidant, anti-fatigue, anti-drunkenness and liver protection activities, and therefore have important application potential in alleviating alcoholic and chemical liver damage. At present, domestic and foreign researchers are developing high F value oligopeptide products involving proteins from three different raw material sources, including plant-based proteins, such as corn, hemp seeds and pine nuts; aquatic-based proteins, such as Antarctic krill, oysters and mussels; and milk-based proteins, such as goat milk and cow milk.

[0003] The key point of high F value peptide preparation technology is to separate aromatic amino acids to the greatest extent while retaining branched amino acids as much as possible. At present, proteolysis is the main way to prepare high F value peptides, which is divided into single enzyme hydrolysis and compound enzyme hydrolysis. Among them, compound enzyme hydrolysis is more popular due to its high efficiency. Compound enzyme hydrolysis first breaks the peptide chain bonds with large molecular weight in the raw material through endonucleases such as neutral protease, pepsin, and alkaline protease to expose aromatic amino acids, and then selects exonucleases such as flavor protease to release aromatic amino acids. The removal of aromatic amino acids is a key point to improve the F value. There are mainly activated carbon and resin adsorption. Activated carbon is a common material for aromatic amino acid adsorption due to its easy operation, low cost and good adsorption. However, in the preparation process of high F value peptides, there are problems such as wide enzyme cleavage sites, difficult to control the hydrolysis process, low release rate of branched and aromatic amino acids, and low protein recovery rate. How to select enzymes that specifically cleave aromatic amino acids to achieve directional preparation and hydrolysis control is an urgent problem to be solved; at the same time, in the process of aromatic amino acid adsorption, differential adsorption means are limited.

[0004] Chlorella is a kind of microalgae with a protein content of up to 50-60%. It is also rich in essential amino acids, such as leucine, arginine and lysine, which account for about 7% of the total protein content; other essential amino acids such as isoleucine, phenylalanine, threonine and valine, usually account for about 4% of the total protein content. Studies have found a variety of biological activities in Chlorella hydrolyzed peptides, such as antioxidant and liver protection activities. Therefore, Chlorella is a good source for preparing high F value peptides, but the effect of preparing high F value peptides from Chlorella is not good at present. Summary of the invention

[0005] To solve the above problems, the present invention provides a high F value peptide of pyrenoid chlorella and a preparation method thereof. The present invention uses a specific endonuclease and an exonuclease to enzymatically hydrolyze pyrenoid chlorella, avoiding the large enzyme consumption caused by the wide range of enzyme cutting sites, saving costs, and preparing high-efficiency, stable and repeatable peptides. At the same time, the high F value peptide successfully prepared using chlorella as a raw material solves the problem of the current lack of raw material sources for preparing high F value peptides. The present invention can be used to prepare medicines, health products and foods with the effects of sobering up and protecting the liver.

[0006] The pyrenoid chlorella high F value peptide provided by the present invention is prepared by crushing the pyrenoid chlorella, mixing it with water, freezing and thawing it, and ultrasonically treating it to prepare a homogenate; adding alkaline protease to perform enzymolysis, and then adding flavor protease to perform enzymolysis; after the enzymolysis product is adsorbed by activated carbon, the supernatant is freeze-dried to obtain the pyrenoid chlorella high F value peptide.

[0007] The pulverizing of Chlorella pyrenoidosa, as one example, is to pulverize Chlorella pyrenoidosa by air flow collision.

[0008] As a preferred embodiment of the present invention, Chlorella pyrenoidosa and distilled water are mixed at a weight-to-volume ratio of 1:20-1:30 (g / ml);

[0009] As a preferred embodiment of the present invention, the pH of the alkaline protease hydrolysis is 8.0-9.0 and the temperature is 50°C.

[0010] The pH of the flavor protease enzymatic hydrolysis system is 7.0-8.0, the temperature is 50° C., and the enzymatic hydrolysis time is 2.5-3.5 hours.

[0011] As a preferred embodiment of the present invention, the activated carbon is a 200-mesh activated carbon (THX-200A) mixed with wood and coconut shell to remove aromatic amino acids.

[0012] In another aspect, the present invention provides a use of the Chlorella pyrenoidosa high F value peptide, which is the use in preparing a product for sobering up or protecting the liver.

[0013] The present invention also provides a product for sobering up or protecting the liver, which contains the pyrenoid chlorella high F value peptide.

[0014] The present invention strictly controls the parameters in the enzymatic hydrolysis and adsorption process. The endo- and exo-enzymatic hydrolysis not only efficiently hydrolyzes the chlorella protein and produces more biologically active peptides, but also solves the high cost and poor stability of industrial production due to the wide range of enzyme cleavage sites. The high F value peptide of the pyrenoid chlorella prepared by the present invention reaches 36.90±8.18, and its F value is increased by 12.72 times compared with the pyrenoid chlorella raw material (2.90±0.07). In addition, the peptides with a molecular weight of <1000Da of the high F value oligopeptide account for 95-99.40%, which meets the characteristics of the oligopeptide, has a small molecular weight, and is easy to digest and absorb. The high F value oligopeptide prepared by the present invention can accelerate the metabolism of ethanol in the liver, protect the liver from alcohol damage, and has the effect of sobering up and protecting the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comparison of the enzymatic effects of the two-step enzyme screening process in the present invention (different enzyme types on DH and PR (A), F value (B) and OD 220 / OD 280 (C) impact);

[0016] Figure 2 Comparison of the enzymatic hydrolysis effects of the composite enzymes in the present invention (different composite enzyme types on DH and PR (A), F value (B) and OD 220 / OD 280 (C) impact);

[0017] Figure 3 A comparison diagram of the enzymatic hydrolysis effect of alkaline protease in the present invention (the influence of different alkaline protease addition amounts (A), hydrolysis time (B), and substrate concentration (C) on the hydrolysis effect of Chlorella vulgaris);

[0018] Figure 4 A comparison diagram of the enzymatic hydrolysis effect of the flavor protease in the present invention (the effect of different flavor protease addition amounts (A) and hydrolysis time (B) on the hydrolysis effect of Chlorella pyrenoidosa);

[0019] Figure 5 A comparison diagram of the activated carbon adsorption effect in the present invention (the effects of activated carbon type (A), pH (B), addition amount (C) and adsorption time (D) on the removal effect of aromatic amino acids);

[0020] Figure 6 The amino acid composition of the high F value oligopeptide of Chlorella pyrenoidosa in the present invention;

[0021] Figure 7 (B) is the reaction kinetic curve of the chlorella pyrenoidosa high F value oligopeptide catalyzing alcohol metabolism in the present invention; (B) is the dose-effect relationship curve of the chlorella pyrenoidosa high F value oligopeptide;

[0022] Figure 8This is the effect of the high F value oligopeptide of Chlorella pyrenoidosa on the liver status of mice with acute alcoholic liver injury. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with embodiments and drawings.

[0024] The definition of high F-value peptides: the molar ratio of branched-chain amino acids (BCAAs, including leucine, isoleucine and valine) to aromatic amino acids (AAAs, tryptophan, tyrosine and phenylalanine) is greater than 20, and the content of AAAs is less than 2%. The calculation formula is as follows:

[0025]

[0026] The definition of protein recovery rate (PR) is: the ratio of the total protein content in the hydrolysate to the total protein content in the raw material. The total protein content in the hydrolysate is determined by the Folin-phenol method, and the total protein content in the raw material is determined by the Kjeldahl method.

[0027] Definition of degree of hydrolysis (DH): the ratio of the amino nitrogen content in the hydrolyzate to the total protein nitrogen content in the hydrolyzate. The amino nitrogen content is determined by the indene tricopper colorimetric method, and the calculation formula is as follows:

[0028]

[0029] Where:

[0030] h-mmol of peptide bonds cleaved per gram of protein after hydrolysis;

[0031] h tot - mmol of peptide bonds per gram of raw material protein, calculated based on the amino acid composition of Chlorella protein tot In this experiment, 8.44mmol:g -1 .

[0032] OD 220 / OD 280 It is the ratio of the absorbance at 220nm and 280nm after the enzymatic solution is diluted several times.

[0033] Example 1: Effect of endonuclease type on the preparation of high F value oligopeptides from Chlorella pyrenoidosa

[0034] The method for treating the pyrenoid chlorella is to pre-crush the pyrenoid chlorella by air flow collision, then mix it with distilled water in a certain ratio, place it in a plate with a diameter of 8 cm and a thickness of 2 cm at -20°C for 12 hours, then thaw it to an ice-water mixture ratio of about 1:10 (v / v), perform power ultrasound for 3 minutes, repeat freezing and thawing 4 times, and use the mixed solution thawed for the first step of enzymatic hydrolysis. The enzyme for the first step of enzymatic hydrolysis is selected from seven enzymes including alkaline protease (Alc), papain (Pap), flavor protease (Fla), pepsin (Pep), trypsin (Try), neutral protease (Neu) and chymotrypsin (Chy), and all enzymatic hydrolysis conditions are enzymatic hydrolysis for 4 hours under the optimal pH and temperature conditions of the corresponding enzymes, and after the enzymatic hydrolysis is completed, the enzyme is inactivated and cooled to room temperature, the pH is adjusted to 4.5, 6g / 100mL of activated carbon is added, and adsorption is carried out at 150rpm for 90 minutes in a 25°C water bath shaker.

[0035] Based on DH, PR, F value and OD 220 / OD 280 The best enzyme was screened for the index, and the results were as follows Figure 1 The DH and PR of alkaline protease were 24.95±4.52% and 48.04±4.01%, respectively, and those of neutral protease were 21.79±1.61% and 47.61±4.01%. The F value and OD value of alkaline protease hydrolysate after dearomatization by activated carbon were 220 / OD 280 The F value and OD value of the neutral protease hydrolysate after dearomatization of activated carbon were 8.59±0.01 and 7.40±0.14, respectively. 220 / OD 280 They were 8.07±0.15 and 10.49±0.75 respectively, which were significantly higher than the enzymatic hydrolysis effects of other enzymes.

[0036] Example 2: Effects of different endo- and exo-enzyme combined hydrolysis on the preparation of high F value oligopeptides from Chlorella pyrenoidosa

[0037] After the enzymes for the first enzymatic hydrolysis are determined (alkaline protease, neutral protease), the enzymes for the second enzymatic hydrolysis are screened. After the first enzymatic hydrolysis is completed, the mixture is cooled to room temperature, and papain, neutral protease, pepsin, trypsin and flavor protease are selected to continue the enzymatic hydrolysis.

[0038] According to DH, PR, OD 220 / OD 280 The best enzyme for the second step was screened by using the F value. The results are as follows Figure 2 The DH and PR of the enzymatic hydrolysate of alkaline protease and flavor protease were 28.17±4.49% and 67.45±2.03%, respectively. The F value and OD value after dearomatization by activated carbon were 220 / OD 280The results are 10.55±0.99 and 14.81±0.66 respectively, which are better than alkaline protease or neutral protease combined with other enzymes. This shows that the combination of alkaline protein and flavor protease is more suitable for the enzymatic hydrolysis and dearomatization of Chlorella vulgaris.

[0039] Example 3: Effects of different factors on the enzymatic hydrolysis of alkaline protease

[0040] (1) After repeated freezing and thawing of Chlorella vulgaris at a mass ratio of 1:20, alkaline protease was added at an enzyme-substrate ratio (E / S) of 1%, 2%, 3%, 4%, and 5% at a reaction temperature of 50°C and a pH of 8. The reaction was carried out for 4 hours. After the hydrolysis was completed, the enzyme was inactivated and cooled to room temperature. The pH of the enzymatic hydrolyzate was adjusted to 4.5, and 6 g / 100 mL of activated carbon was added for adsorption for 90 minutes. The supernatant was obtained by centrifugation at 10,000 rpm for 15 minutes.

[0041] DH, PR, OD 220 / OD 280 When the amount of alkaline protease added was 4%, DH, PR, OD 220 / OD 280 The and F values ​​were 17.82±0.83, 79.75±1.50, 25.46±0.78 and 7.86±0.24, respectively, which were higher than the enzymatic hydrolysis effects of E / S ratios of 1%, 2% and 3%, and equivalent to the enzymatic hydrolysis effect of 5%. Therefore, 4% was selected as the E / S ratio of alkaline protease.

[0042] (2) After repeated freezing and thawing of Chlorella vulgaris at a mass ratio of 1:20, 4% alkaline protease was added at a reaction temperature of 50°C and a pH of 8, and enzymatic hydrolysis was performed for 2-6 hours. After the hydrolysis was completed, the enzyme was inactivated and cooled to room temperature, the pH of the enzymatic hydrolyzate was adjusted to 4.5, 6 g / 100 mL of activated carbon was added for adsorption for 90 minutes, and the supernatant was obtained by centrifugation at 10,000 rpm for 15 minutes.

[0043] DH, PR, OD 220 / OD 280 When the alkaline protease enzymatic hydrolysis time is 5h, DH, PR, OD 220 / OD 280 and F values ​​were 18.34±0.86, 77.57±4.73, 10.47±0.47 and 7.89±0.02 respectively. The enzymatic hydrolysis effect was better than that of 2h, 3h and 4h, and equivalent to that of 6h. Therefore, 5h was selected as the enzymatic hydrolysis time of alkaline protease.

[0044] (3) After repeated freezing and thawing of protein nucleus Chlorella with mass ratios of 1:10, 1:15, 1:20, 1:25 and 1:30, 4% alkaline protease was added at a reaction temperature of 50°C and a pH of 8, and the reaction was carried out for 5 hours. After the hydrolysis was completed, the enzyme was inactivated and cooled to room temperature. The pH of the enzymatic hydrolyzate was adjusted to 4.5, 6 g / 100 mL of activated carbon was added for adsorption for 90 minutes, and the supernatant was collected by centrifugation at 10,000 rpm for 15 minutes.

[0045] DH, PR, OD 220 / OD 280 The and F values ​​were used as indicators to examine the enzymatic effect of alkaline protease. When the substrate concentration ratio was 1:25, DH, PR, OD 220 / OD 280 and F values ​​were 21.60±0.16, 68.29±1.97, 18.48±0.86 and 7.92±1.11, respectively. Comprehensive comparison showed that the enzymatic hydrolysis effect of alkaline protease at 1:25 was better than that at other substrate concentrations.

[0046] Example 4: Effects of different factors on the enzymatic hydrolysis of flavor protease

[0047] (1) After alkaline protease hydrolysis, boil and inactivate the enzyme for 10 minutes, cool to room temperature, and adjust the pH of the hydrolyzate to 7.5. Add flavor protease at an enzyme-substrate ratio (E / S) of 1%, 2%, 3%, 4%, and 5%, shake in a 50°C water bath shaker for 3 hours, inactivate the enzyme, and cool to room temperature. Adjust the pH of the hydrolyzate to 4.5, add 6g / 100mL activated carbon for adsorption for 90 minutes, and centrifuge at 10,000rpm for 15 minutes to obtain the supernatant.

[0048] DH, PR, OD 220 / OD 280 The F value was used as an indicator to examine the enzymatic hydrolysis effect of flavor protease. With the increase of enzyme addition, the DH of the enzymatic hydrolysis product showed an increasing trend, and the F value first increased and then decreased. When the enzyme addition amount was 3%, the F value was 15.47±0.56, and DH tended to stabilize at 35.20±0.03%. Therefore, the enzyme addition amount of flavor protease was determined to be 3%.

[0049] (2) After alkaline proteinase hydrolysis, the enzyme was inactivated by boiling for 10 min, cooled to room temperature, the pH of the hydrolyzate was adjusted to 7.5, 3% flavor protease was added to the hydrolyzate and reacted for 2-6 h, after the hydrolysis was completed, the enzyme was inactivated and cooled to room temperature, the pH of the hydrolyzate was adjusted to 4.5, 6 g / 100 mL of activated carbon was added for adsorption for 90 min, and the supernatant was collected by centrifugation at 10,000 rpm for 15 min.

[0050] DH, PR, OD 220 / OD 280The DH, PR, and OD values ​​of the enzymatic hydrolysis solution of alkaline protease and flavor protease were used as indicators to investigate the enzymatic hydrolysis effect of flavor protease. When the enzymatic hydrolysis time was 3 h, the DH, PR, and OD values ​​of the enzymatic hydrolysis solution of alkaline protease and flavor protease were 220 / OD 280 and F values ​​were 30.90±1.85, 88.69±3.41, 12.50±0.51 and 13.38±0.93, respectively. Taking all factors into consideration, the enzymatic hydrolysis time was selected as 3 h.

[0051] Example 5: Effects of different activated carbon types and conditions on the removal of aromatic amino acids

[0052] (1) After the protein nucleus Chlorella was hydrolyzed by alkaline protease and flavor protease in two steps, the enzymatic solution was inactivated and cooled to room temperature, and the supernatant was obtained by centrifugation at 8000rpm for 20min. The pH of the enzymatic solution was adjusted to 4.5, and five different types of activated carbon were added to the enzymatic solution: THX-200A (Type 1), THX-200B (Type 2), THX-200C (Type 1), Meilun activated carbon (Type 4), and coconut shell activated carbon (Type 5). The amount of each activated carbon (m:v) was 5g / 100mL. The solution was adsorbed at 25℃ for 90min and centrifuged at 10000rpm for 15min to obtain the supernatant.

[0053] F value and OD 220 / OD 280 For the index screening of activated carbon, Type 1 activated carbon has the highest F value and OD among the five activated carbons, which are 13.35±0.46 and 16.51±1.17, respectively. Therefore, THX-200A can adsorb aromatic amino acids and retain branched-chain amino acids to the greatest extent.

[0054] (2) After the protein nucleus Chlorella was hydrolyzed by alkaline protease and flavor protease in two steps, the enzymatic solution was inactivated and cooled to room temperature, and the supernatant was obtained by centrifugation at 8000 rpm for 20 min. The pH of the enzymatic solution was adjusted to 2, 3, 4, 5 and 6 respectively, and THX-200A activated carbon was added at a ratio of 5 g / 100 mL, adsorbed at 25°C for 90 min, and centrifuged at 10000 rpm for 15 min to obtain the supernatant.

[0055] F value and OD 220 / OD 280 To optimize the pH of activated carbon, the F value increases with the increase of pH. When the pH is 6, the F value is the highest at 17.31±2.59, which is the most suitable pH for adsorption of this type of activated carbon.

[0056] (3) After the protein nucleus Chlorella was hydrolyzed by alkaline protease and flavor protease in two steps, the enzymatic solution was inactivated and cooled to room temperature, and the supernatant was collected by centrifugation at 8000 rpm for 20 min. The pH of the enzymatic solution was adjusted to 6, and THX-200A activated carbon was added at a ratio of 1 g / 100 mL, 3 g / 100 mL, 5 g / 100 mL, 7 g / 100 mL and 9 g / 100 mL, respectively, and adsorbed at 25 ° C for 90 min, and the supernatant was collected by centrifugation at 10000 rpm for 15 min.

[0057] F value and OD 220 / OD 280 To optimize the amount of activated carbon added, when the amount of activated carbon used is 1g / 100mL-5g / 100mL, the F value and OD 220 / OD 280 In a growing state, continue to increase the amount of activated carbon, F value and OD 220 / OD 280 Therefore, the amount of activated carbon used was selected to be 5 g / 100 mL.

[0058] (4) After the protein nucleus Chlorella was hydrolyzed by alkaline protease and flavor protease in two steps, the enzymatic solution was inactivated and cooled to room temperature, and the supernatant was obtained by centrifugation at 8000 rpm for 20 min. The pH of the enzymatic solution was adjusted to 6, and THX-200A activated carbon was added at a ratio of 5 g / 100 mL, respectively, and adsorbed at 25°C for 30, 60, 90, 120 and 150 min, and the supernatant was obtained by centrifugation at 10000 rpm for 15 min.

[0059] F value and OD 220 / OD 280 Optimize the activated carbon adsorption time for indicators, the adsorption time is 0-90min, F value and OD 220 / OD 280 As the adsorption time increases, when the adsorption time reaches 90min, the adsorption capacity of aromatic amino acids on activated carbon almost reaches saturation. As time continues to increase, the F value and OD 220 / OD 280 Reduced, therefore, the activated carbon adsorption time was selected as 90min.

[0060] Example 6: Preparation method of high F value peptide from Chlorella pyrenoidosa

[0061] The protein nucleus chlorella was first pre-crushed by air flow collision, then mixed with distilled water at a ratio of 1:25 (g / mL), placed in a plate with a diameter of 8 cm and a thickness of 2 cm at -20°C for 12 hours, then thawed to an ice-water mixture ratio of about 1:10 (v / v) for 3 minutes of power ultrasound, repeated freezing and thawing 4 times, the pH of the homogenate was adjusted to 8, 4% alkaline protease was added, and the enzyme was oscillated in a 50°C water bath for 5 hours, and the enzyme was inactivated in a boiling water bath to obtain the first step hydrolyzate. After cooling, the pH was adjusted to 7.5, 3% flavor protease was added, and the enzyme was oscillated in a 50°C water bath for 3 hours, and the enzyme was inactivated in a boiling water bath to obtain the second step hydrolyzate. After cooling, the pH was adjusted to 6.0, THX-200A activated carbon powder (5g / 100mL) was added, and the mixture was oscillated and adsorbed at 25°C for 90 minutes. The supernatant was taken after centrifugation at 10000rpm for 15 minutes, and the protein nucleus chlorella high F value peptide (CHFO) was obtained after freeze drying.

[0062] The amino acid determination of the high F value peptide of Chlorella pyrenoidosa was carried out by using an amino acid automatic analyzer. The amino acid composition of CHFO is as follows Figure 6 As shown, the relative contents of BCAAs (Val, Ile and Leu) were 6.35%, 3.04% and 11.48%, respectively, accounting for 20.88% of the total amino acid composition. The relative contents of AAAs (Tyr and Phe) in CHFO were significantly reduced, 0.00% and 0.58%, respectively. When the amino acid composition of pyrenoid chlorella powder and pyrenoid chlorella high F value peptide powder was compared, the F value increased from 2.90 to 36.90 (F value is >20). Meet the requirements of high F value peptides.

[0063] The molecular weight of the pyrenoid chlorella high F value peptide powder was determined by gel exclusion chromatography. The molecular weight was mainly concentrated below 1000Da, accounting for 99.40%, of which 500-1000Da accounted for 25.00%, 300-500Da accounted for 54.71, and <300Da accounted for 19.90, which was consistent with the characteristics of oligopeptides, which were more easily absorbed by the body and had higher safety.

[0064] Table 1 CHFO molecular weight distribution

[0065]

[0066] Example 7: Effect of high F value oligopeptide from Chlorella vulgaris on the reaction curve of alcohol dehydrogenase catalyzed alcohol oxidation

[0067] The reaction kinetics curves of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 50 mg / mL CHFO were used to intervene in the alcohol dehydrogenase (ADH) catalyzed alcohol oxidation process. Figure 7As the reaction time increases, NADH continues to rise and the absorbance value (OD 340nm ) continues to increase. As CHFO concentration increases, OD 340nm The rate of increase is faster, indicating that the enzymatic reaction rate is faster. When the CHFO concentration is 0-5mg / mL, the ADH activation rate increases with the concentration, showing a good dose-effect relationship, indicating that CHFO does effectively intervene in the process of ADH catalyzing alcohol oxidation. When the CHFO concentration reaches 5mg / mL, the ADH activity increases to 75%, greatly increasing the rate of alcohol metabolism.

[0068] Case study 8: Effects of high F-value peptides from Chlorella vulgaris on acute alcoholic liver injury in mice

[0069] The following is a mouse model of acute drinking to further illustrate the physiological function of Chlorella high F value oligopeptide in sobering up and protecting the liver.

[0070] Male Kunming mice were selected and fed adaptively for 1 week, and then 10 mice were randomly divided into five groups, namely normal group (NC), model group (MC), positive control group (PC), low-dose group (L-CHFO), and high-dose group (H-CHFO). Each experimental group was gavaged for 14 days according to the set gavage content and dose. Alcohol modeling was performed 4 hours after the last gavage, and food and water were fasted. The second alcohol modeling was performed 12 hours later, and food and water were fasted. After 12 hours, the mice were bled from the eyeballs and killed by dislocating the neck for sampling.

[0071] The H&E staining results of the liver sections of mice in each group are shown in Figure 8 As shown in the figure. The liver surface morphology of the normal group was normal, the hepatocytes had clear outlines and intact structures, while a large number of hepatocytes were necrotic and infiltrated with inflammatory cells in the model group, the hepatic cords were disordered, and vacuoles appeared. In the positive control group and CHFO group, the central veins of the hepatocytes were clear, the cell structure was intact, and there were fewer vacuoles around the nucleus, indicating that the protein nucleus Chlorella high F value oligopeptide can significantly reverse the liver damage caused by alcohol.

Claims

1. A high F value peptide from Chlorella pyrenoidosa, characterized in that: The pyrenoid chlorella high F value peptide is prepared by crushing the pyrenoid chlorella, mixing it with water, freezing and thawing it, and ultrasonically treating it to prepare a homogenate; adding alkaline protease for enzymolysis, and then adding flavor protease for enzymolysis; adsorbing the enzymolysis product with activated carbon, and freeze-drying the supernatant.

2. The high F value peptide of Chlorella vulgaris according to claim 1, characterized in that: The pulverizing of Chlorella pyrenoidosa is to pulverize the Chlorella pyrenoidosa through air flow collision.

3. The high F value peptide of Chlorella pyrenoidosa according to claim 1, characterized in that: The pyrenoid chlorella and distilled water are mixed in a weight-to-volume ratio of 1:20-1:

30.

4. The high F value peptide of Chlorella pyrenoidosa according to claim 1, characterized in that: The pH of the alkaline protease enzymolysis is 8.0-9.0 and the temperature is 50°C.

5. The high F value peptide of Chlorella pyrenoidosa according to claim 1, characterized in that: The pH of the flavor protease enzymatic hydrolysis system is 7.0-8.0, the temperature is 50° C., and the enzymatic hydrolysis time is 2.5-3.5 hours.

6. The high F value peptide of Chlorella pyrenoidosa according to claim 1, characterized in that: The activated carbon is a mixture of wood and coconut shell with a particle size of 200 meshes and is used to remove aromatic amino acids.

7. Use of the high F value peptide of Chlorella vulgaris according to claim 1 in preparing a product for sobering up or protecting the liver.

8. A product for sobering up or protecting the liver, characterized in that: The product contains the pyrenoid chlorella high F value peptide according to claim 1.

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