Preparation method of spirulina polypeptide and its application in alcoholic liver inhibition

Abstract

The present invention provides a preparation method of spirulina polypeptide and its application in alcoholic liver inhibition, the preparation method comprises the following steps: step 1, pre-treating the spirulina powder raw material by colloid mill grinding method and repeated freezing and thawing to obtain a sample solution; step 2, degrading protein by bacterial enzyme synergistic method: adding Bacillus amyloliquefaciens to the sample solution for bacterial hydrolysis, and then enzymolysis with trypsin and papain; step 3, concentrating the sample supernatant after protein degradation, centrifuging, and performing membrane treatment on the supernatant after centrifugation to obtain a spirulina polypeptide liquid with a molecular weight less than 500Da, and then spray drying to obtain a spirulina polypeptide finished product. The spirulina polypeptide preparation method provided by the present invention has low cost, excellent extraction effect, and simple operation, and has high application value and market prospects in alcoholic liver inhibition.

Description

Technical Field

[0001] The invention belongs to the technical field of spirulina polypeptide processing and preparation, and specifically relates to a preparation method of spirulina polypeptide and application of the spirulina polypeptide in alcoholic liver inhibition. Background Art

[0002] As a natural nutritional supplement, Spirulina peptides have potential health benefits, especially in terms of antioxidant, immunomodulatory and anti-inflammatory aspects. However, although some studies have supported its potential in these areas, the specific mechanism of action and clinical application still require more scientific research to further verify and understand its effects and safety.

[0003] Alcoholic liver disease (ALD) is one of the common health problems in the world. Its pathogenesis is a series of liver damage caused by long-term and / or short-term heavy drinking. Alcohol-induced liver inflammation and oxidative stress are one of the important characteristics of alcoholic hepatitis. Existing technologies have shown that oligopeptides have the function of preventing the formation of alcoholic liver disease (such as public articles: Application of a modified peptide of Acanthophora monocyclica in the preparation of drugs for the treatment of alcoholic liver damage, molecular mechanism of intervention of corn peptides on alcohol-induced hepatocyte apoptosis and metabolism and sobering structure-activity relationship of QLLPF, sobering and liver-protecting effects of corn peptide preparations, protective mechanism of corn peptides on acute alcoholic liver damage in mice and ADH activation mechanism, protective effect of selenium-rich peptides of Cardamine violacea on ethanol-induced THLE-2 hepatocyte damage, study on the effect of soybean peptides on alcoholic liver damage in mice, preventive effect of peptides from fruiting bodies of Volvariella volvacea on acute alcoholic liver in mice and influence on intestinal flora). These studies show that bioactive peptides can accelerate the metabolism of ethanol by the liver, thus having a sobering effect and reducing the toxicity of alcohol to the liver. In addition, oligopeptides have strong antioxidant and anti-inflammatory properties, which can effectively inhibit alcohol-induced peroxidative stress and inflammation, and provide liver protection. In addition, bioactive peptides have shown significant protective effects on various types of liver damage, including alcoholic, chemical, drug-induced and immune liver damage. Spirulina is rich in more than 70% protein and is a good raw material for preparing peptides. Currently, peptides prepared with it as a raw material have potential health benefits as natural nutritional supplements, especially in terms of antioxidant, immunomodulatory and anti-inflammatory aspects. However, there have been no reports on related studies on the inhibition of alcoholic liver disease using spirulina peptides as research objects, and there is no sign of the technology for preparing spirulina-based active peptides that can prevent alcoholic liver disease.

[0004] China is the country with the largest production of spirulina in the world, and Otog Banner in Inner Mongolia Autonomous Region is the region with the largest production of spirulina in China. Due to its unique geographical conditions, the protein content of spirulina in Otog Banner can be as high as 70%. The research of the technical solution of this application has been greatly supported by the Spirulina Industrial Park in the region. The industrial production conditions of the park provide suitable conditions for the preparation and industrial production of the Spirulina polypeptide provided by this application.

[0005] In the prior art, the preparation of bioactive peptides is mostly achieved by enzymatic protein hydrolysis, microbial fermentation and chemical methods. The preparation methods of bioactive peptides involved in many Chinese patent applications with publication numbers CN 116200444 A, CN 116212003 A, CN 106701874 B, CN 115970182 A, CN116179638 A, etc. all use enzymatic hydrolysis. However, the enzymatic hydrolysis method has the disadvantages of insufficient purity of the prepared bioactive peptides, long preparation cost and time.

[0006] In addition, some bioactive peptides in the prior art are prepared by using a bacterial enzyme synergistic method (such as the public articles "Purification of angiotensin converting enzyme inhibitory peptides from Spirulina" and "Identification and extraction of persimmon leaf polyphenols and study of in vitro biological activity"), and this preparation method often has problems such as high cost and complicated preparation steps. Therefore, this application intends to design a method for preparing Spirulina polypeptides with high preparation purity, low economic cost and time cost, and simple process. The product prepared by this method meets the GB22492-2008 standard and can be mass-produced in factories. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention intends to provide a method for preparing a spirulina polypeptide and its application in inhibiting alcoholic liver disease. To achieve the above-mentioned purpose, the technical scheme of the present invention is as follows:

[0008] A method for preparing a spirulina polypeptide comprises the following steps:

[0009] Step 1, pre-treating the spirulina powder raw material by using a colloid mill grinding method and repeated freezing and thawing to obtain a sample solution;

[0010] Specifically, the Spirulina is first subjected to repeated freeze-thaw treatment, repeated 2 to 3 times, and then the cell wall is broken according to a solid-liquid ratio of 1:10, ground for 1.5 to 3.5 hours, and after the foam is eliminated, the process is continued to be repeatedly frozen and thawed, repeated 1 to 2 times, to obtain a sample solution.

[0011] Step 2: Degrading protein by bacterial enzyme synergistic method: adding Bacillus amyloliquefaciens to the sample solution for bacterial hydrolysis, and then using trypsin and papain for enzymatic hydrolysis;

[0012] Specifically, firstly, Bacillus amyloliquefaciens is added to the sample solution for bacterial hydrolysis, and the Bacillus amyloliquefaciens is inoculated into the Spirulina sample solution obtained above, with a pH of 6-8, a temperature of 25-35°C, 160-200rpm, and 23-25h, and then a shaking flask culture operation is performed, with a shaking time of 23-25h and 140-160rpm. After the shaking time is over, enzymatic hydrolysis is performed, firstly trypsin is added, and then a composite enzyme of papain is added to continue to degrade the protein, with a solid-liquid ratio of 1:10-1:30, and the amount of enzyme added is 3-7%+3-7% of the Spirulina, and the enzymatic hydrolysis is performed for 5-7h, wherein the temperature of trypsin is 32-42°C, and the pH is 6.5-8.5; the temperature of papain is 45-55°C, and the pH is 5.0-7.0.

[0013] Step 3, concentrating the sample supernatant after protein degradation, centrifuging, and performing membrane treatment on the supernatant after centrifugation to obtain a spirulina polypeptide liquid with a molecular weight less than 500 Da, and then spray drying to obtain a spirulina polypeptide finished product;

[0014] The concentration treatment is carried out using a rotary evaporator at 65-80° C., 2.5-3.5 h, and 25-30 rpm.

[0015] The centrifugal treatment is carried out under the conditions of 8000-12000 rpm, 3-5° C., and 25-35 min. The BCA protein quantitative method (Bicinchoninic Acid Assay) is selected to determine the content of soluble protein in the supernatant after centrifugation.

[0016] The membrane treatment process is: using ultrafiltration to treat the supernatant after centrifugation to obtain two parts with molecular weight greater than 5000Da and molecular weight less than 5000Da, and further treating the part less than 5000Da to obtain spirulina polypeptide liquid with molecular weight less than 500Da.

[0017] The spray drying process uses a spray dryer to spray dry the spirulina polypeptide liquid after the membrane treatment, and the process is carried out under the conditions of a fan temperature of 90-100°C, a heating temperature of 180-220°C, and a peristaltic pump of 25-30 revolutions / min, and finally a spirulina polypeptide finished product with a polypeptide content of 100% below 500Da is obtained.

[0018] The spirulina peptide product prepared by the above method is applied to people who have a drinking habit and need to reduce alcohol damage to the liver, which can effectively inhibit the formation of alcoholic liver. Specifically, the spirulina peptide product with a molecular weight of less than 500Da is applied to alcohol-damaged AML-12 liver cells, and the aspartate aminotransferase and alanine aminotransferase activities of alcohol-damaged AML-12 liver cells are reduced by 46.70%-49.00% and 54.38%-59.24%, respectively. The aspartate aminotransferase and alanine aminotransferase activities are 0.3612U / 10 4 cell-0.3457 U / 10 4 cell and 0.2495U / 10 4 cell-0.2229 U / 10 4 cell, and the obtained Spirulina peptide product significantly reduced the effect of alcohol on AML-12 liver cells.

[0019] The present invention has the following advantages:

[0020] The method of the present invention firstly utilizes the colloid mill grinding method and the pretreatment steps of repeated freezing and thawing, thereby reducing the demand for high-cost equipment, and the amount of enzymes such as Bacillus amyloliquefaciens, trypsin and papain used is relatively small, which helps to reduce the overall production cost.

[0021] The method of the present invention can effectively degrade the protein in spirulina through the bacterial enzyme synergistic method, so that the extraction rate of the polypeptide is significantly improved. After centrifugation, the polypeptide with a molecular weight less than 500Da can be accurately separated through ultrafiltration treatment, which effectively improves the purity and biological activity of the product. In addition, the spray drying process can convert the polypeptide into a powder form that is easy to store and use while retaining the active ingredients, further improving the extraction effect.

[0022] The process design of the method of the present invention is reasonable, the steps are clear, and the operation of the whole process from repeated freeze-thaw treatment, bacterial hydrolysis to enzymatic hydrolysis to centrifugation and spray drying is relatively simple and suitable for large-scale production. At the same time, the required operating parameters (such as temperature, pH, time, etc.) are all within an easily controllable range, so that the method has good operability and industrial application potential.

[0023] The spirulina polypeptide product prepared by the method of the present invention can significantly reduce the effect of alcohol on AML-12 liver cells and effectively inhibit the formation of alcoholic liver.

[0024] In summary, the present invention provides a method for preparing Spirulina polypeptide with low cost, excellent extraction effect and simple operation, which has high application value and market prospect in inhibiting alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The present invention is a flow chart of the preparation process of the Spirulina polypeptide;

[0026] Figure 2 This is a morphological comparison between normal cells and alcohol-damaged cells;

[0027] Figure 3 This is a significance analysis diagram of aspartate aminotransferase activity;

[0028] Figure 4 This is a significance analysis diagram of alanine aminotransferase activity;

[0029] Figure 3 and Figure 4 Medium Ⅰ-below 500 Da; Ⅲ-below 1000 Da; Ⅴ-below 2000 Da;

[0030] VII - below 3000 Da; IX - below 5000 Da;

[0031] Figure 5 The cell status of each experimental group after adding peptides and incubating for 12 hours;

[0032] Figure 6 The cell status of each experimental group after incubation with alcohol for 24 hours. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention is further specifically described below through examples and in conjunction with the accompanying drawings. The following examples 1-3 are all based on Figure 1 The process shown is carried out.

[0034] Example 1

[0035] The specific implementation steps are as follows:

[0036] Spirulina powder was pretreated by colloid mill grinding and repeated freezing and thawing: specifically, Spirulina was first subjected to repeated freezing and thawing treatment, repeated twice, and then the wall was broken according to a solid-liquid ratio of 1:10, ground for 1.5 hours, and continued to freeze and thaw repeatedly after the foam disappeared, repeated twice to obtain a sample solution. Spirulina polypeptide was prepared by bacterial enzyme synergistic method: first, Bacillus amyloliquefaciens was added to the sample solution for bacterial hydrolysis, and Bacillus amyloliquefaciens was inoculated into Spirulina culture medium with different contents, pH 6, temperature 25℃, 160rpm, 23h; then the shaking flask culture operation was carried out, the shaking flask time was 23h, 140rpm, and after the shaking flask time was over, enzymatic hydrolysis was carried out, according to a solid-liquid ratio of 1:10, the enzyme addition amount was 3%+3% of Spirulina, and the compound enzyme of trypsin and papain was added first, and the enzymatic hydrolysis was carried out for 5 hours, wherein the temperature of trypsin was 32℃, pH 6.5; the temperature of papain was 45℃, pH 5.0. The hydrolyzate of spirulina protein was treated by ultrafiltration and separated into two parts: a part with a molecular weight greater than 5000Da and a part with a molecular weight less than 5000Da. The part less than 5000Da was further treated by membrane to obtain a spirulina polypeptide liquid with a molecular weight less than 500Da. The spirulina polypeptide liquid with a molecular weight less than 500Da was concentrated by using a rotary evaporator at 70°C, 3.0h, and 25rpm. The sample was spray dried by a spray dryer to finally obtain a spirulina polypeptide finished product with a polypeptide content of 100%.

[0037] Example 2

[0038] The specific implementation steps are as follows:

[0039] Spirulina powder was pretreated by colloid mill grinding and repeated freezing and thawing. Specifically, the spirulina was first subjected to repeated freezing and thawing treatment, repeated 3 times, and then the wall was broken according to the ratio of solid to liquid of 1:10, and ground for 3.5 hours. After the foam was eliminated, the repeated freezing and thawing was continued, and repeated 2 times to obtain the sample solution. The sample solution was obtained. Spirulina polypeptide was prepared by bacterial enzyme synergy. First, Bacillus amyloliquefaciens was added to the sample solution for bacterial hydrolysis. Bacillus amyloliquefaciens was inoculated into spirulina culture medium with different contents, pH 7, temperature 30℃, 180rpm, 24h. Then the shaking flask culture operation was carried out, the shaking time was 24h, 150rpm, and the shaking time was over. After that, enzymatic hydrolysis was carried out, and the composite enzyme of trypsin and papain was added first according to the ratio of solid to liquid of 1:20 and the enzyme addition amount of 4%+4%, and the enzymatic hydrolysis was carried out for 6h, wherein the temperature of trypsin was 37℃, pH 7.0, and the conditions of papain were 50℃ and pH 6.0. After protein degradation, the experimental sample needs to be centrifuged at 10,000 rpm, 4°C, and 30 min. Spirulina polypeptide liquid is obtained by centrifugation. The BCA method is selected to determine the content of soluble protein in the supernatant after centrifugation. The hydrolyzate of spirulina protein is treated by ultrafiltration and divided into two parts: a molecular weight greater than 5,000 Da and a molecular weight less than 5,000 Da. The part less than 5,000 Da is further treated by membrane to obtain a spirulina polypeptide liquid with a molecular weight less than 500 Da. The spirulina polypeptide liquid with a molecular weight less than 500 Da is concentrated by using a rotary evaporator at 70°C, 3.0 h, and 30 rpm. The concentrated liquid is spray-dried to finally obtain a spirulina polypeptide finished product with a polypeptide content of 100% below 500 Da.

[0040] Example 3

[0041] The specific implementation steps are as follows:

[0042] Spirulina powder was pretreated by colloid mill grinding and repeated freezing and thawing. Specifically, the spirulina was first subjected to repeated freezing and thawing treatment, repeated 3 times, and then the wall was broken according to the solid-liquid ratio of 1:10, ground for 2.5 hours, and continued to freeze and thaw repeatedly after the foam was eliminated, repeated 2 times to obtain the sample solution. Spirulina polypeptide was prepared by bacterial enzyme synergy. First, Bacillus amyloliquefaciens was added to the sample solution for bacterial hydrolysis. Bacillus amyloliquefaciens was inoculated into spirulina culture medium with different contents, pH 8, temperature 35℃, 200rpm, 25h. Then the shaking flask culture operation was carried out, the shaking flask time was 25h, 160rpm, and the shaking flask time was over. After that, enzymatic hydrolysis was carried out, with a solid-liquid ratio of 1:30 and an enzyme addition amount of 6%+6%. Trypsin was added first and then papain was added. The enzymatic hydrolysis was 7h, where the temperature of trypsin was 42℃, pH 8.5, and the conditions of papain were 55℃ and pH 7.0. After protein degradation, the experimental sample needs to be centrifuged at 12000rpm, 5℃, 35min. Spirulina polypeptide liquid is obtained by centrifugation. The BCA method is selected to determine the content of soluble protein in the supernatant after centrifugation. The hydrolyzate of spirulina protein is treated by ultrafiltration and divided into two parts: a molecular weight greater than 5000Da and a molecular weight less than 5000Da. The part less than 5000Da is further treated by membrane to obtain a part with a molecular weight less than 500Da. The part with a molecular weight less than 500Da is concentrated by using a rotary evaporator at 80℃, 3.5h, and 30rpm. The concentrate is spray-dried to finally obtain a spirulina polypeptide finished product with a polypeptide content of 100% below 500Da.

[0043] In order to further verify the inhibitory effect of the Spirulina polypeptide product prepared by the preparation method of the present application on alcoholic liver disease, the present application example further conducted the following experimental research:

[0044] Construction of alcoholic hepatocyte model: To explore the damaging effect of ethanol on AML-12 hepatocytes, hepatocytes were cultured with complete medium containing different concentrations of ethanol for 24 h, and the cck-8 kit was used to detect cell activity. Sorting of cell models in normal group, injury group and inhibition group: After adding spirulina peptide culture medium below 500Da to the independent culture medium for complete culture and incubation for 12 h, the experimental group was added to the alcohol culture medium containing 870 mmol / L for 24 h, which was defined as the inhibition group. The normal group was the normal incubation cells without spirulina peptide and alcohol; the injury group was the normal cells added with alcohol culture medium containing 870 mmol / L for 24 h.

[0045] Verify the inhibitory effect of spirulina peptides: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are two enzymes that are highly present in liver cells, and therefore are also important indicators for liver function testing. The higher the enzyme activity of AST and ALT, the more severe the liver function is damaged. Therefore, in order to explore the effect of spirulina peptides on alcoholic liver injury, the activity of ALT and AST enzymes in AML-12 liver cells after incubation with spirulina peptides and then alcohol injury is measured to verify the effect of spirulina peptides on alcoholic liver injury.

[0046] AML-12 cells were seeded in a 96-well plate and 100 mL of cell suspension was added. The cell density was 1×10 4 After culturing in an incubator for 24 h (37 °C, 5% CO2), the medium was replaced with 100 mL of DMEM / F12 complete medium containing ethanol (1200 mmol / L, 1000 mmol / L, 800 mmol / L) after the cells attached to the wall. The medium was incubated for 24 h. After the incubation, 10 µl of CCK-8 solution was added to each well. The CCK-8 kit is used in the presence of an electron carrier. WST-8 ((2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazole monosodium salt) is oxidized and reduced by dehydrogenases in the cells to generate a water-soluble orange-yellow formazan dye that can be dissolved in the culture medium. The amount of formazan generated is proportional to the cell activity, so the cell activity is detected.

[0047] The cells were subcultured and when they adhered to the wall and the cell confluence reached about 70%, the AML-12 hepatocytes in the normal group, injury group, and inhibition group were tested for aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Figure 3 and Figure 4 The results show that Spirulina peptides with a molecular weight below 500Da can reduce the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in alcohol-damaged AML-12 liver cells by 46.70%-49.00% and 54.38%-59.24%, respectively. The activities of AST and ALT were 0.3612U / 10 4 cell-0.3457 U / 10 4 cell and 0.2495U / 10 4 cell-0.2229 U / 10 4 cell, that is, the spirulina polypeptide product prepared by the method of the present invention can significantly reduce the effect of alcohol on AML-12 liver cells. Figure 5 and Figure 6It can be seen that after adding peptides of different molecular weights to the culture medium, the spirulina peptide finished product has a good inhibitory effect on alcohol-damaged AML-12 liver cells. And compared with other spirulina peptides with different molecular weights, spirulina peptides below 500Da have the best function of inhibiting alcohol-induced liver damage.

[0048] The protection scope of the present invention is not limited to the above-mentioned embodiments. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the scope and spirit of the present invention. If these changes and modifications fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include these changes and modifications.

Claims

1. An application of a Spirulina polypeptide finished product, characterized in that: The spirulina polypeptide product prepared according to the following method is used to prepare a drug for inhibiting the formation of alcoholic liver disease. The preparation method of the spirulina polypeptide product specifically comprises the following steps: Step 1, pre-treating the spirulina powder raw material by using a colloid mill grinding method and repeated freezing and thawing to obtain a sample solution; Step 2: Degrading protein by bacterial enzyme synergistic method: adding Bacillus amyloliquefaciens to the sample solution for bacterial hydrolysis, and then using trypsin and papain for enzymatic hydrolysis; Step 3, concentrating the sample supernatant after protein degradation, centrifuging, and performing membrane treatment on the supernatant after centrifugation to obtain a spirulina polypeptide liquid with a molecular weight less than 500 Da, and then spray drying to obtain a spirulina polypeptide finished product; Specifically, the step 1 comprises first subjecting the spirulina to repeated freeze-thaw treatment, repeating the process 2 to 3 times, and then subjecting the spirulina to cell wall breaking treatment at a solid-liquid ratio of 1:10, grinding the spirulina for 1.5 to 3.5 hours, and continuing to subject the spirulina to repeated freeze-thaw treatment after the foam disappears, repeating the process 1 to 2 times, to obtain a sample solution; The step 2 specifically comprises first adding Bacillus amyloliquefaciens to the sample solution for bacterial hydrolysis, inoculating the Bacillus amyloliquefaciens into the Spirulina sample solution obtained above, wherein the inoculation conditions are pH 6-8, temperature 25-35° C., 160-200 rpm, 23-25 ​​h, and then performing a shake flask culture operation, wherein the shaking time is 23-25 ​​h, 140-160 rpm, and after the shaking time is over, performing enzymatic hydrolysis; The enzymatic hydrolysis process is to first add trypsin and then add a composite enzyme of papain to continue to degrade the protein, with a material-liquid ratio of 1:10-1:30, and the amount of enzyme added being 3-7%+3-7% of the spirulina, respectively. The enzymatic hydrolysis is carried out for 5-7 hours, the temperature of the trypsin is 32-42°C, and the pH is 6.5-8.5; the temperature of the papain is 45-55°C, and the pH is 5.0-7.

0.

2. The use of a Spirulina polypeptide product as claimed in claim 1, characterized in that: The concentration treatment in step 3 is carried out using a rotary evaporator at 65-80° C., 2.5-3.5 h, and 25-30 rpm.

3. The use of a Spirulina polypeptide product as claimed in claim 1, characterized in that: In the step 3, the centrifugation conditions are 8000-12000 rpm, 3-5° C., and 25-35 min. The BCA protein quantitative method is used to determine the content of soluble protein in the supernatant after centrifugation.

4. The use of a Spirulina polypeptide product as claimed in claim 1, characterized in that: The membrane treatment process in step 3 is: the supernatant after centrifugation is subjected to membrane treatment by ultrafiltration to obtain two parts, one with a molecular weight greater than 5000Da and the other with a molecular weight less than 5000Da; the part less than 5000Da is further subjected to membrane treatment to obtain a spirulina polypeptide liquid with a molecular weight less than 500Da.

5. The use of a Spirulina polypeptide product as claimed in claim 1, characterized in that: In step 3, the spray drying treatment adopts a spray dryer to spray dry the spirulina polypeptide liquid after the membrane treatment under the conditions of a fan temperature of 90-100° C., a heating temperature of 180-220° C., and a peristaltic pump of 25-30 rpm, and finally obtain a spirulina polypeptide finished product with a polypeptide content of 100% below 500 Da.

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