Preparation method and application of sturgeon roe liver-protecting peptide

Abstract

The invention discloses a preparation method of a sturgeon roe liver protection peptide, specifically comprising the following steps: (1) adding deionized water to sturgeon roe, homogenizing, and obtaining a sturgeon roe homogenate; (2) adjusting the pH value of the sturgeon roe homogenate, adding pancreatin alkaline protease 1:1 compound enzyme, enzymolysis, enzyme inactivation, cooling, and obtaining a sturgeon roe enzymolysis solution; (3) the sturgeon roe enzymolysis solution is centrifuged, and the supernatant is taken, that is, stored for standby use. Compared with the related products already available on the market, the sturgeon roe liver protection peptide obtained by the present invention has a better effect of protecting alcohol-damaged liver cells and an antioxidant effect.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and more particularly to a preparation method and application of a sturgeon roe liver-protecting peptide. Background Art

[0002] With the development of society, drinking has become more and more popular around the world, and the global drinking content is generally on the rise. Excessive drinking can easily cause alcoholic liver damage. As one of the five internal organs, the liver is an important metabolic and detoxification organ. When the body produces harmful substances or metabolic waste, the liver will actively remove them. Therefore, the liver is an indispensable and important barrier for the human body and helps maintain the body's metabolic homeostasis. Alcoholic liver damage refers to a disease with abnormal liver cell structure and dysfunction caused by long-term and heavy drinking. Its pathogenesis is relatively complex. Studies in recent years have shown that its pathogenesis may be related to multiple factors, including lipid metabolism, oxidative stress, inflammation, cell apoptosis, etc.

[0003] With the rapid development of my country's aquaculture industry, the in-depth development and high-value utilization of freshwater fish resources have become a key research direction. Functional extracts separated from aquatic foods can be used as functional foods and nutritional health products. Peptides obtained from aquatic proteins by biotechnology not only show high nutritional value, but also show biological characteristics for dietary or therapeutic purposes. Aquatic extracts with special activity may become functional foods for human nutrition. Bioactive peptides have always been one of the hotspots of research and development in many countries. Fish is the earliest biological resource that people began to eat. It is rich in protein, vitamins and minerals, and is a high-quality raw material for the development of functional foods such as oligopeptides. At present, a large number of antioxidant, anti-tumor, antibacterial and anti-inflammatory peptides have been obtained from fish such as silver carp, saury, and tilapia by enzymatic separation, and new structures and mechanisms of action have been continuously discovered. Therefore, the rational and efficient development and utilization of freshwater fish resources has broad market prospects and certain social and economic significance.

[0004] Sturgeon is a bony fish with a long history of more than 200 million years. It is one of the oldest species of fish in existence and is regarded as a "living fossil in the water". Sturgeon is a treasure from head to toe. For example, caviar made from sturgeon eggs is rich in protein, contains a variety of essential amino acids and unsaturated fatty acids, inorganic salts, and trace elements such as copper, magnesium, calcium and selenium. It is known as "black gold". Therefore, deep processing of sturgeon can not only make full use of resources and achieve high-value utilization, but also increase the added value of the industry and promote the sustainable and healthy development of sturgeon farming.

[0005] However, current research on liver-protecting peptides is mostly focused on raw materials such as corn and chicken, and there are no reports on the use of sturgeon roe peptides to study the effects of alcohol detoxification and liver protection.

[0006] Therefore, how to prepare a sturgeon roe polypeptide with alcohol sobering and liver protecting effects is a problem that technicians in this field need to solve urgently. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a preparation method and application of sturgeon roe liver-protecting peptide to solve the deficiencies in the prior art.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for preparing sturgeon roe liver-protecting peptide, specifically comprising the following steps:

[0010] (1) adding deionized water to sturgeon roe, homogenizing, and obtaining a sturgeon roe homogenate liquid;

[0011] (2) adjusting the pH value of the sturgeon roe homogenate, adding a 1:1 mixed enzyme of trypsin-alkaline protease, enzymolysis, enzyme deactivation, cooling, and obtaining a sturgeon roe enzymolysis solution;

[0012] (3) centrifuging the sturgeon roe enzymatic hydrolysate, taking the supernatant to obtain the sturgeon roe liver-protecting peptide, and storing it for later use.

[0013] Furthermore, in the above step (1), the mass ratio of sturgeon roe to deionized water is 1:6.

[0014] Furthermore, in the above step (1), the homogenization speed is 8000 rpm and the time is 1 min.

[0015] Furthermore, in the above step (2), the added amount of the 1:1 (mass ratio) pancreatic enzyme-alkaline protease complex enzyme is 0.5% (mass fraction).

[0016] Furthermore, in the above step (2), the enzymatic hydrolysis temperature is 55° C. and the time is 8 h.

[0017] Furthermore, in the above step (2), the enzyme inactivation temperature is 90-100° C. and the time is 10-15 min.

[0018] Furthermore, in the above step (2), the mixture is cooled to room temperature.

[0019] Furthermore, in the above step (3), the centrifugal treatment temperature is 4°C, the rotation speed is 8000 rpm, and the time is 15 min.

[0020] Furthermore, in the above step (3), the storage temperature is -80°C.

[0021] The present invention also claims to protect the use of the sturgeon roe liver-protecting peptide prepared by the above preparation method in the preparation of a medicine for protecting alcoholic liver damage.

[0022] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Compared with related products available on the market, the sturgeon roe liver-protecting peptide prepared by the present invention has better protective effect on liver cells damaged by alcohol and anti-oxidation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of different enzyme types on the antioxidant capacity of ABTS in the enzymatic hydrolysate;

[0025] Figure 2 The effect of different enzymatic hydrolysis time on the antioxidant capacity of ABTS in the enzymatic hydrolyzate;

[0026] Figure 3 The effect of different enzyme addition amounts on the antioxidant capacity of ABTS in the enzymatic hydrolysate;

[0027] Figure 4 The effects of different enzyme types on the activation rate of ADH in vitro;

[0028] Figure 5 The effect of different enzymatic hydrolysis time on the activation rate of ADH in vitro;

[0029] Figure 6 The effect of different enzyme addition amounts on the in vitro ADH activation rate;

[0030] Figure 7 The effect of different concentrations of enzymatic supernatant on the viability of HepG2 cells;

[0031] Figure 8 The effect of different concentrations of alcohol on the viability of HepG2 cells;

[0032] Fig. 9 The effect of different concentrations of enzymatic supernatant on alcohol-damaged HepG2 cells;

[0033] Fig.10 The effect of different concentrations of enzymatic supernatant on the SOD activity of HepG2 cells induced by alcohol;

[0034] Fig.11 The effect of different concentrations of enzymatic supernatant on the GSH content of HepG2 cells induced by alcohol;

[0035] Fig.12 The effect of different concentrations of enzymatic supernatant on the MDA level of HepG2 cells induced by alcohol;

[0036] Fig.13 The effect of different concentrations of enzymatic supernatant on alcohol-induced ALT in HepG2 cells;

[0037] Fig.14The effect of different concentrations of enzymatic supernatant on alcohol-induced AST in HepG2 cells;

[0038] Fig.15 The effects of different concentrations of alcohol on liver cells;

[0039] Fig.16 The effects of different samples on liver cells;

[0040] Fig.17 The effects of different samples on alcohol-damaged liver cells. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] The preparation method of sturgeon roe liver-protecting peptide specifically comprises the following steps:

[0044] (1) adding 6 times the mass of deionized water into the sturgeon roe, and homogenizing at 8000 rpm for 1 min to obtain a sturgeon roe homogenate;

[0045] (2) adjusting the sturgeon roe homogenate to an optimum pH value, adding 0.5% pancreatic enzyme-alkaline protease 1:1 mixed enzyme, placing it in a constant temperature oscillator at 55° C. for enzymolysis for 8 h, then placing it in a boiling water bath at 90° C. for enzyme inactivation for 15 min, and cooling it to room temperature to obtain a sturgeon roe enzymolysis solution;

[0046] (3) The sturgeon roe enzymatic hydrolysate was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was taken to obtain the sturgeon roe liver-protecting peptide, which was stored at -80° C. for later use.

[0047] Example 2

[0048] The preparation method of sturgeon roe liver-protecting peptide specifically comprises the following steps:

[0049] (1) adding 6 times the mass of deionized water into the sturgeon roe, and homogenizing at 8000 rpm for 1 min to obtain a sturgeon roe homogenate;

[0050] (2) adjusting the sturgeon roe homogenate to an optimum pH value, adding 0.5% pancreatic enzyme-alkaline protease 1:1 mixed enzyme, placing it in a constant temperature oscillator at 55° C. for enzymolysis for 8 h, then placing it in a boiling water bath at 95° C. for enzyme inactivation for 12 min, and cooling it to room temperature to obtain a sturgeon roe enzymolysis solution;

[0051] (3) The sturgeon roe enzymatic hydrolysate was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was taken to obtain the sturgeon roe liver-protecting peptide, which was stored at -80° C. for later use.

[0052] Example 3

[0053] The preparation method of sturgeon roe liver-protecting peptide specifically comprises the following steps:

[0054] (1) adding 6 times the mass of deionized water into the sturgeon roe, and homogenizing at 8000 rpm for 1 min to obtain a sturgeon roe homogenate;

[0055] (2) adjusting the sturgeon roe homogenate to an optimum pH value, adding 0.5% pancreatic enzyme-alkaline protease 1:1 mixed enzyme, placing in a constant temperature oscillator at 55° C. for enzymolysis for 8 h, then placing in a boiling water bath at 100° C. for enzyme inactivation for 10 min, and cooling to room temperature to obtain a sturgeon roe enzymolysis solution;

[0056] (3) The sturgeon roe enzymatic hydrolysate was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was taken to obtain the sturgeon roe liver-protecting peptide, which was stored at -80° C. for later use.

[0057] Performance Testing

[0058] 1. Exploratory Experiment

[0059] 1. Materials and Instruments

[0060] Trypsin and alkaline protease were purchased from Yuanye Biotechnology, ADH and NAD+ were purchased from Sigma, sodium pyrophosphate was purchased from Tianjin Damao Chemical Reagent Factory, anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd., SOD, MDA, GSH, ALT, and AST kits were purchased from Nanjing Jiancheng Bioengineering Institute, and BCA kits were purchased from Biyuntian Biotechnology Co., Ltd.

[0061] Homogenizer T25 was purchased from IKA, Germany, constant temperature oscillator SHA-B was purchased from Shanghai Lichen Instrument Technology Co., Ltd., carbon dioxide incubator 3110 Series was purchased from Thermo Corporation, USA, and microplate reader InfiniteM 200Pro was purchased from TECAN, Austria.

[0062] 2. Methods

[0063] 2.1 Optimization of single factor conditions for enzymatic hydrolysis

[0064] Weigh 10g of sturgeon roe, add deionized water at a solid-liquid ratio of 1:6, and homogenize at 8000rpm for 1min. Then adjust to the optimal pH value, add a 1:1 mixed enzyme of pancreatin-alkaline protease, and place the sample in a constant temperature oscillator for enzymolysis at 55°C. Subsequently, place the sample in a boiling water bath and inactivate the enzyme at 95°C for 12min. After cooling to room temperature, centrifuge at 4°C and 8000rpm for 15min, take the supernatant for subsequent index determination, and then store it at -80°C for later use.

[0065] Single factor investigation of enzyme type: the enzyme addition amount was 1%, and the enzymatic hydrolysis time was 4h. The effects of different proteases (trypsin, alkaline protease, and 1:1 mixed enzyme of trypsin-alkaline protease) on the enzymatic hydrolysis effect were tested.

[0066] Single factor investigation of enzymatic hydrolysis time: Under the condition of 1% enzyme addition, compound enzyme was used for enzymatic hydrolysis, and the enzymatic hydrolysis time was set to 2h, 4h, 8h, 12h and 16h respectively.

[0067] Single factor investigation of enzyme addition amount: the compound enzyme was used for 8h enzymatic hydrolysis, and the enzyme addition amounts were 0.2%, 0.5%, 0.8%, 1% and 2% respectively.

[0068] 2.2 Determination of hydrolysis degree and protein recovery

[0069] The degree of hydrolysis was determined by the OPA method, and the protein content of fish roe and enzymatic supernatant was determined by the Kjeldahl method in GB 5009.5-2016 “National Food Safety Standard - Determination of Protein in Food”.

[0070] Protein recovery rate (%) = protein content of enzymatic supernatant / fish roe protein content × 100%.

[0071] 2.3 Antioxidant capacity of ABTS in the supernatant of fish roe hydrolysis

[0072] The ABTS free radical scavenging ability assay is a test method used to evaluate the antioxidant capacity of compounds or samples. ABTS (2,2'-hydrazinobis(3-ethylbenzothiazoline)) is a synthetic free radical compound that can be used to simulate oxygen free radicals in organisms. This method determines the antioxidant properties of a compound by measuring its ability to scavenge ABTS free radicals. During the assay, the ABTS free radical will change color, and the addition of antioxidants will cause the color to fade, the degree of which is proportional to the antioxidant capacity. The basic steps of this method include:

[0073] ① Prepare ABTS solution: react ABTS with hydrogen peroxide to generate a blue free radical solution, which is then diluted with a certain proportion of solvent to obtain a suitable concentration.

[0074] ② Mix the sample to be tested: Mix the compound to be tested with the diluted ABTS solution.

[0075] ③Measure absorbance: Over a period of time, the absorbance change of the mixture is measured to determine the ABTS free radical scavenging ability, that is, the antioxidant capacity.

[0076] This experiment compared the ABTS free radical scavenging ability when the sample protein content was 0.15 mg / mL with reference to the optimized method, and the results were expressed as Trolox equivalent μM TE / g antioxidant.

[0077] 2.4 ADH activation rate

[0078] Take 50 μL of the sample after gradient dilution and 50 μL of ADH (1U / mL) mixed solution and add it to a quartz 96-well plate, use double distilled water as a control, and incubate in a 37°C constant temperature incubator for 20 minutes. Mix 22.4 mM sodium pyrophosphate: 27 mmol / L NAD: 5% ethanol in a ratio of 3:2:1, incubate at 37°C for 5 minutes, and add 150 μL to the quartz plate well. Use an ELISA reader to detect the absorbance value at 340 nm every 40 seconds for 20 minutes. Fit the reaction kinetic curve and calculate the slope.

[0079] ADH activation rate = (K 样品 -K 对照 ) / K 对照 ×100%.

[0080] 2.5 HepG2 cell culture

[0081] HepG2 cells were grown in DMEM cell culture medium containing 10% (volume fraction) fetal bovine serum in a 37°C, 5% CO2 incubator. Subculture was performed when the cells were about 80% confluent.

[0082] 2.6 Establishment of alcohol-induced HepG2 cell injury model

[0083] HepG2 cells in logarithmic growth phase were cultured at 2×10 4 The cells were inoculated at a density of 100 μL in a 96-well culture plate and cultured for 24 hours. The culture medium containing different concentrations (3% to 8%) of alcohol was replaced and cultured for another 24 hours, with 6 replicate wells for each concentration. The supernatant was discarded, and 100 μL of culture medium containing CCK-8 was added respectively. After continuing to culture for 30 minutes, the absorbance (A) was detected at 450 nm using an ELISA reader, and the cell survival rate was calculated using the following formula.

[0084] Cell survival rate = A 酒精组 / A 正常组 ×100%.

[0085] 2.7 Detection of cytotoxicity of fish roe extract on HepG2 cells

[0086] After the cells were inoculated in a 96-well plate and cultured for 24 hours, the culture medium containing different concentrations (50-2000 μg / mL) of fish roe alcohol extract was replaced and cultured for 24 hours. Six replicate wells were set up in each group. The supernatant was discarded, and 100 μL of culture medium containing CCK-8 was added respectively. After continuing to culture for 30 minutes, the absorbance (A) was detected at 450 nm using an enzyme reader.

[0087] 2.8 Cell viability test of fish roe enzymatic hydrolysate against alcohol toxicity

[0088] After the cells were inoculated in 96-well plates and cultured for 24 h, different concentrations of fish roe enzymatic hydrolyzate supernatant were added for pretreatment for 24 h. A final concentration of 6% ethanol was added to each group to induce damage. An equal amount of culture medium was added to the normal control group and incubated at 37°C for 24 h. The supernatant was discarded and 100 μL of culture medium containing CCK-8 was added. After further culture for 30 min, the absorbance (A) was detected at 450 nm using a microplate reader.

[0089] 2.9 Effects of fish roe enzymatic hydrolysate on biochemical parameters of alcohol-induced HepG2 cells

[0090] Hepg2 cells were plated at 2×10 5 / mL was inoculated in a 6-well plate and adhered to the wall for 24 hours, then the supernatant was discarded, the normal group and model group were added with culture medium, and the sample group was pretreated with samples containing different concentrations (0.3mg / mL, 0.8mg / mL, 1mg / mL, 2mg / mL) for 24 hours, and then cultured with culture medium containing 6% ethanol for 24 hours, and then the cells were collected for testing. The ALT and AST kit methods were used to determine the ALT and AST levels in the cell culture medium, and the SOD, MDA, and GSH kit methods were used to determine the SOD, MDA, and GSH levels in the cell culture fluid. The cell protein content was determined using the BCA kit.

[0091] Among them, SOD can eliminate harmful substances produced by organisms during metabolism and is one of the main enzyme systems in cells to resist oxidative stress.

[0092] 3. Test results

[0093] 3.1 Hydrolysis degree and protein recovery rate

[0094] The results are shown in Tables 1-3.

[0095] Table 1 Effect of enzyme type on hydrolysis degree and protein recovery rate

[0096]

[0097]

[0098] Table 2 Effect of enzymatic hydrolysis time on hydrolysis degree and protein recovery rate

[0099] Enzyme hydrolysis time Protein content mg / mL Protein recovery % Degree of hydrolysis % 2h 20.13±0.11 41.84±0.23 39.09±0.40 4h 25.98±0.07 54.01±0.15 38.22±0.42 8h 27.35±0.16 56.87±0.33 44.43±0.97 12h 27.71±0.13 57.61±0.27 45.28±0.39 16h 29.27±1.07 60.85±2.22 46.02±1.21

[0100] Table 3 Effect of enzyme addition on hydrolysis degree and protein recovery rate

[0101] Enzyme addition amount Protein content mg / mL Protein recovery % Degree of hydrolysis % 0.2％ 15.14±0.15 31.47±0.32 53.17±1.37 0.5％ 18.24±0.21 37.93±0.44 52.32±1.02 0.8％ 19.71±0.08 40.97±0.17 53.54±0.21 1％ 21.06±0.63 43.78±1.31 56.02±1.12 2％ 21.75±0.40 45.21±0.84 58.05±0.22

[0102] As shown in Table 1, the protein contents of the enzymatic hydrolysates of the three enzymes are not much different, among which pancreatic enzyme has the highest protein content, followed by the compound enzyme. The trends of the hydrolysis degree and protein recovery rate are consistent with those of the protein content.

[0103] It can be seen from Table 2-3 that with the increase of enzyme addition amount and enzymatic hydrolysis time, the protein content of the enzymatic hydrolysis product also increases.

[0104] 3.2 Antioxidant results of ABTS in enzymatic supernatant

[0105] The results are as follows Figure 1-3 shown.

[0106] Depend on Figure 1-3 It can be seen that the ABTS antioxidant capacity of different enzymatic supernatants is as follows: compound enzyme> pancreatin> alkaline protease, different enzymatic hydrolysis times are 4h>2h>16h>8h>12h, and under the conditions of different enzyme addition amounts, 0.2%>0.8%>0.5%>2%>1%. Under the conditions of different enzymatic hydrolysis times and enzyme addition amounts, the ABTS free radical scavenging rate Trolox equivalent of 0.15mg / mL fish roe enzymatic hydrolysis supernatant is greater than 150, indicating that the fish roe enzymatic hydrolysis supernatant has a good antioxidant effect.

[0107] 3.3 Effect of enzymatic hydrolysis conditions on in vitro ADH activation rate

[0108] The results are as follows Figure 4-6 shown.

[0109] Depend on Figure 4-6It can be seen that the enzymatic hydrolysates obtained by different enzyme types, enzymatic hydrolysis time and enzyme addition amount have certain differences in the in vitro ADH activation effect at different concentrations. Among them, the enzymatic hydrolysates of different enzyme types have good activation effects at different concentrations. When the concentration is 1mg / mL and 2.5mg / mL, the ADH activation rate of the composite enzyme is the highest. When the concentration increases to 5mg / mL, the ADH activation rate of alkaline protease is the highest. When the sample concentration is 1mg / mL, the ADH activation rate increases with the increase of enzymatic hydrolysis time. There is no significant difference between the effects of 8h and 12h, and the ADH activation rate of 16h is the best. When the sample concentration is 2.5mg / mL, the ADH activation rate of the enzymatic hydrolysis time is 12h is the best. When the sample concentration is 5mg / mL, the ADH activation rate of the enzymatic hydrolysis time is 8h is the best. And with the increase of sample concentration, the ADH activation rate also shows an upward trend.

[0110] The compound enzyme was comprehensively selected, and the enzyme addition amount was 0.5% and the enzymatic hydrolysis time was 8h as the optimal enzymatic hydrolysis conditions for in vitro cell experiments.

[0111] 3.4 Effects of different concentrations of enzymatic supernatant and alcohol on HepG2 cell viability

[0112] The results are as follows Figure 7-8 shown.

[0113] Depend on Figure 7 It can be seen that different concentrations of enzymatic hydrolysates can promote the proliferation of HepG2 cells.

[0114] Depend on Figure 8 It can be seen that compared with the normal group cells, when the alcohol concentration is 3%, the cell viability of HepG2 can be significantly reduced. When the alcohol concentration is 6%, the relative cell viability is about 50%. Therefore, the 6% alcohol concentration is selected to damage the cells.

[0115] 3.5 Protective effect of fish roe enzymatic hydrolysate on alcohol-damaged HepG2 cells

[0116] The results are as follows Fig. 9 shown.

[0117] Depend on Fig. 9 It can be seen that with the increase of enzyme addition amount, the relative cell activity also increases.

[0118] 3.6 Effects of fish roe enzymatic hydrolysate on biochemical parameters of alcohol-induced HepG2 cell damage

[0119] The results are as follows Figure 10-14 shown.

[0120] Depend on Fig.10It can be seen that the SOD enzyme activity in the model group was significantly lower than that in the control group (P<0.01), the antioxidant mechanism in the cells was out of balance, and the cells were in a state of oxidative damage. The SOD enzyme activity in the sample treatment group was increased to varying degrees compared with the model group, indicating that the sample treatment group could resist alcohol-induced oxidative stress damage and restore the oxidative stress mechanism of HepG2 cells by overexpressing SOD enzyme activity. The sample addition groups with different concentrations were significantly different from the model group (P<0.01). The sample treatment could not completely resist the cell damage caused by alcohol, but it had a protective effect to a certain extent.

[0121] Depend on Fig.11 It can be seen that compared with the normal control group, the GSH content in the alcohol model group was significantly reduced (P<0.01). Compared with the alcohol model group, the GSH content in the different concentration groups was increased to varying degrees. This shows that sturgeon enzymatic supernatant can promote GSH regeneration and enhance the liver's free base clearance ability, thereby reducing the "secondary strike" damage to the liver caused by the accumulation of peroxidation products in the body and protecting liver function.

[0122] Depend on Fig.12 It can be seen that compared with the normal group, the MDA level in the alcohol model group was significantly increased (P<0.01), indicating that 6% alcohol can induce severe lipid peroxidation in cells. Compared with the alcohol model group, different concentration sample groups can reduce the content of MDA in damaged cells.

[0123] Depend on Figure 13-14 It can be seen that compared with the normal group, HepG2 cells treated with 6% alcohol can significantly induce ALT and AST leakage (P<0.01), and pretreatment with different concentrations of fish roe hydrolysate can significantly reduce the ALT and AST release of HepG2 cells induced by 6% alcohol (P<0.01). It can be seen that sturgeon roe hydrolysate supernatant plays a protective role in liver cells by reducing the increase in ALT and AST activity caused by alcohol damage.

[0124] 2. Comparative Test

[0125] 1. Test methods

[0126] 1.1 Sample and alcohol film concentration screening

[0127] When the cell density exceeds 80%, HepG2 cells are digested and the cell density is 2 × 10 5 / mL was spread on a 96-well plate, adhered to the wall for 24 hours, and the supernatant was discarded. Different concentrations (protein concentrations were 0.1mg / mL, 0.2mg / mL, 0.5mg / mL, and 1mg / mL) of alcohol detoxification drink (commercially available), corn peptide (commercially available), fish roe enzymatic hydrolyzate (Example 2) and alcohol (2% to 10%) were prepared respectively. The samples were incubated for 24 hours and the alcohol was incubated for 12 hours. CCK-8 was used to detect cell viability.

[0128] 1.2 Sample protection Alcohol damage to cells

[0129] Digestion and plating, the cell density is 2×10 4 / well, adhered to the wall for 24 hours, samples were pre-protected for 24 hours, and cell viability was detected by CCK-8 after 7% alcohol damage for 12 hours.

[0130] 2. Test results

[0131] 1.1 Alcohol modeling

[0132] The results are shown in Table 15.

[0133] Depend on Fig.15 It can be seen that different concentrations of alcohol have different degrees of damage to liver cells, with a cell survival rate of nearly 60% at around 7%. Therefore, subsequent experiments selected an alcohol concentration of 7% for sample protection tests.

[0134] 2.2 Sample toxicity

[0135] Before conducting the sample protection test, the sample toxicity is evaluated first. Fig.16 shown.

[0136] Depend on Fig.16 It can be seen that the fish roe enzymatic hydrolysate of the present invention and the commercially available liver protection products (alcohol detoxification drink and corn peptide) are non-toxic to liver cells and have a certain effect of promoting liver cell proliferation.

[0137] 2.3 Protective effect of samples on alcohol-damaged liver cells

[0138] The results are as follows Fig.17 shown.

[0139] Depend on Fig.17 It can be seen that compared with commercially available liver protection products (alcohol detoxification drink and corn peptide), the fish roe enzymatic hydrolysate of the present invention can improve the cell survival rate of alcohol-damaged liver cells at different protein concentrations, and 1 mg / mL protein concentration has the highest cell protection effect. Therefore, the fish roe enzymatic hydrolysate of the present invention has a good alcohol detoxification and liver protection effect.

[0140] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing sturgeon roe liver-protecting peptide, characterized in that: The specific steps include: (1) adding deionized water to sturgeon roe, homogenizing, and obtaining sturgeon roe homogenate; (2) adjusting the pH value of the sturgeon roe homogenate, adding a 1:1 mixed enzyme of trypsin-alkaline protease, enzymolysis, enzyme deactivation, cooling, and obtaining a sturgeon roe enzymolysis solution; (3) centrifuging the sturgeon roe enzymatic hydrolysate, taking the supernatant to obtain the sturgeon roe liver-protecting peptide, and storing it for later use.

2. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (1), the mass ratio of the sturgeon roe to deionized water is 1:

6.

3. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (1), the homogenization speed is 8000 rpm and the time is 1 min.

4. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (2), the added amount of the 1:1 pancreatic enzyme-alkaline protease complex is 0.5%.

5. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis temperature is 55° C. and the time is 8 h.

6. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (2), the enzyme is inactivated at a temperature of 90-100°C for a time of 10-15 minutes.

7. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (2), the cooling is to room temperature.

8. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (3), the centrifugal treatment temperature is 4°C, the rotation speed is 8000 rpm, and the time is 15 min.

9. The method for preparing a sturgeon roe hepatoprotective peptide according to claim 1, characterized in that: In step (3), the storage temperature is -80°C.

10. Use of the sturgeon roe liver-protecting peptide prepared by the preparation method according to any one of claims 1 to 9 in the preparation of a drug for protecting alcoholic liver damage.

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