Preparation method of sturgeon roe peptide freeze-dried powder with anti-aging effect

Abstract

The invention discloses a preparation method of sturgeon roe peptide freeze-dried powder with an anti-aging effect. The preparation method specifically comprises the following steps: (1) unfreezing sturgeon roes, adding water, uniformly stirring, grinding and crushing, and adjusting the pH value; (2) heating and naturally cooling; (3) adding alkaline protease, uniformly stirring, carrying out enzymolysis, and adding starch sodium octenylsuccinate and vitamin E powder; (4) carrying out enzyme inactivation, naturally cooling, and adjusting the pH value; (5) carrying out rough filtration and centrifugal treatment, taking supernate, adsorbing, filtering and taking filtrate; and (6) carrying out plate frame filtration, taking filtrate, carrying out vacuum freeze drying, screening and packaging, thereby obtaining the product. The preparation method disclosed by the invention has the advantages of simplicity and convenience in operation, low cost, high embedding yield and the like, and has important significance in improving the stability and biological activity of the sturgeon roe peptide. Moreover, the sturgeon roe peptide freeze-dried powder has an anti-aging effect, and can be widely applied to oral liquid, solid beverages, tablet candies and the like in common foods.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and more particularly to a method for preparing sturgeon roe peptide freeze-dried powder with anti-aging effects. Background Art

[0002] Sturgeon roe is a nutritious food resource, rich in protein, unsaturated fatty acids, peptides, amino acids, trace elements and vitamins and other active ingredients. Among them, sturgeon roe peptides have multiple biological activities such as anti-oxidation, anti-fatigue and immunity enhancement. However, the collection of sturgeon roe is limited by the breeding season and number of sturgeons, and the processing process is relatively complicated, requiring multiple steps to obtain edible sturgeon roe products. In addition, the nutrients in sturgeon roe are easily affected by factors such as temperature and humidity and lost. Therefore, how to maintain the integrity of the nutritional components of sturgeon roe is also a technical challenge.

[0003] Vacuum freeze drying (freeze drying) is a drying technology that uses the principle of sublimation to dehydrate materials. After the material is quickly frozen, it is heated in a vacuum environment (lower than the triple point pressure of water). The freeze-dried sturgeon roe retains its original flavor and nutritional active ingredients, and is convenient for long-term storage. However, since sturgeon roe contains a large amount of oil, it is easy to clump during the freeze-drying process, which affects the quality and taste of the freeze-dried powder.

[0004] Encapsulation technology is an effective method to protect active substances. It can prevent the interaction of active substances with the external environment by encapsulating them, thereby improving their stability, water solubility and biological activity.

[0005] Currently, preparation methods for sturgeon roe peptide encapsulation have been reported, but these methods generally have problems such as complex preparation process, high cost, and low encapsulation yield.

[0006] Therefore, how to develop a method for preparing sturgeon roe peptide freeze-dried powder that is simple to operate, low in cost, and has a high embedding yield is an urgent problem that those skilled in the art need to solve. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy 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 peptide freeze-dried powder with anti-aging effect, specifically comprising the following steps:

[0010] (1) thawing the sturgeon roe, adding water, stirring, grinding and crushing, adjusting the pH value, and obtaining a sturgeon roe solution;

[0011] (2) the sturgeon roe solution is heated up and then cooled naturally;

[0012] (3) firstly adding alkaline protease to the sturgeon roe solution after cooling, stirring evenly for enzymolysis, and then adding sodium starch octenyl succinate and vitamin E powder;

[0013] (4) enzyme deactivation, natural cooling, and pH adjustment to obtain sturgeon roe enzymolyzate;

[0014] (5) coarsely filtering and centrifuging the sturgeon roe hydrolysate, taking the supernatant, adsorbing it through an activated carbon series column, filtering, and taking the filtrate to obtain a sturgeon roe peptide solution;

[0015] (6) plate and frame filtering, taking the filtrate, vacuum freeze drying, sieving, and packaging to obtain sturgeon roe peptide freeze-dried powder with anti-aging effects.

[0016] Furthermore, in the above step (1), the mass ratio of sturgeon roe to water is 1:6.

[0017] Furthermore, in the above step (1), the grinding equipment is a colloid mill, and the number of times is 1; the operation of adjusting the pH value is specifically: adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8.

[0018] Furthermore, in the above step (2), the temperature is raised to 80°C and maintained for 10 minutes; and then naturally cooled to 55°C.

[0019] Furthermore, in the above step (3), the amount of alkaline protease added is 1% of the weight of sturgeon roe protein.

[0020] Furthermore, in the above step (3), the enzymatic hydrolysis temperature is 55°C and the time is 8h;

[0021] Furthermore, in the above step (3), the amount of sodium starch octenylsuccinate added is 10 g / L; the content of vitamin E in the vitamin E powder is 50%, and the amount added is 12 mg / L;

[0022] The above method has the further beneficial effect that the embedding agent is a key component for preparing the sturgeon roe peptide freeze-dried powder. The embedding agent prepared by the present invention can effectively embed the sturgeon roe peptide by selecting food-grade embedding agent raw materials with good film-forming properties, water resistance and stability.

[0023] Furthermore, in the above step (4), the temperature for enzyme inactivation is 90-100°C for 10-15 min; the mixture is naturally cooled to room temperature; and the operation for adjusting the pH value is specifically as follows: hydrochloric acid solution is added, stirred evenly, and the pH value of the material is adjusted to 7.

[0024] Furthermore, in the above step (5), the tools used for coarse filtration are filter bags and 200-mesh screens; the rotation speed of the centrifugal treatment is 5000-20000 r / min; and the number of times of activated carbon series column adsorption is 3 times.

[0025] Furthermore, in the above step (6), the filter paper precision of the plate and frame filtration is 0.1 μm, and the number of filtrations is 3 times; the vacuum degree of the vacuum freeze-drying is 100 Pa, the temperature is -45°C, and the time is 2000 min; the mesh size of the sieving is 80 mesh; and the packaging material is a PE bag.

[0026] The above method has the further beneficial effect that homogenizing the sturgeon roe peptide solution and the embedding agent solution in a certain proportion is an important step in preparing the sturgeon roe peptide freeze-dried powder. The present invention ensures the uniformity and stability of the homogenized liquid by selecting appropriate process parameters such as homogenization method, temperature and time.

[0027] Vacuum freeze drying is a key step in preparing sturgeon roe peptide freeze-dried powder. The present invention ensures the quality and stability of sturgeon roe peptide freeze-dried powder by selecting appropriate process parameters such as vacuum degree, temperature and time.

[0028] Post-processing such as screening and packaging of the obtained sturgeon roe peptide freeze-dried powder is an important step to ensure product quality and stability. The present invention ensures that the product meets relevant standards by selecting appropriate process parameters such as the mesh size of the sieve and the packaging material.

[0029] 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:

[0030] 1. The preparation method of the present invention has the advantages of simple operation, low cost, high embedding yield, etc., and is of great significance for improving the stability and biological activity of sturgeon roe peptide.

[0031] 2. The sturgeon roe peptide freeze-dried powder prepared by the present invention has anti-aging effects and can be widely used in liquid beverages, solid beverages, compressed candies, etc. in ordinary foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effect of different H2O2 concentrations on HSF cell viability;

[0033] Figure 2 The effect of each sample on the content of type III collagen in HSF cells;

[0034] Figure 3 The effect of each sample on the content of type IV collagen in HSF cells;

[0035] Figure 4 is the effect of each sample on the expression of β-galactosidase in HSF cells;

[0036] Figure 5 is the effect of each sample on the relative length of telomeres in HSF cells;

[0037] Figure 6 The effect of each sample on the NAD+ content in HSF cells. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1

[0040] The preparation method of sturgeon roe peptide freeze-dried powder with anti-aging effect specifically comprises the following steps:

[0041] (1) thawing the sturgeon roe, taking 1 kg, adding 6 kg of deionized water, stirring evenly, grinding once with a colloid mill, adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8 to obtain a sturgeon roe solution;

[0042] (2) heating the sturgeon roe solution to 80° C. and maintaining it for 10 min, and then naturally cooling it to 55° C.;

[0043] (3) firstly adding alkaline protease with a content of 1% of sturgeon roe protein to the sturgeon roe solution after cooling, stirring evenly, performing enzymolysis at 55° C. for 8 h, and then adding 10 g of sodium octenyl succinate starch and 12 mg of 50% vitamin E powder to each L of sturgeon roe solution;

[0044] (4) inactivating the enzyme at 90° C. for 15 min, cooling naturally to room temperature, adding hydrochloric acid solution, stirring evenly, and adjusting the pH value of the material to 7 to obtain sturgeon roe enzymatic hydrolyzate;

[0045] (5) firstly coarsely filter the sturgeon roe enzymatic hydrolysate through a filter bag and a 200-mesh screen, then centrifuge at a speed of 5000 r / min, take the supernatant, adsorb it three times through a series of activated carbon columns, filter, and take the filtrate to obtain a sturgeon roe peptide solution;

[0046] (6) First, the sturgeon roe peptide solution is filtered three times through a plate frame with a filter paper precision of 0.1 μm, and the filtrate is taken and sterilized at high temperature; then, vacuum freeze-dried for 2000 min under the conditions of a vacuum degree of 100 Pa and a temperature of -45°C, sieved through a sieve with a mesh number of 80, and finally packaged in a PE bag to obtain a sturgeon roe peptide freeze-dried powder with anti-aging effect.

[0047] Example 2

[0048] The preparation method of sturgeon roe peptide freeze-dried powder with anti-aging effect specifically comprises the following steps:

[0049] (1) thawing the sturgeon roe, taking 1 kg, adding 6 kg of deionized water, stirring evenly, grinding once with a colloid mill, adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8 to obtain a sturgeon roe solution;

[0050] (2) heating the sturgeon roe solution to 80° C. and maintaining it for 10 min, and then naturally cooling it to 55° C.;

[0051] (3) firstly adding alkaline protease with a content of 1% of sturgeon roe protein to the sturgeon roe solution after cooling, stirring evenly, performing enzymolysis at 55° C. for 8 h, and then adding 10 g of sodium octenyl succinate starch and 12 mg of 50% vitamin E powder to each L of sturgeon roe solution;

[0052] (4) inactivating the enzyme at 95° C. for 12 min, cooling naturally to room temperature, adding hydrochloric acid solution, stirring evenly, and adjusting the pH value of the material to 7 to obtain sturgeon roe enzymatic hydrolyzate;

[0053] (5) firstly filter the sturgeon roe enzymatic hydrolyzate through a filter bag and a 200-mesh screen, then centrifuge at a speed of 10,000 r / min, take the supernatant, adsorb it three times through a series of activated carbon columns, filter, and take the filtrate to obtain a sturgeon roe peptide solution;

[0054] (6) First, the sturgeon roe peptide solution is filtered three times through a plate frame with a filter paper precision of 0.1 μm, and the filtrate is taken and sterilized at high temperature; then, vacuum freeze-dried for 2000 min under the conditions of a vacuum degree of 100 Pa and a temperature of -45°C, sieved through a sieve with a mesh number of 80, and finally packaged in a PE bag to obtain a sturgeon roe peptide freeze-dried powder with anti-aging effect.

[0055] Example 3

[0056] The preparation method of sturgeon roe peptide freeze-dried powder with anti-aging effect specifically comprises the following steps:

[0057] (1) thawing the sturgeon roe, taking 1 kg, adding 6 kg of deionized water, stirring evenly, grinding once with a colloid mill, adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8 to obtain a sturgeon roe solution;

[0058] (2) heating the sturgeon roe solution to 80° C. and maintaining it for 10 min, and then naturally cooling it to 55° C.;

[0059] (3) firstly adding alkaline protease with a content of 1% of sturgeon roe protein to the sturgeon roe solution after cooling, stirring evenly, performing enzymolysis at 55° C. for 8 h, and then adding 10 g of sodium octenyl succinate starch and 12 mg of 50% vitamin E powder to each L of sturgeon roe solution;

[0060] (4) inactivating the enzyme at 100° C. for 10 min, cooling naturally to room temperature, adding hydrochloric acid solution, stirring evenly, and adjusting the pH value of the material to 7 to obtain sturgeon roe enzymatic hydrolyzate;

[0061] (5) firstly filter the sturgeon roe enzymatic hydrolyzate through a filter bag and a 200-mesh screen, then centrifuge at a speed of 20,000 r / min, take the supernatant, adsorb it three times through a series of activated carbon columns, filter, and take the filtrate to obtain a sturgeon roe peptide solution;

[0062] (6) First, the sturgeon roe peptide solution is filtered three times through a plate frame with a filter paper precision of 0.1 μm, and the filtrate is taken and sterilized at high temperature; then, vacuum freeze-dried for 2000 min under the conditions of a vacuum degree of 100 Pa and a temperature of -45°C, sieved through a sieve with a mesh number of 80, and finally packaged in a PE bag to obtain a sturgeon roe peptide freeze-dried powder with anti-aging effect.

[0063] Comparative Example 1

[0064] The preparation method of sturgeon roe peptide freeze-dried powder with anti-aging effect specifically comprises the following steps:

[0065] (1) thawing the sturgeon roe, taking 1 kg, adding 6 kg of deionized water, stirring evenly, grinding once with a colloid mill, adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8 to obtain a sturgeon roe solution;

[0066] (2) heating the sturgeon roe solution to 80° C. and maintaining it for 10 min, and then naturally cooling it to 55° C.;

[0067] (3) first adding alkaline protease with a content of 1% of sturgeon roe protein to the cooled sturgeon roe solution, stirring evenly, and performing enzymolysis at 55° C. for 8 h;

[0068] (4) inactivating the enzyme at 95° C. for 12 min, cooling naturally to room temperature, adding hydrochloric acid solution, stirring evenly, and adjusting the pH value of the material to 7 to obtain sturgeon roe enzymatic hydrolyzate;

[0069] (5) firstly filter the sturgeon roe enzymatic hydrolyzate through a filter bag and a 200-mesh screen, then centrifuge at a speed of 10,000 r / min, take the supernatant, adsorb it three times through a series of activated carbon columns, filter, and take the filtrate to obtain a sturgeon roe peptide solution;

[0070] (6) First, the sturgeon roe peptide solution is filtered three times through a plate frame with a filter paper precision of 0.1 μm, and the filtrate is taken and sterilized at high temperature; then, vacuum freeze-dried for 2000 min under the conditions of a vacuum degree of 100 Pa and a temperature of -45°C, sieved through a sieve with a mesh number of 80, and finally packaged in a PE bag to obtain a sturgeon roe peptide freeze-dried powder with anti-aging effect.

[0071] Comparative Example 2

[0072] Sturgeon roe peptide NLPL, the synthesis process is as follows:

[0073] 1.1 Experimental equipment: 1 / 10,000 electronic balance; vertical reactor (20*250mm, No. 1 sand core); 50mL centrifuge tube; centrifuge, HPLC semi-preparative, 2cm C18 column, disposable pipette; nitrogen; circulating water vacuum pump; washing bottle; test tube; long-necked pipette; constant temperature heater

[0074] Experimental materials and reagents: 2-CL resin; DIC, DIEA, methanol; DCM; DMF; 20% piperidine / DMF; detection reagent A (5 g ninhydrin-100 mL anhydrous ethanol), B (analytical grade pyridine); HOBT; acetonitrile, nitrogen, TFA

[0075] 1.2 Check sequence: AA amino acid: FMOC-LEU-OH, FMOC-PRO-OH, FMOC-ASN(TRT)-OH

[0076] 1.3 Select a reactor (20*250mm) and write the name of the polypeptide on the reactor as a mark. This process uses FMOC solid phase chemical synthesis. In the following process, AA represents amino acid. Use an electronic balance to weigh 0.5g of 2-cl resin, put it into the reactor, and add DCM to soak for 30 minutes.

[0077] 1.4 Weigh AA (starting from the first position at the C-terminal end, FMOC-LEU-OH) and add it to a centrifuge tube, dissolve it with DCM and add DIEA, shake well. Use a disposable pipette to draw the solution and add it to the reactor in the previous step, bubbling nitrogen for 90 minutes, and add a mixture of methanol and DCM for 20 minutes after the reaction is completed.

[0078] 1.5 Washing: After the liquid in the reactor is drained with a circulating water vacuum pump, add industrial-grade DMF into the reactor with a wash bottle, wash with nitrogen bubbling for 30 seconds, and then drain the liquid in the reactor with a circulating water vacuum pump. Repeat this operation 4 times.

[0079] 1.6 Removal of Fmoc: Add 20% piperidine / DMF solution into the reactor using a wash bottle and react with nitrogen bubbling for 20 minutes.

[0080] 1.7 Washing: Refer to 1.5, and replace the industrial grade DMF with analytical grade DMF during the fifth washing process.

[0081] 1.8 Resin detection: Use a long-necked pipette to take 10-20 resins from the reactor and place them at the bottom of the test tube. Then use a rubber-tipped dropper to take two drops of detection reagent A and B and drop them into the test tube to allow the resin and the detection reagent to fully contact. Then place the test tube in a constant temperature of 100℃ and heat for 2 minutes. Observe the color of the resin. If the resin shows color, it means that 1.6 Fmoc removal is successful. If it does not show color, repeat 1.6-1.8 operations.

[0082] 1.9 Coupling: Weigh AA (FMOC-PRO-OH at the second position of the C-terminus) and HOBT in a centrifuge tube, fully dissolve them with DMF, then add DIC, mix and add to the dried resin, and react with nitrogen bubbling for 1 hour.

[0083] 1.10 Resin detection: Refer to 1.8 and observe the color of the resin. If it is colorless, it means the connection is complete, and proceed to 1.11; if there is color, repeat 1.9.

[0084] 1.11 Washing, same as 1.5.

[0085] 1.12 Repeat steps 1.6-1.11 until the peptide grafting is completed and Fmoc is finally removed (see 1.6), followed by washing (see 1.5)

[0086] 2. Operational procedure added:

[0087] 2.1 Pay attention to the cleanliness of the medicine spoon when weighing.

[0088] 3. Cleavage of the Peptide from the Resin

[0089] 3.1 Preparation of cutting reagent

[0090] Prepare cutting reagent (taking 100mL cutting reagent as an example)

[0091] Cutting reagent A: 95 mL TFA + 1 mL water + 2 mL EDT + 2 mL Tis, for later use.

[0092] 3.2 Preparation for sedimentation

[0093] Precool anhydrous ether at -20°C. Use 10 mL of ether to precipitate the peptide for 1 mL of cleavage reagent.

[0094] 3.3 Peptide cleavage

[0095] The resin was drained and weighed, and then cut with cutting fluid for 2 h. It was then precipitated with ice ether.

[0096] 4. Peptide Purification

[0097] Chromatographic column: 20*250mm daisogel 8 micron

[0098] Mobile phase: A: 0.1% TFA water; B: 0.1% TFA acetonitrile

[0099] Flow rate: 10mL / mLin

[0100] The sample was loaded via pump A, and then 5% acetonitrile water was equilibrated for 5 min and the gradient was then run: 0, 5%; 5, 5%; 45, 45%.

[0101] The sample peaks collected and tested were prepared with an analytical purity greater than 98%.

[0102] 5. Freeze-drying of samples

[0103] The sample was transferred into a freeze drying dish and placed in a freeze dryer for 24 hours to obtain a pure product.

[0104] Performance Testing

[0105] 1. Stability test

[0106] The sturgeon roe peptide freeze-dried powders obtained in Example 2 (embedded) and Comparative Example 1 (unembedded) were dissolved in water at a concentration of 100-200 mg / 25 mL, respectively, and placed at 37° C. for a period of time and then observed.

[0107] The results showed that the unembedded sturgeon roe peptide solution of Comparative Example 1 had oily floating on the surface and agglomerated precipitation in the middle; while the sturgeon roe peptide raw material after embedding in Example 2 had good overall stability, with basically no surface floating and no agglomerated precipitation. The reason is that the unembedded sturgeon roe raw material of Comparative Example 1 contains water-soluble components and oily components, and its solubility in water is not as good as that of Example 2 after embedding.

[0108] 2. Anti-aging test

[0109] The sturgeon roe enzymatic hydrolyzate (XM) prepared in step (4) of Example 2 and the sturgeon roe peptide NLPL (XT) prepared in Comparative Example 2 were respectively used for anti-aging tests.

[0110] 2.1 Cell culture

[0111] Purchase HSF primary cells and culture them in HSF-specific medium. When the cell coverage rate reaches about 80%, cell passage is performed. Subsequent experiments are started after the sixth generation of cells are in good condition.

[0112] 2.2 Modeling concentration screening

[0113] HSF cells were seeded into 96-well plates at an appropriate concentration, 100 μL per well, and incubated at 37°C, 5% CO2 for 24 hours, and the culture medium was discarded. The cells were divided into a normal group and a H2O2 injury group (0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 2.0 mmol / L), with six replicate wells in each group, incubated at 37°C, 5% CO2 for 2 hours, and the OD value of each group was determined by CCK-8 method, and the relative cell viability was calculated.

[0114] 2.3 Sample toxicity test

[0115] HSF cells in the logarithmic growth phase were taken, adjusted to an appropriate cell concentration and inoculated into a 96-well plate. The cells were divided into a normal group and different concentration gradient sample groups. The normal group was given blank culture medium. Six parallels were set for each concentration and cultured for 24 hours. The OD value of each group was determined by CCK-8 method, and the relative cell viability was calculated.

[0116] 2.4 Type III and IV collagen detection

[0117] According to the appropriate cell concentration, six-well plates were plated and divided into normal group, model group, low, medium and high sample groups. Collect the cell culture supernatant and centrifuge at 2000-3000rpm / min for 20min to carefully collect the supernatant for testing. Dilute the biotin antigen, standard product, and avidin-HRP. Add the standard product, control product, and sample to be tested to the enzyme-labeled plate respectively, and then add the working solution. After the reaction is completed, add the stop solution to complete the reaction and measure the absorbance value. Repeat the test three times.

[0118] 2.5SA-β-gal staining

[0119] According to the appropriate cell concentration, six-well plates were plated and divided into normal group, model group, low, medium and high sample groups. After the cell culture modeling was completed, the cell culture medium was aspirated, washed once with PBS, 1mL of staining fixative was added, and fixed at room temperature for 15min. The fixative was aspirated, and the cells were washed 3 times with PBS, 3min each time. PBS was aspirated, and 1mL of SA-β-gal staining solution was added to each well. Incubate at 37℃ overnight and seal with plastic wrap to prevent the staining solution from evaporating. Observe and take pictures under an ordinary optical microscope, and repeat the experiment three times.

[0120] 2.6 TeloTAGGG TM Telomere length test

[0121] According to the appropriate cell concentration, six-well plates were plated and divided into normal group, model group, low, medium and high sample groups. After the cell culture modeling and sampling were completed, the cell culture medium was aspirated to collect the cells for testing. The cells were broken to determine the protein concentration. 2μL of enzyme digestion buffer (10X), 0.5μL HinfI and 0.5μL Rsa I were added, and the total system was 20μL. Take 15μL of control DNA, add 2μL of nuclease-free water, and then add 2μL of enzyme digestion buffer (10X), 0.5μL HinfI and 0.5μL Rsa I, and the total system was 20μL DIG marker: marker 4μL+4μL 5×loading buffer+12μL nuclease-free water. Incubate in a 37℃ water bath for 2h, and add 5μL 5×loading buffer to terminate the reaction. Prepare 1×TAE buffer; prepare 0.8% agarose gel. The samples were spotted into the corresponding lanes, and the DNA fragments were separated by electrophoresis at 70V for 3h, and then transferred to a nylon membrane by Southern blotting. The transferred DNA fragments were hybridized with a digoxigenin (DIG)-labeled probe specific for telomeric repeat sequences, and then incubated with a DIG-specific antibody covalently linked to alkaline phosphatase. Finally, the fixed telomeric probe was visualized using CDP-Star, a highly sensitive chemiluminescent substrate for alkaline phosphatase. The average TRF length can be determined by comparing the signal with a molecular weight standard. The experiment was repeated three times.

[0122] 2.7 NAD+ content detection

[0123] According to the appropriate cell concentration, six-well plates were plated and divided into normal group, model group, low, medium and high sample groups. When collecting the plates, the culture medium was aspirated, 200 μL of NAD+ / NADH extract was added with a pipette, and gently blown to promote cell lysis; the lysis process can be performed at room temperature or on ice. Then centrifuged at 12,000g, 4℃ for 5-10min, and the supernatant was taken as the sample to be tested.

[0124] Prepare NADH standard, set up NADH standard curve, and prepare alcohol dehydrogenase working solution. Pipette 20μl of the sample to be tested into a 96-well plate, determine the total amount of NAD+ and NADH in the sample, and set up duplicate wells for the sample. Determine the content of NAD+, NADH or NAD+ / NADH ratio in the sample: Pipette 50-100μL of the sample to be tested into a centrifuge tube and heat it in a 60℃ water bath or on a PCR instrument for 30 minutes to decompose NAD+. If insoluble matter is produced after heating, centrifuge at 10,000g at room temperature or 4℃ for 5 minutes, and pipette 20μL of the supernatant as the sample to be tested into a 96-well plate, and set up duplicate wells for the sample. Set up blank control, standard, and sample groups in the 96-well plate, and add the sample to be tested, NAD+ / NADH extract, and alcohol dehydrogenase working solution as required. Incubate at 37℃ in the dark for 10 minutes (to convert NAD+ in the sample into NADH). Mix the colorimetric solution appropriately, then add 10 μL of the colorimetric solution to each well, mix well, and incubate at 37°C in the dark for 30 minutes. Orange-yellow formazan will be formed. Measure the absorbance at 450 nm. Repeat the experiment three times.

[0125] 2.8 Experimental results and discussion

[0126] 2.8.1 Effects of different H2O2 concentrations on HSF cell viability

[0127] Cell survival rate is one of the intuitive indicators to measure the degree of cell damage. Figure 1 shown.

[0128] As shown in Table 1, with the increase of H2O2 concentration, the relative cell viability showed a downward trend, and H2O2 concentration ≥ 800μM had a significant inhibitory effect on cell proliferation. The inhibition rate of cell proliferation by 900μM H2O2 reached (61.42±0.019)%, the inhibition rate of 1000μM H2O2 reached (50.06±0.020)%, and the inhibition rate of 1200μM H2O2 was as high as (37.66±0.025)%. To construct a cell aging model, a concentration with cell viability between 50% and 70% should be selected as the modeling concentration. If the cell survival rate is too high, the damage is not obvious. If the survival rate is too low, the cells may suffer from severe irreversible damage, which is not conducive to later experiments. Therefore, this experiment selected 1000μM H2O2 for 2h to induce HSF cell aging damage.

[0129] 2.8.2 Effect of sample treatment on type III collagen production

[0130] Collagen is an important structural protein, mainly found in the connective tissue of animals, and is essential for maintaining the integrity of tissue structure and function. Its structural feature is that three left-handed helical α peptide chains are twisted into a right-handed super triple helix. Collagen composed of three identical α chains is called type III collagen, which is mostly found in blood vessels, new skin and scar tissue; type IV collagen is involved in the composition of basement membrane. The primary structure of collagen is "Gly-XY" (X is mostly proline Pro, Y is mostly hydroxyproline Hyp), and the hydroxyproline hydroxyl group can stabilize the collagen helical structure through intermolecular hydrogen bonds. Therefore, changes in collagen in the skin can indirectly reflect the true state of the skin.

[0131] Effects of sturgeon roe hydrolysate (XM) and sturgeon roe peptide NLPL (XT) on the content of type III collagen in HSF cells damaged by H2O2 Figure 2 shown.

[0132] As shown in Table 2, compared with the control group, the type III collagen content in the Damage group was significantly reduced (p<0.05). Compared with the Damage group, the type III collagen content of fibroblasts in the four samples after treatment was significantly increased (p<0.05).

[0133] The results showed that both sturgeon roe hydrolysate and sturgeon roe peptide NLPL can promote the expression of type III collagen in fibroblasts. The sturgeon roe hydrolysate group (9.66±0.06) ng / mL was significantly higher than the sturgeon roe peptide NLPL group (9.15±0.06) ng / mL, and the ability of the sturgeon roe hydrolysate group to promote fibroblast collagen was better than that of the sturgeon roe peptide NLPL group at the same concentration.

[0134] 2.8.3 Effect of sample treatment on type IV collagen production

[0135] Effects of sturgeon roe enzymatic hydrolysate and sturgeon roe peptide NLPL on the content of type IV collagen in HSF cells damaged by H2O2 Figure 3 shown.

[0136] Depend on Figure 3 It can be seen that compared with the control group, the content of type IV collagen in the model group treated with H2O2 was significantly reduced (p<0.05), while each sample treatment group significantly increased the content of type IV collagen in damaged HSF cells (p<0.05), specifically 22.32 (XM) and 17.02 (XT) ng / mL. Among them, the content of sturgeon roe enzymatic hydrolysate group increased most significantly.

[0137] 2.8.4 Effect of sample treatment on the expression of β-galactosidase

[0138] Most normal cells are believed to have limited ability to divide, and enter a state of senescence when they cannot divide. At this time, the cells are still alive, but the expression profiles of the cells' genes and proteins have changed greatly. When cells age, the activity level of senescence-associated β-galactosidase (SA-β-Gal) increases due to the accumulation of enzymes. The colorless X-Gal substrate generates a dark blue product under the catalysis of senescence-specific β-galactosidase, making it easy to observe senescent cells expressing β-galactosidase under an optical microscope.

[0139] Effects of sturgeon roe enzymatic hydrolysate and sturgeon roe peptide NLPL on the expression of β-galactosidase in HSF cells damaged by H2O2 Figure 4 shown.

[0140] Depend on Figure 4 It can be seen that compared with the control group, the color of cells in the Damage group was dark blue, indicating that the expression of β-galactosidase in HSF cells increased after H2O2 damage, while the expression of β-galactosidase in each sample was reduced after treatment.

[0141] 2.8.5 Effect of sample processing on telomere relative length

[0142] Telomere is a DNA sequence at the end of a eukaryotic chromosome composed of multiple repeated nucleotide elements (TTAGGG) in series. It is the most important and accurate indicator of a person's aging rate. Its initial length is determined by genetic and environmental factors and will decrease over time. When the telomere shortens to a certain extent, the cell can no longer replicate and divide, and the telomere becomes shorter and shorter, eventually causing the regenerative aging of the cell. Studies have shown that telomere length is closely related to DNA repair, aging, apoptosis, and tumorigenesis. In this experiment, relative quantitative qPCR was mainly used to directly compare the average telomere length of the samples, that is, the copy number ratio (T / S) of the telomere repeat sequence (Tel) to the copy number of the single copy gene (SCR) in the genome was used as the relative telomere length.

[0143] Protective effects of sturgeon roe hydrolysate and sturgeon roe peptide NLPL on H2O2-induced HSF cell damage Figure 5 shown.

[0144] Depend on Figure 5 It can be seen that compared with the control group, the relative telomere length in the model group damaged by H2O2 treatment was significantly reduced by 9.64% (p<0.05); compared with the model group, the shortening of the relative telomere length was inhibited after adding each sample for treatment, and the relative telomere length of each sample was 9.97% (XM) and 9.43% (XT) higher than that of the model group, respectively, which was statistically significant.

[0145] 2.8.6 Effect of sample treatment on NAD+ content

[0146] NAD+ (Nicotinamide adenine dinucleotide) is a coenzyme present in all living cells. Its synthesis and degradation and its products are involved in cell apoptosis, metabolic regulation, and gene expression regulation, etc., playing important functions in cells and the body.

[0147] Effects of sturgeon roe enzymatic hydrolysate and sturgeon roe peptide NLPL on NAD+ content in HSF cells damaged by H2O2 Figure 6 shown.

[0148] Depend on Figure 6 It can be seen that the NAD+ content in the model group was significantly lower than that in the normal control group (P<0.01). The results showed that 1000μMH2O2 would destroy the body's antioxidant system and accelerate the aging process. After sample treatment, the NAD+ content in the sample group increased significantly (P<0.05).

[0149] 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 peptide freeze-dried powder with anti-aging effect, characterized in that: The specific steps include: (1) thawing the sturgeon roe, adding water, stirring, grinding and crushing, adjusting the pH value, and obtaining a sturgeon roe solution; (2) the sturgeon roe solution is heated up and then cooled naturally; (3) firstly adding alkaline protease to the sturgeon roe solution after cooling, stirring evenly for enzymolysis, and then adding sodium starch octenyl succinate and vitamin E powder; (4) enzyme deactivation, natural cooling, and pH adjustment to obtain sturgeon roe enzymolyzate; (5) coarsely filtering and centrifuging the sturgeon roe hydrolysate, taking the supernatant, adsorbing it through an activated carbon series column, filtering, and taking the filtrate to obtain a sturgeon roe peptide solution; (6) plate and frame filtration, taking the filtrate, sterilizing at high temperature, vacuum freeze drying, screening, and packaging to obtain the sturgeon roe peptide freeze-dried powder with anti-aging effect.

2. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (1), the mass ratio of the sturgeon roe to water is 1:

6.

3. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (1), the grinding equipment is a colloid mill, and the number of times is 1; the operation of adjusting the pH value is specifically: adding a saturated NaOH solution, stirring evenly, and adjusting the pH value of the material to 8.

4. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (2), the temperature is raised to 80°C and maintained for 10 minutes; and the temperature is naturally lowered to 55°C.

5. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (3), the amount of alkaline protease added is 1% of the weight of sturgeon roe protein.

6. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (3), the enzymatic hydrolysis temperature is 55° C. and the time is 8 h.

7. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (3), the amount of sodium starch octenylsuccinate added is 10 g / L; the content of vitamin E in the vitamin E powder is 50%, and the amount added is 12 mg / L.

8. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (4), the temperature for enzyme inactivation is 90-100°C for 10-15 minutes; the natural cooling to room temperature; and the operation of adjusting the pH value are specifically as follows: adding hydrochloric acid solution, stirring evenly, and adjusting the pH value of the material to 7.

9. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (5), the tools used for coarse filtration are filter bags and 200-mesh screens; the rotation speed of the centrifugal treatment is 5000-20000 r / min; and the number of times the activated carbon series column is adsorbed is 3 times.

10. The method for preparing a sturgeon roe peptide freeze-dried powder with anti-aging efficacy according to claim 1, characterized in that: In step (6), the filter paper precision of the plate and frame filtration is 0.1 μm, and the number of filtrations is 3 times; the vacuum degree of the vacuum freeze-drying is 100 Pa, the temperature is -45°C, and the time is 2000 min; the mesh size of the sieving is 80 mesh; and the packaging material is a PE bag.

Images