WO2015100692A1

WO2015100692A1 - Method of preparing queen bee embryo peptides by enzymolysis

Info

Publication number: WO2015100692A1
Application number: PCT/CN2014/000972
Authority: WO
Inventors: 赵树民; 鲍勇刚; 石松传
Original assignee: 赵树民; 于利
Priority date: 2013-12-31
Filing date: 2014-11-05
Publication date: 2015-07-09
Also published as: CN103725742A; CN103725742B

Abstract

Provided is a method of preparing queen bee embryo peptides by enzymolysis, comprising: pretreating a sample, removing lipids from the queen bee larvae powder, preparing queen bee larvae powder sample liquid, conducting ultrasonic treatment, conducting enzymolysis, placing the enzymatic hydrolysates into boiling water for 20 minutes to deactivate the enzyme, cooling to room temperature, adjusting the PH value of the enzymatic hydrolysates to 7.0, removing the supernatant after centrifuging, obtaining hydrolyzed queen bee embryo peptide powder after drying. The queen bee embryo polypeptides of the invention can be used to produce health care products for improving functions of the human body, and screen biologically active polypeptides effective for the human body.

Description

Method for preparing queen bee embryo peptide by enzymatic hydrolysis

Technical field

The present invention relates to a method for preparing a royal jelly embryo peptide by enzymatic hydrolysis.

Background technique

The queen bee larva, also known as the queen bee or the queen bee, is a bee fertilized egg that grows into a larva body by feeding the fresh royal jelly, and is a larva taken from the surface of the royal jelly of Wangtai. The queen bee larvae feed on royal jelly, and the larvae themselves are extremely rich in nutrients, and their body surface also adheres to the royal jelly. It is a by-product of the artificial production of royal jelly. The queen bee larva is a living animal that is rich in nutrients and rich in enzymes and biologically active substances. The queen bee larvae not only have high protein content (more than 50% of dry matter), but also 18 kinds of amino acids, which are rich in 8 kinds of essential amino acids. The content of aspartic acid and glutamic acid is relatively high, and these two amino acids have a good brain-building effect on the human body. In addition, it also contains taurine. The queen bee larva contains a large number of essential and trace elements (mg/100g dry matter): copper 2.402, potassium 1489.3, sodium 81.5, zinc 11.5, magnesium 152.5, aluminum 78.3, selenium 18.1 (micrograms / 100 grams), calcium 84.6, Iron 4.2, molybdenum 0.26 (ng/g). The queen bee larvae are rich in vitamins, especially vitamin A, much more than beef or eggs, second only to cod liver oil; vitamin D is also very rich, 1g fresh larva contains 6100 ~ 7340 international units, and is considered to contain high vitamin D Cod liver oil contains only 100 to 600 international units per 1 g. In addition, the queen larvae also contain high-activity and high-bio-mixed hormones, of which juvenile hormones and ecdysone are abundant. At the same time of nutrient-rich, the cholesterol content of the queen bee larvae is relatively low. Studies have shown that the cholesterol content of the queen bee larvae is less than 10mg/100g, and its cholesterol content is extremely low compared with foods such as beef, milk and eggs, so the queen bee larva is a low Cholesterol food resources are extremely valuable for development. Especially noteworthy is that it contains more rare Trace elements such as selenium, cobalt, chromium, etc. The queen bee larva contains a variety of biological hormones, enzymes (superoxide dismutase 5670 μg / 100 g) and other highly biological substances. The queen bee larva is a very precious natural product. It is rich in protein, amino acids, polysaccharides, vitamins, minerals and many other nutrients required by the human body. It plays an important role in supplementing nutrition and regulating immunity.

The queen bee larvae have anti-fatigue effects. It indicated that the larvae of the queen bee can significantly improve the anti-stress ability of mice and show obvious anti-fatigue effects. The queen bee larva is a nutrient-rich natural substance that promotes growth. The queen bee larva can regulate the function of the central nervous system, and has the functions of enhancing physical strength, calming the nerves, refreshing the spirit, improving sleep and enhancing memory. Applicable to Shen Shen forgetfulness, insomnia, dreams, mental disorders, upset and so on. The queen bee larva improves cardiovascular function and has the functions of enhancing cardiac contractility, balancing blood pressure, improving microcirculation, promoting tissue metabolism, and increasing white blood cells. Applicable to shortness of breath, qi and blood, sallow complexion, pale face, weakness of limbs. In particular, it has a liver-protecting effect, which can improve function and restore damaged liver cells. The queen bee larva has a good regulation effect on the digestive system, can increase appetite, help digestion, and benefit from bowel movements. It has a good effect on sputum bloating, stomach anorexia and constipation, and has an auxiliary effect on chronic superficial gastritis. The queen bee larva has a regulating effect on the endocrine system, which can adjust menstruation, improve menopausal discomfort, and enhance sexual desire between men and women. There is a significant improvement in the number of male sperm, poor mobility, and prolonged liquefaction time. For menstrual period is not allowed, the amount is large, the amount is small, menopausal syndrome, impotence, premature ejaculation, female ovulation disorders, infertility and other auxiliary effects. Practice has proved that the queen bee larva can be beauty and beauty, strong blood, spleen and stomach, and benefit the mind. It is the most ideal health care product for qi and nourishing, supplementing nutrition, especially for the elderly, frail, nourishing after pregnancy, nursery during pregnancy and adolescent growth retardation. In summary, the role of the queen bee larvae mainly includes: 1, the role of brain and brain; 2, enhance the body's immunity; 3, anti-tumor effect; 4 anti-oxidation and beauty; 5, improve the digestive system.

Now the annual output of single group of royal jelly is over 10kg. The annual output of royal jelly in China exceeds 3,000 tons. According to the production of 1kg of royal jelly and 0.2kg-0.3kg of bee larvae, the annual production of queen bee larvae can reach 600-900. Ton. However, the annual larvae used for processing in the country are only about 50 tons, and the rest are directly disposed of by beekeepers. This waste is amazing.

The queen bee larvae is a food-rich resource with extremely rich nutritional value in nutrition, health care, and beauty. The aspect has good research and development significance, but due to the long-term scientific research conditions, technical conditions, and economic development constraints, the current development and utilization is far from enough, and the research investment and industrial development are not enough. The queen larvae research and industrial development have not yet entered practicality. Stage. The development and utilization of the queen bee larvae is imperative.

Bioactive peptides are a class of functional factors that have the special functions of scavenging free radicals, improving immunity, delaying aging, and lowering blood pressure. These peptides are not active in the original protein sequence. When the protein is treated with intestinal enzymes or in vitro enzymes, these peptides will be released from the original sequence and exhibit special activities such as immunopeptides, casein phosphopeptides, Antibacterial peptides and blood pressure lowering peptides.

The main products of the queen bee larvae are the following: lyophilized powder, protein extract, fresh larvae processing solution. Freeze-dried powder is one of the most common products of the queen bee larva. It has been reported in the literature that various types of proteins of the queen bee larvae are extracted from the lyophilized powder of the queen bee larvae. At present, there is no patent report on the preparation of hydrolyzed polypeptides from the source of the larvae, which is a widely used and inexpensive protein source.

Summary of the invention

In order to solve the above technical problems, the present invention provides a method for preparing a royal jelly embryo peptide using a bee larva.

The invention is a method for preparing a royal jelly embryo peptide by enzymatic hydrolysis, comprising:

Step 1: Sample pretreatment: The queen bee larvae are rapidly frozen at -80 ° C during the survival period, and dried, and the dried samples of the queen bee larvae are ground into powder;

Step 2: Degreasing of the queen bee larva powder: The pretreated powder of the queen bee larvae is degreased in a supercritical extraction device. The degreasing conditions are: extraction pressure 35 MPa, extraction temperature 35 ° C, extraction time 200 minutes, separation under normal pressure, collection of degreased The queen bee larva protein is used for enzymatic hydrolysis;

Step 3: Preparation of the sample liquid of the queen bee larvae powder: using the above-mentioned degreased queen bee larvae powder as the raw material, the protein content of the raw material is more than 70%, and the raw material of the queen bee larvae powder is 1:10 (w/v according to the ratio of material to liquid). Adding deionized water at 50 ° C and stirring and mixing for 4 hours to obtain a sample liquid of the queen bee larvae powder;

Step 4: Ultrasonic treatment of the queen bee larva powder sample liquid: pretreatment of the bee larva powder sample liquid using pulsed ultrasonic waves with a power of 250 W to 1250 W, ultrasonic treatment time of 30 minutes, corresponding to one pulse of ultrasonic wave emission time of 2 to 4 seconds, gap time 2 second;

Step 5: Enzymatic hydrolysis of the sample of the queen bee larvae powder: adjust the pH of the sample of the defatted queen bee larvae powder after ultrasonic treatment to 7.5-8.5, and heat it to 40 °C-60 °C in a constant temperature water bath. According to the enzyme and the defatted queen bee larvae powder sample The liquid mass ratio is 0.02-0.04:1, and the complex protease is added. The complex protease consists of a neutral protease and a flavor protease in a mass ratio of 1:1, and the reaction is stopped when the degree of hydrolysis reaches 20%;

Step 6: The enzymatic hydrolysate is heated in a boiling water bath for 20 minutes to kill the enzyme, cooled to normal temperature, the pH of the enzymatic hydrolysate is adjusted to 7.0, and the supernatant is taken after centrifugation;

Step 7: The above supernatant is dried to obtain a hydrolyzed queen bee embryo peptide powder.

The queen bee larva is a 2 to 3 day old queen bee larva.

The invention relates to a method for determining a polypeptide content in a queen bee embryo peptide prepared by enzymatic hydrolysis, which uses 10% trichloroacetic acid (TCA) to precipitate a macromolecular protein in a hydrolyzate, and after centrifugation, a double is added to the supernatant. The urea reduction reagent was used to measure the OD value at 540 nm, and then a Gly-Gly-Tyr-Arg tetrapeptide standard solution was prepared, standard peptide data was determined, a standard curve was prepared, and the polypeptide content in the sample was looked up on the curve.

A method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide includes a free radical scavenging ability assay, a superoxide anion radical scavenging ability assay, and a liposome peroxidation inhibiting ability assay.

The free radical scavenging ability is determined by measuring the removal force of the DPPH free radical by the hydrolyzed royal jelly embryo peptidase sample solution.

The superoxide anion radical scavenging ability was measured by using a riboflavin-photo-nitroblue tetrazolium system.

The liposome peroxidation inhibition ability is determined by adding 0.2 ml of liposome solution to a 10 m test tube, 50 ul of different volume of sample and 50 mmol/L FeSO ₄ solution, and then adding phosphate buffer solution to 3 mL without sample. The reaction tube was used as a model. The test tube without FeSO ₄ solution was used as a blank. The reaction was placed in a 37 ° C water bath for 40 minutes. Finally, 10 ml of 10% trichloroacetic acid and 1 ml of 0.8% thiobarbituric acid (TBA) were added. , placed in boiling water for 15 minutes, cooled in ice water, centrifuged at 8000 r / min for 10 minutes, the supernatant was taken at 532 nm to measure the absorbance (A), the inhibition rate of lipid peroxidation was calculated according to the following formula: lipid over Oxidation inhibition rate (%) = (A model - A sample) / (A model - A blank) × 100%.

Advantageous technical effects of the present invention are:

1. The present invention utilizes a complex protease (Protamex) (neutral protease: flavor protease = 1:1) to hydrolyze the queen bee embryo larvae. As an endoprotease, neutral protease has pure natural, safe and non-toxic, strong hydrolysis ability and wide range of action. The flavor protease is natural and safe, and can be applied to the hydrolysis of various animal and vegetable proteins, and the flavor optimization at the later stage to remove bitterness. Improve the taste, can produce good animal and plant hydrolysis products, improve product quality and reduce costs. Appropriate flavor proteases are added during food processing to hydrolyze the flavor precursors, thereby releasing flavor substances and enhancing and improving the flavor of the food. The enzymatic hydrolysis of the composite protease not only ensures sufficient hydrolysis of the queen larvae of the queen bee, but also enables the hydrolyzed product to have a flavor suitable for eating, and is guaranteed to be natural, safe and non-toxic.

2. The bio-enzymatic technology of the invention utilizes the biocatalytic decomposition of different proteases to decompose the macromolecular protein molecules into skin-like small molecular substances to a certain extent, so that the nutrients can be more beneficial to the human body to digest and absorb, and may also be improved. Its biological activity. The enzymatic hydrolysate can be used for biological activity research or multi-intestinal separation and purification research, which lays a foundation for further improving the product utilization value.

3. The free amino acid of the queen bee embryo peptide powder prepared by the invention is obviously increased, especially the proportion of essential amino acids is increased, and the essential amino acid is an amino acid which cannot be synthesized by human beings, and must be obtained from food, so that the present invention The small molecule queen bee embryo polypeptide which is easy to be absorbed and has good anti-oxidation effect can greatly improve the nutritional value of the queen bee embryo.

4. The invention applies ultrasonic technology to proteolytic preparation of queen bee embryo polypeptide, which can significantly shorten the proteolytic time of the defatted queen bee embryo larva, so that the effective small molecule polypeptide is fully decomposed.

5. The invention is a biological enzymatic method, which can effectively release the small molecule polypeptide in the queen bee embryo through the monitoring of the enzymatic hydrolysis process, thereby improving the utilization rate of the raw material and improving the health care value thereof;

6. The raw materials used in the present invention are widely available and inexpensive, and the added value of the queen bee embryo larvae can be greatly improved by the technology of the present invention.

DRAWINGS

Figure 1: Supercritical extraction device for degreasing of queen bee larvae powder.

1, cylinder 2, filter 3, compressor 4, buffer tank 5, pressure regulating valve 6, preheater

7, check valve 8, extraction kettle 9, pressure reducing valve 10, sampling bottle 11, accumulating flow meter

Figure 2: Gly-Gly-Tyr-Arg tetrapeptide standard curve.

Figure 3: Comparison of DPPH free radical scavenging ability of queen bee embryo peptides and control BHT.

Figure 4: Comparison of the scavenging ability of the queen bee embryo peptide to superoxide anion radical (O ₂ ·) and the control group BHT.

Figure 5: Determination of liposome peroxidation inhibition ability.

Figure 6: Effect of different protease hydrolysis of queen bee embryo proteins on peptide yield.

detailed description

The specific embodiments of the present invention are further described in detail below with reference to the drawings and embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Step 1: Sample pretreatment: 2 to 3 days old queen bee larvae (bee queen embryos, 2 to 3 days old queen bee larvae did not grow and the respiration rate was very similar, and then they began to differentiate into bee worker bees as the feeding conditions changed. It is the basis for selecting the larvae of the queen bee 2 to 3 days. It is rapidly frozen at -80 ° C during the survival period of the queen bee larvae, freeze-dried with a freeze dryer, and the dried sample of the queen bee larva is ground into a powder.

Step 2: Degreasing of the queen bee larvae powder: Put the pretreated sample of the queen bee larvae into the supercritical extraction device as shown in Fig. 1 to degrease, and put the queen bee larvae powder into the extraction kettle to open the cylinder, the CO ₂ in the cylinder 1 After filtering through the filter 2, it enters the compressor 3 (diaphragm type), is sent to the buffer tank 4 after the pressure reaches the required pressure, enters the preheater 6 through the pressure regulating valve 5 from the buffer tank, and heats the CO ₂ to the desired temperature. Then, it enters the extraction kettle 8 to extract oil and fat. Degreasing conditions: extraction pressure 35 MPa, extraction temperature 35 ° C, extraction time 200 minutes, separation at atmospheric pressure. The degreased queen bee larvae protein was collected for enzymatic hydrolysis.

Step 4: Ultrasonic treatment of the queen bee larva powder sample liquid: using the pulsed ultrasonic wave with a power of 250W to 1250W to pretreat the sample liquid of the queen bee larva powder, the ultrasonic treatment time is 30 minutes, the ultrasonic wave emitting time corresponding to one pulse is 2 to 4 s, and the gap time is 2 s;

In step 5, the degree of hydrolysis in the enzymatic hydrolysis process is determined by the pH-stat method:

The formula for calculating the degree of hydrolysis is as follows:

formula,

h: the number of peptide bonds (mmol/g) that are cleaved per unit mass of protein;

h _tot : the total number of peptide bonds in the unit mass of raw material protein (mmol/g);

In the formula: M _α is the average molecular weight of AA in the hydrolyzed protein.

M _α =∑p _i ×M _i

Where: P _i is the amino acid content of a certain constituent protein;

Mi is the amino acid molecular weight of a certain constituent protein.

It can be seen from the above two formulas that h _tot of a particular protein is a fixed value, and the h _tot of the queen beetle larva protein can be calculated, and its value is 7.57.

When the solution pH ≥ 6, the quantitative relationship between the amount of peptide bonds broken during proteolysis and the amount of added alkali is as follows:

Where: B-NaOH consumption, mL;

N _b -NaOH concentration, mol / L;

Mp-amount of protein hydrolyzed, g;

1/α-correction coefficient;

The average degree of dissociation of α-α-NH ₂ in the protein substrate can be calculated by the following formula:

Wherein, the pH of the solution when the protein is subjected to enzymatic hydrolysis;

The dissociation constant of pK-NH ₃ +; the proteolytic conditions are different, and the α value is different. The specific conversion is as follows. Therefore, the hydrolysis degree (DH) calculation formula can be evolved into the following formula:

From the above derivation formula, the pH-stat method only needs to record the amount of alkali consumed in the enzymatic hydrolysis process to calculate the DH value during proteolysis.

Table 1 correction factor 1 / α under different conditions

From the start of the addition of the enzyme, the hydrolysis was detected every 10-12 minutes, and the hydrolysis was stopped when the degree of hydrolysis reached 20%.

Step 7: using a conventional freeze-drying method, drying the above supernatant, and adding an auxiliary material, such as adding 10%-30% of the supernatant to the edible starch, etc., and spray-drying to obtain a hydrolyzed queen bee embryo peptide in a pale yellow or white powder form. powder.

In order to determine the content of the polypeptide in the hydrolyzed peptide of the queen bee larvae (the queen bee embryo), the ability of the method to hydrolyze the queen beetle larvae protein can be determined by the following method.

The macromolecular protein in the hydrolyzate was precipitated by 10% trichloroacetic acid (TCA). After centrifugation, the biuret reagent was added to the supernatant, and the OD value was measured at 540 nm, and then Gly-Gly-Tyr-Arg was prepared. Peptide standard solution, standard peptide data was determined, and a standard curve of Gly-Gly-Tyr-Arg tetrapeptide was prepared, as shown in Fig. 2, and the content of the polypeptide in the sample was looked up on the curve.

The specific determination procedure of the polypeptide content is as follows. 2.5 ml of the sample solution is added to 2.5 ml of a 10% (w/v) aqueous solution of trichloroacetic acid (TCA), mixed on a vortex mixer, uniformly allowed to stand for 10 minutes, and then centrifuged at 4000 rpm. Transfer the supernatant to a 50 ml volumetric flask for 15 minutes and dilute to the mark with 5% TCA. Then take 6.0 ml of the above solution and add another tube to the biuret reagent 4.0 ml (sample solution: biuret reagent). =3:2V/V), uniformly mixed on a vortex mixer for 10 minutes, centrifuged at 2000 rpm for 10 minutes, and the supernatant was taken. The OD value was measured at 540 nm. The concentration of the polypeptide in the sample solution was determined by the standard curve C (mg/ Ml) in turn to determine the polypeptide content of the sample.

The standard curve was prepared as follows: Take 10 10 ml volumetric flasks and prepare 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.6 and 1.8 mg/ml Gly-Gly-Tyr-Arg tetrapeptide standard solution with 5% TCA and then take 6.0. Ml standard solution was added to 4.0 ml of biuret reagent and mixed on a vortex mixer for 10 minutes at 2000 r/min for 10 minutes. The supernatant was taken at 540 nm to determine the OD value (with the first tube as a blank control) to the concentration of the peptide. A standard curve (bottom) was prepared for the ordinate of the abscissa X (mg/ml) OD as the ordinate Y (the lower graph) to obtain a regression equation y=0.3681x+0.0013, R ² =1. In this example, the measured data is as follows: OD ₅₄₀ = 0.634, and the polypeptide concentration calculated according to the formula Y = 0.3681X + 0.0013 is 1.72 mg / ml.

The invention relates to a method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide, which comprises the determination of free radical scavenging ability, the determination of superoxide anion radical scavenging ability and the determination of liposome peroxidation inhibition ability, the purpose of which is to detect hydrolyzed queen bee embryo peptide Antioxidant and anti-aging ability.

1. The free radical scavenging capacity is determined by measuring the removal force of the DPPH free radical (DPPH·) by the hydrolyzed royal jelly embryo peptidase sample solution. The specific determination method is:

Dissolve in 2 ml of DPPH powder with 32 ml of absolute ethanol, and store at 4 ° C in the dark. The ability of the hydrolyzed royal jelly embryo peptidase sample solution to remove DPPH free radicals (DPPH·) was determined. According to the method of Table 2, the reaction was carried out for 10 minutes, centrifuged at 5000 r/min for 3 minutes, 200 ul of each reaction solution was taken in a 96-well plate, and the absorbance A was measured at 517 nm with a microplate reader; BHT (2,6-di-tert-butyl) Base p-cresol) as a positive control

Table 2

In the table, A is the absorbance value.

The clearance rate is calculated according to the following formula:

Clearance rate (%) = [1-(Ai-Aj)/A] × 100%

Note: In the formula, A is the absorbance value, A ₀ is the negative control, and the blank sample absorbs the light; A _i is the absorbance after adding the sample reaction solution.

DPPH· is a relatively stable nitrogen-centered aromatic free radical. Studying the direct capture of antioxidant samples or the combination with DPPH· can predict the ability of antioxidants to scavenge aromatic free radicals. BHT can be used both as a hydrogen donor and as a free radical capture agent in the antioxidant process. Used as an antioxidant in food processing. As shown in Figure 3, the ability of the queen bee embryo polypeptide to scavenge DPPH· is very strong, which is significantly higher than that of the control group. In the concentration range of the study, as the concentration increases, the scavenging ability increases gradually, showing a better amount. Effective relationship. Therefore, the queen bee embryo polypeptide has a good ability to scavenge DPPH.

2. The superoxide anion radical scavenging ability is determined by using riboflavin-photo-nitroblue tetrazolium system (NBT method) measurement. Using a riboflavin-photo-nitroblue tetrazolium system (NBT method); the specific steps, 0.5mL 50mmol / LTris-HCl buffer solution (pH 8.2), 0.4mL deionized water in a 2mL tube after mixing In a 37 ° C water bath for 20 minutes, immediately after removal, add 0.1mL of pre-heated 3mmol / L pyrogallol at 37 ° C, shake quickly and then take 200ul to 96-well plate reaction for 3-5 minutes, the reaction was measured at 325nm Front and rear absorbance. BHT was used as a positive control.

The clearance rate is calculated according to the following formula:

Clearance rate (%) = (ΔA1 - ΔA2) / ΔA1 × 100%

Among them, ΔA1 is the increase value of the auto-oxidation absorbance of pyrogallol, and ΔA2 is the absorbance after adding the sample reaction solution.

Superoxide anion radicals (O ₂ ·) not only have important biological functions, but also are closely related to various diseases. It is the first oxygen radical formed by the ground state oxygen after receiving an electron, and it can be formed into a series of reactions to generate other oxygen radicals, so it is of special significance. As shown in Figure 4, the clearance rate of (O ₂ ·) of the queen bee embryo peptide increased with the increase of the polypeptide concentration in the detection concentration range; at the same concentration, its ability to scavenge (O ₂ ·) was much higher than that of the sample. BHT. Therefore, the queen bee embryo peptide pair has a better ability to scavenge superoxide anion.

3. The liposome peroxidation inhibition ability is determined by adding 0.2 ml liposome solution to a 10 m test tube, 50 ul of a different volume of the sample and 50 mmol/L FeSO4 solution, and then adding a phosphate buffer solution. 3 mL of sample tube without sample was used as a model. The tube without FeSO ₄ solution was used as a blank. The reaction was placed in a 37 ° C water bath for 40 minutes, and finally 10% trichloroacetic acid 1 ml, 0.8% thiobarbituric acid (TBA) was added. 1 ml, uniformly mixed, placed in boiling water for 15 minutes, cooled in ice water, centrifuged at 8000 r/min for 10 minutes, and the supernatant was taken to measure absorbance at 532 nm (A).

Calculate the inhibition rate of lipid peroxidation according to the following formula:

Inhibition rate of lipid peroxidation (%) = (A model - A sample) / (A model - A blank) × 100%

Free radical theory believes that human aging and disease are closely related to liposome peroxidation, and liposome peroxidation is caused by free radical attack. To reduce or prevent peroxidation of liposomes, antioxidants must be used. Therefore, the inhibitory activity of the queen bee embryo peptide on liposome peroxidation can reflect the antioxidant activity of the sample. As shown in Fig. 5, it can be seen that as the concentration of the queen bee embryo peptide increases, the inhibition ability of liposome peroxidation also increases. When the concentration reaches 250 mg/ml, (w/v), the inhibition rate reaches the highest. The ability of 60.71% to inhibit liposome peroxidation also reflects the ability of the queen bee embryo peptide to scavenge free radicals.

The above results indicate that the queen bee embryo peptide prepared by the invention has good antioxidant capacity and ability to scavenge free radicals, and has good application prospects in the food industry and health care products industry.

In the present invention, the hydrolase is selected from a complex protease consisting of a neutral protease and a flavor protease in a mass ratio of 1:1.

Trypsin (trypsin), Pepsin (pepsin), Papain (papain), Flavourzyme (flavor protease), Alcalase (alkaline protease) and Protamex (complex protease), Alcalase enzymatic hydrolysis of royal jelly embryo protein extract, Under the optimal enzymatic conditions of the selected enzyme, the experimental conditions were as follows: the ratio of material to liquid was 1:50, the amount of enzyme was 4 mg/ml, the hydrolysis time was 4 hours, the enzymatic hydrolysis temperature was 40 °C, pepsin was hydrolyzed to pH 2.5, and the base was hydrolyzed. The protease Alcalase hydrolyzed the remaining enzymes of pH 8.0 to dilute pH 7.0, and compared the activity of the enzymatic hydrolysates of various enzymes with the sensory quality.

The table below shows the effect of comparing the proteases on the bioactivity, multiply yield and sensory evaluation of the enzymatic hydrolysate under these conditions.

Table 3 Enzymatic hydrolysis effect of several proteases on queen bee embryo protein powder

It can be seen from the above table that, from the flavor of the enzymatic hydrolysate, the enzymatic hydrolysate of papain is too poor in flavor and has astringent odor. The enzymatic hydrolysate of trypsin and pepsin is large, but active. The highest, complex protease and flavor protease action of the enzymatic hydrolysate is the best. From the biological activity of the enzymatic hydrolysate, the enzymatic hydrolysate of trypsin has the highest DPPH·free radical scavenging activity, while the complex protease hydrolysate has the highest scavenging activity and lipase inhibitory activity against O ₂ ·free radicals. . As shown in Figure 6, the yield of polypeptides produced by enzymatic hydrolysis of the queen bee embryo protein was shown. As shown in Fig. 6, under the same enzymatic hydrolysis time, the complex protease produced the highest yield of the polypeptide obtained by enzymatic hydrolysis of the queen bee embryo protein. Therefore, the present invention selects a composite protease as an optimal protease for enzymatically decomposing the embryonic protein of the queen bee.

The above is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention. It should also be considered as the scope of protection of the present invention.

Claims

A method for preparing a royal jelly embryo peptide by enzymatic hydrolysis, comprising:

Step 1: Sample pretreatment: The queen bee larvae are rapidly frozen at -80 ° C during the survival period, and dried, and the dried samples of the queen bee larvae are ground into powder;

Step 2: Degreasing of the queen bee larva powder: The pretreated powder of the queen bee larvae is degreased in a supercritical extraction device. The degreasing conditions are: extraction pressure 35 MPa, extraction temperature 35 ° C, extraction time 200 minutes, separation under normal pressure, collection of degreased The queen bee larva protein is used for enzymatic hydrolysis;

Step 3: Preparation of the sample liquid of the queen bee larvae powder: using the above-mentioned degreased queen bee larvae powder as the raw material, the protein content of the raw material is more than 70%, and the raw material of the queen bee larvae powder is 1:10 (w/v according to the ratio of material to liquid). Adding deionized water at 50 ° C and stirring and mixing for 4 hours to obtain a sample liquid of the queen bee larvae powder;

Step 4: Ultrasonic treatment of the queen bee larva powder sample liquid: using the pulsed ultrasonic wave with a power of 250W to 1250W to pretreat the sample liquid of the queen bee larva powder, the ultrasonic treatment time is 30 minutes, the ultrasonic wave emitting time corresponding to one pulse is 2 to 4 s, and the gap time is 2 s;

Step 5: Enzymatic hydrolysis of the sample of the queen bee larvae powder: adjust the pH of the sample of the defatted queen bee larvae powder after ultrasonic treatment to 7.5-8.5, and heat it to 40 °C-60 °C in a constant temperature water bath. According to the enzyme and the defatted queen bee larvae powder sample The liquid mass ratio is 0.02-0.04:1, and the complex protease is added. The complex protease consists of a neutral protease and a flavor protease in a mass ratio of 1:1, and the reaction is stopped when the degree of hydrolysis reaches 20%;

Step 6: The enzymatic hydrolysate is heated in a boiling water bath for 20 minutes to kill the enzyme, cooled to normal temperature, the pH of the enzymatic hydrolysate is adjusted to 7.0, and the supernatant is taken after centrifugation;

Step 7: The above supernatant is dried to obtain a hydrolyzed queen bee embryo peptide powder.
A method for preparing a royal jelly embryo peptide by the enzymatic hydrolysis method according to claim 1, wherein The queen bee larvae are 2 to 3 day old bee larvae.
A method for determining the content of a polypeptide in a queen bee embryo peptide prepared by the enzymatic hydrolysis method according to claim 1, wherein the macromolecular protein in the hydrolyzate is precipitated by using 10% trichloroacetic acid (TCA), and after centrifugation, in the supernatant The biuret reagent was added to the solution, and the OD value was measured at 540 nm. Then, a Gly-Gly-Tyr-Arg tetrapeptide standard solution was prepared, standard peptide data was determined, a standard curve was prepared, and the polypeptide content in the sample was looked up on the curve.
A method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide, which comprises the determination of free radical scavenging ability, the determination of superoxide anion radical scavenging ability and the determination of liposome peroxidation inhibition ability.
The method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide according to claim 4, wherein the radical scavenging ability is determined by measuring a DPPH free radical removal by hydrolyzing the queen bee embryo peptidase sample solution. force.
The method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide according to claim 4, wherein the superoxide anion radical scavenging ability is measured by using a riboflavin-photo-nitroblue tetrazolium system.
The method for detecting the antioxidant activity of a hydrolyzed queen bee embryo peptide according to claim 4, wherein the liposome peroxidation inhibiting ability is determined by adding 0.2 ml of a liposome solution to a 10 m test tube. Different volumes of sample and 50 mmol / L FeSO4 solution 50 ul, then add phosphate buffer solution to 3 mL of sample tube without sample as a model, test tube without FeS04 solution as a blank, the reaction was placed in a 37 ° C water bath for 40 minutes, and finally Add 1 ml of 10% trichloroacetic acid, 1 ml of 0.8% thiobarbituric acid (TBA), mix well, place in boiling water for 15 minutes, cool in ice water, centrifuge at 8000 r/min for 10 minutes, and take the supernatant at 532 nm. The absorbance (A) was measured, and the inhibition rate of lipid peroxidation was calculated according to the following formula: inhibition rate of lipid peroxidation (%) = (A model-A sample) / (A model-A blank) × 100%.