Active peptide for promoting bone growth, composition as well as preparation method and application of active peptide

Abstract

The invention discloses an active peptide for promoting bone growth, a composition as well as a preparation method and application of the active peptide. The active peptide for promoting bone growth comprises at least one of Leu-Arg, Leu-Leu, Phe-Leu and Ala-Phe. In order to overcome the defects of lack of traditional application and traditional Chinese medicine theory support, insufficient activity, large peptide fragment molecular weight and the like of existing food-borne polypeptides for promoting bone growth, the invention prepares polypeptides for promoting bone growth by utilizing an enzyme hydrolysis technology based on the traditional application of embryonated eggs, clarifies the structure of active polypeptides, performs quantitative analysis, and provides a basis for the food-borne polypeptides for promoting bone growth. Finally, the active peptide capable of promoting bone cell proliferation is provided, and the active peptide is natural, safe, free of toxic and side effects and capable of being taken for a long time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an active peptide for promoting bone growth, a composition, and a preparation method and application thereof. BACKGROUND

[0002] Bone growth plays a key role in promoting the growth and development of children and is an important link in the prevention and treatment of osteoporosis. The problem of slow growth and development of children is increasingly concerned, which is mainly related to multiple factors such as genetics, nutrition, and environment, especially malnutrition and endocrine disorders. Appropriate supplementation of growth-promoting factors has become a key measure to prevent and treat the growth and development of children. However, growth-promoting factors such as growth hormone (GH) and insulin-like growth factor-1 (IGF-1) have safety risks, and their side effects include susceptibility to infection, endocrine disorders, and metabolic disorders. They are mostly used as drugs, and lack long-term safety.

[0003] Therefore, other methods for promoting bone growth are increasingly valued, such as nutrient supplementation, lactoferrin, collagen polypeptide, and milk-derived polypeptide. Bioactive peptides can also regulate the proliferation and differentiation of growth plate chondrocytes and play an important role in promoting the growth and development of children. In particular, exogenous growth-promoting active peptides prepared by biological enzymolysis or fermentation from animals, plants, or microorganisms, such as cartilage collagen peptides, bovine bone peptide chelates, and deer horn peptides. These food-derived polypeptides are green and natural in production process, and have mild effects after being ingested by the human body, no toxicity and side effects to the human body, and are considered as an ideal choice for prevention and health care or daily treatment, which is widely favored by consumers. It is a hot spot of scientific research in recent years. However, the above-mentioned food-derived polypeptides also have problems such as large molecular weight (mostly more than 1 kDa) difficult to absorb, and non-traditional food sources (such as deer horn), therefore, it is still the direction of scientific research and an important demand of industrial application to find short peptides with the function of regulating bone growth and development, especially 2-4 peptides, which have the characteristics of easy absorption and good activity.

[0004] Chicken embryo eggs, also known as live beads, are rich in nutrients and have various types, delicious taste and good taste, and are the traditional folk tonic and recuperation of excellent products, which are loved by consumers. Live beads are traditional food with a history of hundreds of years. Li Shizhen recorded in "Compendium of Materia Medica": "chicken embryo eggs, warm and non-toxic, main to add essence, help qi and blood, support deficiency, treat male and female deficiency, arrow and confused". In addition, chicken embryo eggs also have the functions of treating headache, migraine, headache and limb madness. Therefore, chicken embryo eggs are widely used in promoting growth in folk, and have good support of traditional Chinese medicine theory, and have good application basis for bone growth and development. During the incubation process, the embryo in the egg gradually develops, and the nutrients in the yolk and egg white are absorbed by the embryo and transformed into new substances. This process not only changes the physical structure of the egg, but also causes significant changes in its chemical composition. However, there are few studies on the specific components and mechanisms of chicken embryo eggs and their enzymatic polypeptides. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art, overcome the shortcomings of the existing food-derived polypeptide for promoting bone growth, such as lack of traditional application and support of traditional Chinese medicine theory, insufficient activity, large molecular weight of peptide segment, etc., based on the traditional application of chicken embryo eggs, using enzyme hydrolysis technology, preparing polypeptides with promoting bone growth, determining the structure of active polypeptides, and performing quantitative analysis, ultimately providing an active peptide for promoting bone growth, composition and preparation method and application.

[0006] Therefore, the present application provides an active peptide for promoting bone growth, which is a safe natural substance and can be used as a raw material for daily life, and is widely used in diet or medicine.

[0007] The present application also provides a composition for promoting bone growth.

[0008] The present application also provides a preparation method of the above composition.

[0009] The present application also provides an application.

[0010] The present application also provides a product for promoting bone growth.

[0011] According to the first aspect of the present application, an active peptide for promoting bone growth is provided, which includes at least one of Leu-Arg, Leu-Leu, Phe-Leu and Ala-Phe.

[0012] According to the second aspect of the present application, a composition for promoting bone growth is provided, which comprises the active peptide for promoting bone growth according to the first aspect of the present application.

[0013] In some embodiments of the present application, the active peptide for promoting bone growth in the composition is 0.6% to 2% by weight.

[0014] In some embodiments of the present application, the composition further comprises at least one of the following active peptides:

[0015] Leu-Gly, Leu-Ser, Ala-Leu, Leu-Thr, Leu-Glu, Leu-Arg, Phe-Gly, Asp-Ala, Leu-Asn-Glu-Leu-Leu-Asp, Lys-Ala, Leu-Gly-Gly, Ala-Arg, Ser-Leu, Val-Arg, Glu-Ala, Leu-Lys, Val-Phe, Val-Lys, Val-Met.

[0016] According to a third aspect of the present application, a preparation method of a composition for promoting bone growth is provided, comprising the following steps:

[0017] S1: crushing the shelled cooked chicken embryo egg and homogenizing with water to obtain a homogenate;

[0018] S2: adjusting the pH of the homogenate obtained in step S1 to 6 to 9, adding protease and mixing to perform enzymolysis for 16 to 36 hours;

[0019] S3: performing temperature increase to inactivate the enzyme activity in the enzymolysis system in step S2, removing oil, centrifuging to remove the precipitate, and taking the supernatant to obtain the composition for promoting bone growth.

[0020] In some embodiments of the present application, the weight ratio of water to cooked chicken embryo egg in the homogenate in step S1 is (6 to 10) to 1.

[0021] In some embodiments of the present application, the weight ratio of water to cooked chicken embryo egg in the homogenate in step S1 is (7 to 9) to 1.

[0022] In some embodiments of the present application, the protease in step S2 is at least one selected from papain, neutral protease, alkaline protease, bromelain and trypsin.

[0023] In some embodiments of the present application, the amount of the protease is 0.5% to 3% of the weight of the cooked chicken embryo egg in step S1.

[0024] In some embodiments of the present application, the amount of the protease is 1% to 2.5% of the weight of the cooked chicken embryo egg in step S1.

[0025] In some embodiments of the present application, the temperature of the enzymolysis in step S2 is 37°C to 65°C.

[0026] In some embodiments of the present application, the temperature of the enzymatic hydrolysis in step S2 is 50-55°C.

[0027] In some embodiments of the present application, the time of the enzymatic hydrolysis in step S2 is 18-24h.

[0028] In some embodiments of the present application, the condition of the enzyme activity inactivation in step S3 is heating at 75-100°C for 5-30min.

[0029] In some embodiments of the present application, the condition of the enzyme activity inactivation in step S3 is heating at 90-100°C for 10-20min.

[0030] In some embodiments of the present application, the condition of the centrifugation in step S3 is centrifugation at 5000-10000rpm for 15-45min.

[0031] In some embodiments of the present application, the condition of the centrifugation in step S3 is centrifugation at 7000-9000rpm for 20-40min.

[0032] In some embodiments of the present application, the step S3 further comprises freeze-drying the supernatant.

[0033] According to a fourth aspect of the present application, it is proposed the use of any one of (1)-(3) in the preparation of a product for promoting bone growth:

[0034] (1) the active peptide for promoting bone growth according to the first aspect of the present application;

[0035] (2) the composition for promoting bone growth according to the second aspect of the present application;

[0036] (3) the composition for promoting bone growth prepared by the preparation method according to the third aspect of the present application.

[0037] According to a fifth aspect of the present application, it is proposed a product for promoting bone growth, which contains acceptable adjuvants and at least one of (1)-(3) below:

[0038] (1) the active peptide for promoting bone growth according to the first aspect of the present application;

[0039] (2) the composition for promoting bone growth according to the second aspect of the present application;

[0040] (3) the composition for promoting bone growth prepared by the preparation method according to the third aspect of the present application.

[0041] In some embodiments of the present application, the product comprises food and / or drugs.

[0042] In some embodiments of the present application, the acceptable adjuvant in the medicine includes at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent, and a surfactant.

[0043] In some embodiments of the present application, the binder is at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose, or polyvinyl pyrrolidone.

[0044] In some embodiments of the present application, the disintegrant is at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or alginic acid.

[0045] In some embodiments of the present application, the lubricant is at least one of microfine silica, magnesium stearate, calcium stearate, stearic acid, talc, or anhydrous silica gel.

[0046] In some embodiments of the present application, the coating agent includes at least one of hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, cellulose acetate phthalate, polyvinyl alcohol phthalate, ethyl cellulose, or cellulose acetate.

[0047] In some embodiments of the present application, the suspending agent includes at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose, or aluminum stearate gel.

[0048] In some embodiments of the present application, the surfactant is at least one of lecithin, sorbitan monooleate, or glycerol monostearate.

[0049] In some embodiments of the present application, the acceptable adjuvant in the food includes at least one of starch, magnesium stearate, stevia, or sorbitol.

[0050] The present application has at least the following beneficial effects:

[0051] (1) The active peptide for promoting bone growth provided by the present application has the effect of promoting bone cell proliferation, can be used for preparing products with the effect of promoting bone growth, and is natural, safe, non-toxic, and can be taken for a long time.

[0052] (2) The composition for promoting bone growth provided by the application can reach the content of active dipeptides Leu-Arg (0.78%), Leu-Leu (0.02%), Phe-Leu (0.01%) and Ala-Phe (0.05%) by optimizing the preparation process, and the survival rate of osteoblasts treated by the composition is close to 125% (1.0 mg / mL).

[0053] (3) The preparation method of the composition for promoting bone growth provided by the application has a nitrogen recovery rate of 75% and the highest proportion of small molecular peptides with a molecular weight less than 1 kDa in the preparation process, and the preparation method is simple, easy to operate and convenient for industrialization.

[0054] (4) The composition for promoting bone growth provided by the application can be used as a dietary supplement, and the dietary supplement has the activity of promoting the proliferation of osteoblasts, is natural, safe, non-toxic and side effect-free, and can be taken for a long time. BRIEF DESCRIPTION OF DRAWINGS

[0055] The application will be further described below in combination with the drawings and examples, in which:

[0056] Figure 1 Figure 1 is a cell proliferation detection result diagram of bone cells treated by different concentrations of enzyme hydrolysate in Example 1 of the application;

[0057] Figure 2 Figure 2 is a nitrogen recovery rate result diagram of enzyme hydrolysate under the action of different enzymes in Example 2 of the application;

[0058] Figure 3 Figure 3 is a polypeptide length distribution result diagram of enzyme hydrolysate under the action of different enzymes in Example 2 of the application; wherein, the relative peak area refers to the ratio of the ion chromatogram peak area of the polypeptide to the total ion chromatogram peak area of all identified polypeptides;

[0059] Figure 4 Figure 4 is a cell proliferation detection result diagram of cells treated by enzyme hydrolysate under the action of different enzymes in Example 2 of the application; wherein, the Con group is a control group without enzyme hydrolysate treatment;

[0060] Figure 5 Figure 5 is a nitrogen recovery rate result diagram of enzyme hydrolysate under the action of dominant protease complex in Example 3 of the application;

[0061] Figure 6 Figure 6 is a polypeptide length distribution characteristic result diagram of enzyme hydrolysate under the action of dominant protease complex in Example 3 of the application;

[0062] Figure 7 Figure 7 is a cell proliferation detection result diagram of cells treated by enzyme hydrolysate under the action of dominant protease complex in Example 3 of the application;

[0063] Figure 8Figure of the growth promoting activity evaluation results of the growth promoting dipeptides with different concentrations in Example 4 of the present application;

[0064] Figure 9 Figure of the release curve of the active dipeptides under different enzymolysis conditions in Example 5 of the present application;

[0065] The different lowercase letters marked in the histogram part of the above figure represent significant differences between different groups. DETAILED DESCRIPTION

[0066] The concept and technical effects of the present application will be described below in combination with examples for a clear and complete description, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0067] Example 1: Exploring the biological activity of chicken embryo enzymolysis product

[0068] This example explores the growth promoting ability of the chicken embryo enzymolysis product obtained by enzymolysis. The preparation method, test method and results of the chicken embryo enzymolysis product are as follows:

[0069] 1. Preparation of chicken embryo enzymolysis product:

[0070] The boiled and shelled chicken embryo egg was crushed and homogenized, and the homogenate liquid was diluted with ultrapure water to a chicken embryo protein concentration of 1wt%. The specific pH value of the solution was measured by a pH meter, and the pH of the system was adjusted to 7.5±0.1. The papain was accurately weighed and added to the reaction system, and the amount of papain was 1wt% of the chicken embryo protein. The reaction system was placed in a constant temperature water bath shaker with a shaking frequency of 120rpm and a temperature of 55±0.5℃ for continuous enzymolysis for 24h.

[0071] When the reaction was terminated, the reaction system was quickly placed in a 95℃ metal bath for heating for 15min (heating rate 5℃ / min). After cooling, the oil-water separator was used for liquid-liquid separation and degreasing, and the high-speed refrigerated centrifuge was used for centrifugation at 8000rpm and 4℃ for 30min to separate the supernatant. Finally, the obtained supernatant was freeze-dried and placed in a-20℃ refrigerator for standby, and the chicken embryo enzymolysis product was obtained.

[0072] 2. Test the cell proliferation promoting ability of chicken embryo enzymolysis product:

[0073] MC3T3-E1 (Subclone 14) cells were seeded at a density of 5000 cells / well in 96-well plates, with 100 μL of culture medium per well. The 96-well plates were incubated for 24 h at 37°C, 5.0% CO2, and 90% humidity to allow for full cell adhesion. The culture medium was then removed from the 96-well plates, and different concentrations of chicken embryo digest were added, with six replicates for each concentration. The labeled 96-well plates were then incubated for another 24 h. After incubation, 90.0 μL of complete culture medium (without samples) was added, followed by 10.0 μL of CCK-8 reagent. A blank group (containing only complete culture medium and CCK-8 reagent without cells) and a control group (sample concentration of 0 mg / mL) were also established. After incubation for 3 h, the OD value was measured at 450.0 nm using a multi-mode microplate reader. Cell proliferation activity was calculated using the following formula:

[0074] The results are as follows Figure 1 As shown.

[0075] Depend on Figure 1 It can be seen that when treated with 0.25–1.0 mg / mL chicken embryo enzymatic hydrolysate, the cell proliferation activity of the treated groups was significantly improved compared with that of the control group, showing a certain dose-dependent effect. The cell proliferation activity was the highest at a concentration of 1.0 mg / mL (132.2%).

[0076] 3. Sequence identification of proteins contained in chicken embryo enzymatic hydrolysate

[0077] The chicken embryo enzymatic hydrolysate was separated and detected using X500 LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry. The mobile phase was 0.1% (v / v) formic acid aqueous solution (A) and LC-MS grade acetonitrile (B). The elution method was as follows: 0-4.0 min 95% A; 4.0-6.0 min 95%-90% A; 6.0-30.0 min 90%-60% A; 30.0-34.0 min 60%-10% A; 34.0-40.0 min 10% A; 40.0-42.0 min 10%-95% A; 42.0-52.0 min 95% A. The liquid chromatography conditions were set as follows: flow rate 0.05 mL / min, injection volume 1.0 μL, and column temperature 40.0°C. The mass spectrometry analysis used an electrospray ion source (ESI) with the following working parameters: scan interval 0.642 s, ion source temperature 500°C, positive ion detection mode, and spray voltage 5500V. The time-of-flight mass spectrometry (TOF-MS) was set to double-stage scan range (primary 100-1200 Da and secondary 50-1200 Da), and the information-dependent acquisition (IDA) mode was used with a maximum of 4 candidate ions and the dynamic exclusion function enabled. The remaining parameters were set according to the standard experimental settings for proteomics.

[0078] The mass spectrometry raw data file format (*.wiff2) was converted to *.mgf format using the software ProteoWizard 3.2. The Fasta sequence was screened and downloaded from the Uniprot protein database (https: / / www.uniprot.org / ) using the keywords "Egg", "Chicken", and "Embryo".

[0079] Based on the Fasta sequence obtained above, the theoretical amino acid composition of the proteins contained in the chicken embryo enzymatic hydrolysate was analyzed, and the specific data are shown in Table 1.

[0080] Table 1 Amino acid composition of proteins in chicken embryo enzymatic hydrolysate

[0081]

[0082] From Table 1, the amino acid residues L, K, E, R and the like in the sequence have high recurrence frequency and content ratio, more than 8%; in addition, leucine (L, 10.17%) and lysine (K, 9.97%) have the highest ratio, followed by glutamic acid (E, 9.33%), arginine (R, 7.99%) and valine (V, 6.72%). Leucine (L) and valine (V) are branched chain amino acids, which can promote protein synthesis and energy metabolism of embryonic cells by activating the mTOR pathway, thereby promoting the growth and repair of muscle cells; lysine (K, 9.97%) is an essential amino acid required by the human body, which can support bone and overall growth by promoting the secretion of growth hormone and calcium absorption; arginine (R, 7.99%) can regulate blood circulation and enhance vasodilation by generating nitric oxide (NO), thereby promoting cell growth and repair, and it can also promote the secretion of growth hormone, indirectly producing the effect of promoting the growth and development of the body. At the same time, glutamic acid (E, 9.33%) can also stimulate cell proliferation and growth by providing energy and promoting amino acid metabolism, and has a promoting effect on the growth of the brain and nervous system. These amino acids play an important role in embryonic growth and development and cell proliferation, and have important guiding significance for the subsequent discovery and preparation of growth-promoting peptides.

[0083] Example 2 Screening of the most suitable protease species

[0084] In this example, the nitrogen recovery rate, polypeptide molecular weight distribution, free amino acid composition and cell proliferation activity in the chicken embryo hydrolysate obtained by testing different proteases were tested to screen the most suitable protease species. The specific experimental methods and results are as follows:

[0085] 1. Sample preparation:

[0086] The chicken embryo was crushed and homogenized, and the homogenate was diluted with ultrapure water to a chicken embryo protein concentration of 1%; the specific pH value of the solution was measured by a pH meter and the pH of the system was adjusted to the optimum pH of each enzyme, the corresponding protease was added, and the enzyme was allowed to act under the optimum enzyme hydrolysis conditions of each protease for 24 hours. After the end, the enzyme was inactivated (95℃, 15min), and after cooling to room temperature, centrifugation was performed (8000r / min, 4.0℃, 30min), and the supernatant was freeze-dried. The sample was placed in a-20℃ refrigerator for standby. The optimum enzyme hydrolysis conditions of the proteases used are shown in Table 2.

[0087] Table 2 Optimum enzyme hydrolysis conditions of different proteases

[0088]

[0089] 2. Nitrogen recovery rate analysis:

[0090] Referring to the protein determination method in national standard GB 5009.5-2016, the nitrogen content of the enzymatic hydrolysate was determined using the Kjeldahl method, and the nitrogen recovery rate was calculated. The formula for calculating the nitrogen recovery rate is as follows:

[0091] The results are as follows Figure 2 As shown.

[0092] Depend on Figure 2 It can be seen that papain had the highest nitrogen recovery rate (85.25%), significantly better than other enzymes. Furthermore, the nitrogen recovery rates of alkaline protease (63.42%), trypsin (65.41%), neutral protease (63.51%), and bromelain (59.28%) were similar. These four proteases all achieved nitrogen recovery rates of around 60% for chicken embryo protein, indicating relatively thorough enzymatic hydrolysis. It is noteworthy that although the acidic protease treatment of chicken embryo protein lasted for 24 hours, theoretically it should have been fully hydrolyzed, but its recovery rate was only 27.72%, reflecting the poor hydrolytic efficiency of this enzyme on chicken embryo protein. This may be because the pH conditions of the acidic protease's action environment are acidic, under which the solubility of chicken embryo protein is poor, and fewer enzyme cleavage sites are exposed, resulting in poor hydrolysis.

[0093] Overall, the samples treated with different proteases were fully hydrolyzed under their optimal conditions, laying a good foundation for further analysis. Papain showed a significant advantage in the hydrolysis of chicken embryos and should be given priority in the subsequent preparation and screening of growth-promoting peptides.

[0094] 3. Polypeptide molecular weight distribution:

[0095] The molecular weight distribution of peptides in chicken embryo enzymatic hydrolysate was determined according to the national standard GB / T 22492-2008. The chromatographic conditions were set as follows: SKgel G2000SWXL gel chromatography column (7.8 mm × 300 mm), mobile phase V(water):V(acetonitrile) = 80:20, trifluoroacetic acid (TFA) added to the aqueous phase at 0.1%, isocratic elution mode, time set at 40 min, flow rate at 0.5 mL / min, injection volume at 10 μL, and monitoring wavelength at 214 nm. Molecular weight standards were used: GGG (189 Da), GGYR (451 Da), bacitracin (1450 Da), insulin (5808 Da), and cytochrome C (12384 Da). After the determination, the molecular weight distribution of peptides was expressed as logarithm (lg) of the peptide molecular weight. MW A linear regression equation was performed on the retention time (Rt), and the results are shown in Table 3.

[0096] Table 3. Polypeptide molecular weight distribution of chicken embryo hydrolysates treated with different proteases

[0097]

[0098] From Table 3, we can see that:

[0099] 1) The enzymatic hydrolysates obtained by the six proteases have some differences in the distribution of polypeptide molecular weight, but they are mainly distributed in the small molecular weight section of <3kDa, indicating that the enzymatic hydrolysis of each sample is sufficient. This is mainly due to the long enzymatic hydrolysis time (24h), which gives the enzyme and substrate protein sufficient contact opportunities, thereby releasing enough small molecular polypeptides.

[0100] 2) For the differences between different samples, the proportion of <1kDa polypeptide molecular weight section in the enzymatic hydrolysates corresponding to papain, alkaline protease, trypsin and bromelain is relatively high, all above 60%, especially the corresponding components of trypsin and alkaline protease are 79.01% and 75.20%, respectively, which reflects the full advantage of these two proteases in the release of small molecular polypeptides, which can create favorable conditions for the release of short-chain active peptides.

[0101] 3) The molecular weight distribution of the enzymatic hydrolysates corresponding to acid protease and neutral protease shows different characteristics. Although the <1kDa molecular weight section is still the main distribution range, the proportion is relatively low, only 55.41% and 59.06%, respectively. On the contrary, the proportion of >5kDa large molecular weight section in the enzymatic hydrolysate corresponding to acid protease is more than 8%, which is much higher than that of other enzymatic hydrolysates, which directly reflects the weak position of this enzyme in the enzymatic hydrolysis of chicken embryo protein. This trend is consistent with the nitrogen recovery rate results. It is worth noting that the proportion of neutral protease in the 3-10kDa section (11.19%) is significantly higher than that of acid protease (9.85%), but its nitrogen recovery rate is also higher than that of acid protease. The analysis reason may be that neutral protease has obvious advantages in the initial degradation of large molecular chain chicken embryo protein to improve the protein nitrogen hydrolysis release rate, but it is not suitable for deep hydrolysis of peptide chain, so it presents the situation of high nitrogen recovery rate and more 3-10kDa molecular weight components.

[0102] 4. Polypeptide length distribution:

[0103] The collected mass spectrometry data was converted into format using ProteoWizard 3.0, the relevant protein sequence database was downloaded from the Uniport tool website (keywords: "Egg" & "Chicken" & "Embryo"), and polypeptide sequence statistical analysis was performed using the peptidomics software PepOSX3.5.0 (Guangdong, Guangzhou Zhi Peptide Biological Technology Co., Ltd.). Parameter settings: use Exhaust and Search dual engines for spectrum resolution, polypeptide identification length is set to 2-25, primary ion deviation is 0.005 Da, secondary ion deviation is 0.02 Da, parallel core number is 32, sub-ion cluster matching types a, b and y, Bayesian score threshold is 60, and other complete sub-ion cluster matching spectrum results are retained, and the results obtained are shown in Figure 3 .

[0104] As can be seen from Figure 3 , the enzymatic products produced by the action of six non-specific proteases on chicken embryo proteins are mainly short peptides, especially polypeptides with lengths concentrated in the range of 3 to 6. The reason for this is that the enzymatic products produced by the action of non-specific proteases on chicken embryo proteins are mainly short peptides, especially polypeptides with lengths concentrated in the range of 3 to 6, which is consistent with the theoretical expectation. Non-specific proteases have a wide range of enzyme action sites, and the degree of protein hydrolysis is deep, so the enzymatic products are mainly distributed as short peptides. It should be emphasized that although the enzyme cleavage sites are widely distributed, different proteases still exhibit their own unique action characteristics. Specifically, the samples treated with papain are mainly concentrated in tripeptides, while the samples treated with alkaline protease and trypsin are mainly concentrated in dipeptides; the peptide segment distribution of the sample corresponding to acid protease is relatively balanced, but most of them are mainly in the length range of 2-3. The identified peptide segments of the samples corresponding to neutral protease and bromelain are relatively few, but from their relative abundance, they are basically mainly distributed in the length range of 5-7, indicating that the action sites of these two enzymes on chicken embryo proteins are relatively single and the degree of hydrolysis is relatively shallow.

[0105] As can be seen from Figure 3 b, the distribution of peptide length and peak area in the enzymatic products of each group of samples is similar to the distribution of peptide length and number, but in individual cases, there may be certain differences, for example, although dipeptides in the sample corresponding to trypsin have a low proportion in the number distribution (about 25%), their peak area accounts for as high as 42%, reflecting that the actual abundance of dipeptides in the sample may dominate. Therefore, taking into comprehensive consideration, the relative peak area distribution of polypeptides can more accurately reflect the composition proportion characteristics of polypeptides in the enzymatic products at each polypeptide length, while the length-number distribution is more inclined to show the actual identification species richness of polypeptides at each length in the sample.

[0106] 5. Free amino acid composition:

[0107] Take 200 μL of the enzyme sample, add 100 μL of 0.1 mol / L triethylamine acetonitrile solution and 100 μL of 0.2 mol / L PITC acetonitrile solution, mix well by vortex oscillation, and incubate at room temperature in the dark for 1 hour. Then add 600 μL of n-hexane, vortex again for 1 minute, and collect the lower aqueous solution after 10 minutes of static layering. Finally, dilute the sample to 4 times the original volume with 0.05 mol / L sodium acetate buffer. The liquid phase method is analyzed by machine analysis on a Thermo UltiMate 3000 HPLC system, and the chromatographic column is Elite-AAP amino acid special column (250x4.6mm), the mobile phase A is acetonitrile-methanol-water (3:1:1) solution, B is the special component solution of this method, gradient elution: 0-39min 5.0%-45.0% A, 39-40min 45.0%-100.0% A, 40-50min 100.0% A, 50-51min 100.0%-5.0% A, 51-70min 5.0% A, liquid chromatography conditions: flow rate 1.0mL / min, column temperature 38.0℃, injection volume 10.0μL, detection wavelength 254.0nm, and the results are shown in Table 4.

[0108] Table 4 Free amino acid composition of chicken embryo enzyme hydrolysate treated with different proteases (unit: mg / 100g)

[0109]

[0110]

[0111] As can be seen from Table 4, the amino acid composition of the enzyme hydrolysate obtained by different proteases is relatively rich, and contains 8 essential amino acids for human body and histidine and arginine (amino acids necessary for the growth of infants), reflecting that the chicken embryo protein hydrolysate has high nutritional value. However, it is worth noting that the amino acid composition and content of different enzyme hydrolysate samples also show significant differences. Among them, the total amount of free amino acids (TAA) of chicken embryo protein treated with trypsin is the highest, followed by neutral and alkaline proteases, indicating that these enzymes have relatively stronger universality in the enzymatic hydrolysis of chicken embryo protein, have more enzyme cutting sites, and therefore have more amino acid residues that can be released. In addition, from the distribution of free amino acid composition, the content of leucine (Leu) and lysine (Lys) in each sample is relatively high, which is significantly higher than that of other amino acids, which is consistent with the amino acid composition results obtained by the theoretical complete degradation analysis of chicken embryo protein, to some extent, it confirms the reliability of the sequence analysis of chicken embryo protein in the foregoing, and further suggests the possibility of the existence of high abundance dipeptide.

[0112] 6. Cell proliferation activity:

[0113] The test method is described in Example 1, and the results are as follows: Figure 4 As shown.

[0114] Depend on Figure 4 It was found that, except for acidic protease, the products of the other five proteases all exhibited varying degrees of proliferative activity. Among them, neutral protease and papain showed the most significant growth-promoting activities, reaching a maximum of 117.30% and 112.34%, respectively, followed by bromelain (with a maximum of 112.30%). Looking at the effects of individual samples at different concentrations, the growth-promoting activity of some enzymatic hydrolysates showed a certain concentration dependence, with low concentrations promoting proliferation and high concentrations inhibiting proliferation (e.g., neutral protease), indicating that their function may be closely related to the concentration and structural characteristics of the peptides. Notably, acidic protease hydrolysates exhibited strong cell-inhibiting effects under high concentration conditions. This is speculated to be because the hydrolysis of embryonic proteins in a strongly acidic environment (pH 3.0) may have produced certain inhibitory peptides or aggregated hydrophobic peptides. These peptides may have strong cytotoxicity or inhibit proliferation signaling pathways, thereby leading to apoptosis.

[0115] Example 3: Investigation of the enzymatic hydrolysis effect of the composite enzymatic hydrolysis system

[0116] This embodiment, based on the investigation of the effect of a single protease on the enzymatic hydrolysis of chicken embryos in Example 2, further tested the enzymatic hydrolysis effects of different composite enzymatic hydrolysis systems, including nitrogen recovery rate, peptide length distribution, and cell proliferation activity. The specific experimental methods and results are as follows:

[0117] 1. Sample preparation:

[0118] Based on the research results of the above embodiments, and taking into account nitrogen recovery rate, protease hydrolysis characteristics, and growth-promoting activity evaluation data, papain, neutral protease, and bromelain were selected as the dominant enzyme preparations. Following the experimental protocol shown in Table 5, chicken embryonic protein (concentration 5%, w / w, on a dry basis) underwent combined enzymatic hydrolysis (including simultaneous compounding and segmented enzymatic hydrolysis). Sample preparation parameters not mentioned in Table 5 were the same as those in Example 2.

[0119] Table 5. Compound Enzymatic Hydrolysis Scheme

[0120]

[0121] Note: Enzyme 1 refers to papain, enzyme 2 refers to neutral protease, and enzyme 3 refers to bromelain; "enzyme 1 + enzyme 2" and "enzyme 1 + enzyme 3" refer to the simultaneous enzymatic hydrolysis of two enzymes, while "→" in "enzyme 1 → enzyme 2" indicates that the two enzymes are enzymatically hydrolyzed in stages. 2. Nitrogen recovery rate analysis:

[0122] The nitrogen recovery rate test method is described in Example 2, and the results are as follows:Figure 5 As shown in Table 1.

[0123] As shown in Table 1. Figure 5 It can be seen that the nitrogen recovery efficiency is not as expected when chicken embryo proteins are treated by using a double-enzyme synergistic hydrolysis system, and the enzymatic hydrolysis efficiency is lower than that of single-enzyme enzymolysis; from Figure 5 a, among the three complex enzymolysis schemes of papain and neutral protease combination, the nitrogen recovery rate of complex enzymolysis scheme 2 is significantly lower than that of papain, and the enzymolysis effect of complex enzymolysis scheme 1 and complex enzymolysis scheme 3 is also slightly lower than that of papain. Figure 5 b, the nitrogen recovery rates of the three complex enzymolysis schemes of papain and bromelain combination are much lower than that of papain, and the enzymolysis efficiency decreases to a greater extent.

[0124] The reason for the decrease of nitrogen recovery rate after complex enzymolysis is that different proteases compete with each other for the same substrate, and the substrate site may be saturated. When neutral protease, papain and bromelain cut the amino terminal and carboxyl terminal of amino acid residues, they all show significant cutting preference for hydrophobic amino acids such as leucine (L). When two enzymes act together (such as complex enzymolysis scheme 1), their competitive binding to the same substrate region may cause the cutting site to be saturated too early, causing mutual inhibition of enzyme activity. In addition, in the segmented enzymolysis (such as scheme 2 and scheme 3), the preferential cutting of L residues by the front-stage enzymolysis may destroy the key action site of the subsequent enzymolysis, resulting in the inability of the latter to effectively release the target peptide segment. At the same time, the intermediate products generated by the front-stage enzymolysis (such as hydrophobic short peptides rich in L) may also hinder the recognition and binding of the subsequent enzyme to the remaining L residues through steric hindrance or charge shielding effect, thereby reducing the enzymolysis effect.

[0125] 3. Polypeptide length distribution:

[0126] The influence of various complex enzymolysis processes on the length distribution of polypeptide chains in protein hydrolysis products was systematically analyzed by using polypeptidomics technology, and the results are shown in Table 2. Figure 6

[0127] As shown in Table 2. Figure 6 a, the length of the peptide segments obtained by different single-enzyme enzymolysis is mainly distributed in 4-6 amino acid residues, and the total number of identified peptide segments in the products of neutral protease and bromelain is also less than that of papain. After different complex enzymolysis, the number of polypeptides with a length of 2-6 amino acid residues decreases to a certain extent, and the number is between papain and neutral protease. The research results show that double-enzyme synergistic hydrolysis may lead to excessive degradation of substrate proteins, and promote the generation of various types of peptides (2-6 amino acid residues) to decrease. This effect may be related to the excessive cutting of complex enzyme system to protein backbone.

[0128] Figure 6 ​​bIt can be seen that the double-enzyme synergistic hydrolysis system did not significantly increase the relative abundance of dipeptide components in the final product, but the number of tripeptides and above, especially tetrapeptides, increased significantly, indicating that after using the complex enzyme hydrolysis system, proteins and longer peptide chains were further hydrolyzed into tetrapeptide components. It is worth noting that the number of identified tetrapeptides did not increase significantly Figure 6 a), indicating that complex enzyme hydrolysis mainly enhances the degree of hydrolysis of the substrate protein rather than producing novel peptide segments. The number of peptides with a length of ≥9 in the neutral protease hydrolysate was only 74, but the relative composition accounted for 20%, which was similar to the peptide molecular weight distribution results of the neutral protease sample in Table 3, indicating that there were many large molecular fragment residues that were not fully cut in the hydrolysate. In the papain product, the number of peptides with a length of 2-6 showed a higher number of identifications, and the relative composition mainly consisted of dipeptides, even higher than the complex enzyme hydrolysis, indicating that papain was more advantageous than complex enzyme hydrolysis in improving the release of dipeptides in the sequence.

[0129] 4. Cell proliferation activity:

[0130] The test method is described in Example 1, and the results are shown in Figure 7 .

[0131] It can be seen from Figure 7 that:

[0132] 1) At a low concentration (0.25 mg / mL), most of the hydrolysates showed a certain degree of proliferation, and some samples (such as complex scheme 2) even significantly improved the cell survival rate (p<0.05), indicating that they may have potential activity to promote cell proliferation. However, there are also some samples (such as complex 3 and complex 6) whose cell survival rate is lower than that of the control group (Ctrl), indicating that they may lack proliferation activity at this concentration, or even have a slight inhibitory effect on cell growth.

[0133] 2) When the concentration increased to 0.5 mg / mL and 1.0 mg / mL, the proliferation of some samples was weakened, and even began to show a certain degree of cell inhibition. Among them, at 0.5 mg / mL, the survival rate of most samples was close to 110%, but the statistical difference was not significant, indicating that the proliferation promotion effect was limited. However, at a high concentration of 1.0 mg / mL, some samples began to show more obvious proliferation or inhibition, such as the cell survival rate of the papain product increased significantly to nearly 125%, while complex schemes 5 and 6 showed more significant inhibition.

[0134] Based on the nitrogen recovery rate, amino acid composition, polypeptide composition analysis results and the evaluation results of the growth-promoting activity of the enzyme hydrolysate obtained in Example 2 and Example 3, it is preferred to use papain single enzyme hydrolysis as the preferred enzyme hydrolysis scheme for chicken embryo growth-promoting active peptides.

[0135] Example 4 Active ingredient study of chicken embryo protein peptides prepared by the advantage enzymolysis scheme

[0136] Based on the papain monoenzyme enzymolysis scheme screened from the test results of Example 2 and Example 3, the polypeptide composition in the enzymolysis product obtained by the monoenzyme 1 (papain) scheme in Example 3 was explored, and it was found that the active dipeptide was a relatively high proportion of peptide segments in the enzymolysis product. The growth-promoting ability of the above-mentioned active dipeptide was further evaluated, and the specific experimental method and results are as follows:

[0137] 1. Polypeptide composition analysis of enzymolysis product:

[0138] Based on the foregoing basis, the polypeptide composition of the enzymolysis product obtained by papain hydrolysis of chicken embryo protein was analyzed by polypeptidomics analysis means. For all identified polypeptides and the above-identified potential high-abundance dipeptide components, the ion flow chromatogram peaks were extracted and area integration was performed, the relative content distribution of each peptide segment was calculated and sorted according to the area, and the top 25 polypeptides were extracted. The results are shown in Table 6.

[0139] Table 6 Analysis of Top 25 Peptide Segments in Enzymolysis Product Treated by Papain

[0140]

[0141]

[0142]

[0143] Note: In the table, the relative composition ratio refers to the relative peak area ratio of the polypeptide in all identified polypeptides; " / " indicates that no known biological activity is matched; Peptide Ranker score refers to the potential activity score of the peptide segment, and a value greater than 0.5 indicates that it is more likely to be an active peptide.

[0144] As can be seen from Table 6, dipeptide substances occupy a dominant position in the enzymolysis product, especially the relative composition ratio of LG and LA dipeptides is relatively high, reaching more than 1.5%. This phenomenon is consistent with the polypeptide relative peak area ratio-length distribution results measured in Example 3 Figure 6 b), and in-depth analysis of the amino acid composition characteristics of the target dipeptides found that these short peptide molecules are generally rich in leucine (L) residues. This phenomenon may be due to two factors: first, the catalytic characteristics of the used protease tend to hydrolyze leucine-related sites; second, the leucine itself has a relatively high content in the primary sequence of the substrate protein (embryo protein), which is also consistent with the theoretical speculation analysis of the potential high-abundance dipeptides in the sequence of chicken embryo protein in Example 2.

[0145] It is worth noting that the relative abundance data of polypeptides shown in Table 6 is derived from the mass spectrometry response intensity of identified peptide segments, and this relative quantitative result can be affected by the following factors: incomplete polypeptide identification and interference of non-peptide components in the sample. Therefore, there can be a certain difference between the actual content and the current estimated value.

[0146] Further, the 25 screened polypeptides were subjected to bioactivity evaluation by using the PeptideRanker online prediction tool and the BIOPEP-UWM database, and the results are shown in Table 6.

[0147] As can be seen from Table 6, these dipeptide molecules generally exhibit multiple physiological activity characteristics, most of which are hypoglycemic and hypotensive; although no peptide segment with direct growth-promoting activity is found through bioactivity matching, this is mainly because the related database has not yet included “growth-promoting activity”. Therefore, in the preparation of growth-promoting activity peptides, the relative composition proportion of polypeptides and the Peptide Ranker score (it is generally believed that a value greater than 0.5 indicates a higher possibility of being an active peptide) are mainly referred to. In addition, according to related reports, DPP-IV (dipeptidyl peptidase IV) inhibition activity can be related to growth promotion, so it can be associated with it in screening.

[0148] 2. Activity evaluation of active dipeptides

[0149] On the basis of the foregoing, by comprehensively investigating the secondary mass spectrometry identification results of each polypeptide and its bioactivity characteristics, 7 high-abundance potential growth-promoting dipeptides are screened as target analytes in this embodiment (as shown in Table 7). According to the PeptideRanker score, it is found that the scores of FG, FL and AF are all greater than 0.95, indicating that they have a higher possibility of being active peptides. At the same time, considering that the above-mentioned polypeptide components all exhibit high relative abundance in the enzyme hydrolysate, this characteristic suggests that they have good development potential in targeted preparation research. In addition, the peptide segments VF, LL and LR not only have a score greater than 0.50, but also belong to the polypeptide sequences with the highest theoretical content ranking TOP 15 in the chicken embryo protein sequence, and have high potential for targeted release. According to the rapid hatching and development characteristics of chicken embryos, it is speculated that these polypeptides are most likely to be the main growth-promoting active ingredients, so they are also screened as potential growth-promoting active peptides for further experiments.

[0150] Table 7 Target Peptide Segment Parameter Table

[0151]

[0152] Note: PI refers to isoelectric point, and GRAVY refers to average hydrophobicity.

[0153] The potential growth-promoting dipeptides screened above were chemically synthesized, and standard solutions with concentrations of 0.04, 0.2, 1.0 and 5.0 mM were prepared for evaluating the proliferation effect on MC3T3-E1 osteoblast precursor cells. The results are shown in Table 1. Figure 8

[0154] It can be seen from Figure 8 that:

[0155] 1) At low concentrations (0.04 and 0.2 mM), most of the peptides can significantly improve cell survival rate, especially the FL, VF and AF groups, which have significantly higher proliferation-promoting effect than the control group (p<0.05), indicating that these peptides may have good growth-promoting activity. However, the proliferation-promoting effect of some peptides (such as FG, LQ) is relatively mild, suggesting that the biological activity of different peptides may be closely related to their amino acid composition and sequence characteristics.

[0156] 2) With the increase of concentration to 1.0 mM, some peptides still maintain a high cell survival rate (such as VF, AF), especially the cell survival rate of LL and LR test groups is significantly improved compared with the low concentration test group, indicating that the biological activity of different peptides has obvious concentration dependence.

[0157] 3) When the concentration is further increased to 5.0 mM, the proliferation-promoting effect of most peptides is significantly weakened, and some peptides even show certain cytotoxicity. Among them, the cell survival rate of LQ is significantly lower than that of the control group (p<0.05), which is speculated to be related to the increase of peptide hydrophobicity, the change of cell membrane permeability or the activation of apoptosis-related signal pathways. In contrast, the LL, LR, AF and FL groups still maintain a high cell survival rate under high concentration conditions, indicating that they may have good biocompatibility and potential to promote the growth of osteoblasts.

[0158] Example 5: Investigation of the effect of different enzymatic conditions on the release of growth-promoting dipeptides

[0159] This example is based on the QQQ technique to quantitatively analyze the effect of enzymatic parameters on the release of growth-promoting dipeptides in embryonic proteins. The specific experimental methods and experimental results are as follows:

[0160] 1. Quantitative detection method of growth-promoting dipeptides

[0161] 1) Preparation of standard solution:

[0162] Precisely weigh the polypeptide (LL, LR, LQ, VF, FL, FG, AF) standard powder and prepare standard solutions with concentrations of 2, 4, 6, 8 and 10 μg / mL.

[0163] 2) Confirmation of target ion cluster and establishment of MRM detection method: ​

[0164] The highest concentration of peptide standard solution (50.0 μg / mL) was drawn up using a syringe pump and injected directly into the mass spectrometer at a flow rate of 10.0 μL / min. The precursor ion was scanned using Q1 (100–1200 Da). After confirming the mass-to-charge ratio of the precursor ion, the product ion fragments in the range of 50–1200 m / z were scanned using Product Ions (Q2) mode. The bombardment energy was manually adjusted until the precursor ion signal value was reduced to about 1 / 3 of that of the product ion. High-abundance secondary fragment ions were selected as monitoring targets, and their declustering voltage (DP value), spray voltage (EP value), and collision energy (CE value) were optimized to establish the MRM detection method.

[0165] 3) Quantitative determination method for the target growth-promoting dipeptide:

[0166] Column: ACQUITY UPLC HSS T3 (1.8μm, 1×100mm, (Waters, USA); Chromatographic conditions: Mobile phase: 0.1% (v / v) formic acid solution (A) and acetonitrile (B); Gradient elution program: 0–1.0 min 95.0% A, 1.0–1.5 min 95.0%–90.0% A, 1.5–7.5 min 90.0%–60.0% A, 7.5–8.5 min 60.0%–10.0% A, 8.5–10.0 min 10% A, 10.0–10.5 min 10.0%–95.0% A, 10.5–13.0 min 95.0% A; Flow rate: 0.10 mL / min; Column temperature: 40.0℃; Mass spectrometry conditions: ESI source, positive ion mode; Operating mode: Multiple Reaction Monitoring (MRM); Other parameters were set to the instrument's default optimal values.

[0167] 2. Effects of different enzymatic hydrolysis conditions on the release of the target active dipeptide

[0168] 1) Enzyme addition amount:

[0169] Following the optimal enzymatic hydrolysis scheme selected in Example 3, and with the enzymatic hydrolysis pH, temperature, and time fixed, five variable groups were set with enzyme addition amounts of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% to investigate the effect of different enzyme addition amounts on the release of growth-promoting dipeptides.

[0170] 2) Enzymatic hydrolysis pH:

[0171] According to the optimal enzymolysis scheme screened in Example 3, the corresponding enzymolysis conditions were fixed, the enzyme addition amount, the enzymolysis temperature and the enzymolysis time were unchanged, and five variable groups were set respectively with the enzymolysis pH being 6.5, 7.0, 7.5, 8.0 and 8.5 to explore the release of the growth-promoting dipeptides under different enzymolysis pH conditions.

[0172] 3) Enzymolysis temperature:

[0173] According to the optimal enzymolysis scheme screened in Example 3, the corresponding enzymolysis conditions were fixed, the enzyme addition amount, the enzymolysis pH and the enzymolysis time were unchanged, and five variable groups were set respectively with the enzymolysis temperature being 45, 50, 55, 60 and 65°C to explore the release of the growth-promoting dipeptides under different enzymolysis temperatures.

[0174] 4) Enzymolysis time:

[0175] According to the optimal enzymolysis scheme screened in Example 3, the corresponding enzymolysis conditions were fixed, the enzyme addition amount, the enzymolysis pH and the enzymolysis temperature were unchanged, and five variable groups were set respectively with the enzymolysis time being 6, 12, 18, 24 and 30 h to explore the release of the growth-promoting dipeptides under different enzymolysis times.

[0176] The active dipeptide content detection results obtained under different enzymolysis conditions are shown in Table 2. Figure 9

[0177] It can be seen from Table 2 that: Figure 9

[0178] 1) The content of the LR dipeptide under each condition is significantly higher than that of other peptide segments, fully demonstrating the dominant abundance of this polypeptide in the composition of the enzymolysis polypeptide, which is expected to be used as a characteristic recognition factor of the chicken embryo protein enzymolysis product in the later stage, and to provide a good foundation for the identification and quality control of the chicken embryo growth-promoting active peptide raw material.

[0179] 2) The content of different dipeptides changes significantly with the change of the enzymolysis conditions. Among them, with the increase of the enzyme addition amount from 0.5% to 2.5%, the content of the dipeptide LR presents a trend of first increasing and then decreasing, and reaches the maximum at the enzyme addition amount of 1.0%, while the release amounts of other several peptide segments present a trend of gradually increasing or slightly decreasing, and finally reach the maximum at 2.0% to 2.5%. The main reason for the different change trends of these dipeptides with the increase of the enzyme addition amount is that part of the polypeptides is released from the source protein with the increase of the enzyme addition amount, but at the same time, another part of the polypeptides is degraded in the enzymolysis process, thus presenting different change trends.

[0180] ​​3) Each polypeptide also presents a differential trend in the curve of pH change, in which LR reaches the maximum value at pH 7.0, and the maximum values of the other two peptides are mainly concentrated around pH 7.0, presenting a downward trend at both ends, which is mainly related to the action characteristics of papain under acidic and alkaline conditions. The optimal pH value of papain is 7.0-7.5, and the test results obtained in the present application are consistent with the related literature reports.

[0181] 4) At different temperatures, the content of LR reaches the maximum value at 50°C, and the maximum values of the contents of the other two peptides are mainly concentrated in the interval of 50°C-55°C, also presenting a downward trend at both ends, which is mainly caused by the decline of protease activity at low temperature and the partial denaturation and inactivation of protease at high temperature.

[0182] 5) At different enzymolysis times, the content change trend of LR is similar to that of the other polypeptides, and the content reaches a larger value at an enzymolysis time of 18h, and the content of the dipeptide gradually decreases when the enzymolysis time exceeds 18h. This phenomenon is obviously different from the change trend of the degree of hydrolysis and the nitrogen recovery rate in the traditional enzymolysis process. The analysis reason is that when the enzymolysis time is too long, the original released dipeptide may be further hydrolyzed into free amino acids, so that the content decreases.

[0183] Therefore, suitable enzymolysis conditions are crucial for efficient release of growth-promoting active dipeptides in chicken embryo proteins. Considering the industrial production cost and the content of dipeptides, the addition amount of enzyme 2.0%, pH 7.0, temperature 50°C and time 18h are selected as the preferred preparation conditions of the growth-promoting active dipeptides in chicken embryos. Under the conditions, the target peptide is prepared by targeted enzymolysis release, and the absolute contents of the seven dipeptides are LL (0.02%), LR (0.78%), LQ (0.09%), VF (0.04%), FL (0.01%), FG (0.10%) and AF (0.05%) respectively.

[0184] Example 6 An active peptide for promoting bone growth and a preparation method thereof

[0185] The present embodiment provides a preparation method of an active peptide for promoting bone growth, and the preparation conditions of the preparation method are determined based on the test and screening results of Examples 1-5, and specifically include the following steps:

[0186] 1) Cook the chicken embryo egg with water, remove the shell, weigh (1.0 kg is weighed), crush, and add an appropriate amount of water for homogenization to obtain a chicken embryo egg homogenate.

[0187] 2) The homogenate of the chicken embryo eggs obtained in step 1) was mixed with water in a ratio of 1:8 by weight and added to an enzyme hydrolysis tank, the pH was adjusted to 7.0, 2% of the mass of the cooked chicken embryo eggs was added to papain, and the enzyme hydrolysis system was placed in a constant temperature water bath shaker with a shaking frequency of 120 rpm and a temperature of 50±0.5°C for continuous enzyme hydrolysis for 18 hours.

[0188] 3) When the reaction was terminated, the enzyme hydrolysis system of step 2) was removed and quickly placed in a 95°C metal bath for 15 minutes of continuous heating (temperature rise rate of 5°C / min).

[0189] 4) After cooling, oil-water separation was performed to remove fat, a high-speed refrigerated centrifuge was used to remove the precipitate at 8000 rpm and 4°C for 30 minutes, and the supernatant was obtained. Finally, the obtained supernatant was freeze-dried to obtain the product.

[0190] Pilot production research in Example 7

[0191] This example was based on the process parameters optimized by screening in Examples 1-5 and carried out a pilot study:

[0192] Take 1000 chicken embryo eggs (weight 51.2 kg), cook and remove the shell (weight 43.75 kg), add an appropriate amount of water to break and homogenize to obtain a homogenate. Pour the homogenate into a 500L fermentation tank, add 8 times the weight of water to 400L, adjust the pH to 8.0, add 875 grams of papain (purchased from Nanning Pangbo, 2 million units), and hydrolyze at 55°C for 18 hours. Inactivate the enzyme at 95°C for 15 minutes, remove the upper oil, filter out the residue, obtain the supernatant, and freeze-dry after appropriate concentration to obtain chicken embryo protein peptides; the yield is 16.87%, and the quality inspection report is shown in Table 8.

[0193] Table 8 Quality inspection report of pilot test

[0194]

[0195]

[0196] Example 8 Preparation of chicken embryo protein peptide tablet candy

[0197] Take 80.0g of chicken embryo protein peptides obtained in the pilot test of Example 7, mix with 500g of starch, add 10% starch aqueous solution 10g to make soft material, add 0.1g of magnesium stearate, 5g of steviol glycoside, and 3g of sorbitol, mix uniformly, and press into 2000 tablets to obtain chicken embryo protein peptide tablet candy, each tablet containing 40mg of chicken embryo protein peptides.

[0198] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. An active peptide for promoting bone growth, characterized in that, The active peptide for promoting bone growth comprises at least one of Leu-Arg, Leu-Leu, Phe-Leu and Ala-Phe.

2. A composition for promoting bone growth, characterized by, The composition comprises the active peptide for promoting bone growth according to claim 1.

3. The composition for promoting bone growth according to claim 2, wherein The active peptide for promoting bone growth in the composition is 0.6% to 2% by weight.

4. A method of preparing a composition for promoting bone growth, characterized by, The preparation method comprises the following steps: S1: crushing the shelled cooked chicken embryo egg and homogenizing with water to obtain a homogenate; S2: adjusting the pH of the homogenate obtained in step S1 to 6 to 9, adding a protease and mixing to perform enzymolysis for 16 to 36 hours; S3: performing temperature elevation to inactivate the enzyme activity in the enzymolysis system in step S2, removing oil, centrifuging to remove the precipitate, and taking the supernatant to obtain the composition for promoting bone growth.

5. The preparation method according to claim 4, characterized in that, In step S1, the weight ratio of water to the cooked chicken embryo egg in the homogenate is (6 to 10):

1.

6. The preparation method according to claim 4, characterized in that, In step S2, the protease is at least one selected from papain, neutral protease, alkaline protease, bromelain and trypsin; Preferably, the amount of the protease is 0.5% to 3% of the weight of the cooked chicken embryo egg in step S1.

7. The preparation method according to claim 4, characterized in that, In step S2, the temperature of the enzymolysis is 37°C to 65°C.

8. The preparation method according to claim 4, characterized in that, In step S3, the inactivation conditions of the enzyme activity are heating at 75°C to 100°C for 5 to 30 minutes; Preferably, in step S3, the centrifugation conditions are 5000 to 10000 rpm for 15 to 45 minutes.

9. Use of any one of (1) to (3) in the preparation of a product for promoting bone growth: (1) the active peptide for promoting bone growth according to claim 1; (2) the composition for promoting bone growth according to claim 2 or 3; (3) the composition for promoting bone growth prepared by the preparation method according to any one of claims 4 to 8.

10. A product for promoting bone growth, characterized by, The product contains acceptable adjuvants and at least one of (1) to (3) below: (1) the active peptide for promoting bone growth according to claim 1; (2) the composition for promoting bone growth according to claim 2 or 3; (3) the composition for promoting bone growth prepared by the preparation method according to any one of claims 4 to 8.

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