An industrial preparation process and application of hydrolyzed egg yolk powder that can promote tibia growth

Abstract

The present invention relates to the technical field related to proteolysis technology, and discloses an industrial preparation process and application of a hydrolyzed egg yolk powder that can promote tibial growth. This preparation technology has high repeatability and can achieve industrial production. By using a high-pressure enzymolysis tank and through high-temperature and high-pressure treatment, volatile odor substances in egg yolk protein are evaporated, improving the flavor and taste of the product, and opening the protein structure to expose the protease hydrolysis sites, making it easier to bind with proteases. Proteases derived from papaya, pineapple, and Bacillus subtilis are selected for composite directional enzymolysis to improve the enzymolysis efficiency, shorten the enzymolysis time, and increase the protein yield; among them, papain and alkaline protease derived from Bacillus subtilis are endopeptidases, which can more effectively hydrolyze from the inside of the protein with an opened spatial structure to obtain more peptide components, and then through the end-cutting action of bromelain and flavorzyme, the proportion of hydrophobic amino acids at the peptide ends is reduced, improving the product flavor.

Description

Technical Field

[0001] The present invention relates to the technical field related to proteolysis technology, and specifically to an industrial preparation process and application of hydrolyzed egg yolk powder that can promote tibia growth. Background Art

[0002] Egg yolk protein is mainly composed of 4 structural proteins, namely low-density lipoprotein, phosvitin, vitellin and high-density lipoprotein. Hydrolyzed egg yolk powder is prepared from defatted egg yolk protein as raw material, and by using the proteolytic action of protease, macromolecular proteins (or peptides) are converted into polypeptides or oligopeptides with relatively low molecular weights. Research shows that hydrolyzed egg yolk powder can not only provide basic nutritional needs for humans, but also has multiple functional activities, such as antibacterial, antioxidant, immune regulation and regulation of blood pressure, blood sugar and other activities, and plays an important role in human health care and disease prevention. In addition, taste and smell are the main application indicators for evaluating hydrolyzed egg yolk powder. Research shows that different raw material pretreatment methods and enzymatic hydrolysis processes have significant effects on the efficacy and application of hydrolyzed egg yolk powder. Therefore, selecting an appropriate preparation process is crucial for the application of hydrolyzed egg yolk powder.

[0003] The preparation methods of hydrolyzed egg yolk powder in the prior art mainly use degreasing and hydrothermal treatment to pretreat the raw materials and then use proteases from different sources for enzymatic hydrolysis or fermentation processes. The main defects are low enzymatic hydrolysis efficiency, low product yield, relatively high molecular weight of the enzymatic hydrolysis product hydrolyzed egg yolk powder, and obvious fishy smell of the product, resulting in limited market application.

[0004] In view of the above technical disadvantages, the present invention proposes a preparation method of hydrolyzed egg yolk powder, and this preparation technology has high repeatability and can realize industrial production. Summary of the Invention

[0005] The purpose of the present invention is to provide an industrial preparation process and application of hydrolyzed egg yolk powder that can promote tibia growth, so as to overcome the above-mentioned defects in the prior art.

[0006] The present invention is realized through the following technical solutions.

[0007] An industrial preparation process of hydrolyzed egg yolk powder that can promote tibia growth, the specific steps are as follows:

[0008] Step 1: Take defatted egg yolk powder and add it to water, and adjust the pH of the material-liquid to 8.0 - 8.5 with 7.5% sodium hydroxide aqueous solution;

[0009] Step 2: After stirring and mixing evenly, use a high-pressure cooking reactor to raise the temperature to 110 - 120 °C, carry out high-temperature and high-pressure cooking, and continue to keep warm;

[0010] Step 3: After the heat preservation, the material liquid is homogenized by a colloid mill;

[0011] Step 4: After homogenization, compound protease is added to the material liquid, and the temperature is raised to 45 - 60 °C, and enzymatic hydrolysis is continued for 6 - 12 hours;

[0012] Step 5: After the enzymatic hydrolysis is completed, it is separated by a disc separator;

[0013] Step 6: To the separated clear liquid, activated carbon and diatomaceous earth are added. After adsorption, it is filtered through a plate and frame filter;

[0014] Step 7: The sugar content of the clear liquid after filtration is concentrated to 20 - 30° by a vacuum low - temperature thin - film evaporator;

[0015] Step 8: The concentrated material liquid is sterilized by UHT instant high - temperature sterilization;

[0016] Step 9: After sterilization, the material liquid is spray - dried and packaged to obtain the finished product of hydrolyzed egg yolk powder.

[0017] Further technical solution: In the above - mentioned Step 1, the mass ratio of defatted egg yolk powder to water is 1 - 3:10 - 20.

[0018] Further technical solution: In the above - mentioned Step 2, the duration of high - temperature and high - pressure cooking should be maintained at 3 hours or more.

[0019] Further technical solution: In the above - mentioned Step 2, the heat preservation duration should be maintained at more than 5 hours.

[0020] Further technical solution: In the above - mentioned Step 4, the compound protease mainly includes papain, bromelain, alkaline protease derived from Bacillus subtilis, and flavorzyme.

[0021] Further technical solution: In the above - mentioned Step 4, the concentration of the compound protease should be 0.5 - 3.0%.

[0022] Further technical solution: In the hydrolyzed egg yolk powder prepared by the above - mentioned preparation process, the proportion of protein peptide segments with a relative molecular weight ≤ 2000 Da reaches more than 95%, and the lysine content ≥ 7.0%.

[0023] Further technical solution: The flavor and taste of the hydrolyzed egg yolk powder product prepared by the above - mentioned preparation process are significantly improved.

[0024] Advantages of the present invention:

[0025] The present invention provides a preparation method of hydrolyzed egg yolk powder. This preparation technology has high repeatability and can realize industrial production.

[0026] The egg yolk protein mainly has a globulin structure. Using a high-pressure enzymatic hydrolysis tank, through high-temperature and high-pressure treatment, the volatile odor substances in the egg yolk protein are evaporated to improve the flavor and taste of the product. At the same time, the high-temperature and high-pressure treatment opens the protein structure, exposes the protease hydrolysis sites, and makes it easier to bind with proteases. Proteases derived from papaya, pineapple, and Bacillus subtilis are selected for composite directional enzymatic hydrolysis to improve the enzymatic hydrolysis efficiency, shorten the enzymatic hydrolysis time, and increase the product yield. Among them, papain and the alkaline protease derived from Bacillus subtilis are endopeptidases, which can hydrolyze more from the inside of the protein with an open spatial structure to obtain more peptide components, and then through the end-cutting action of bromelain and flavor enzyme, reduce the proportion of hydrophobic amino acids at the peptide ends and improve the product flavor.

[0027] Using high-temperature and high-pressure cooking pretreatment and then preparing hydrolyzed egg yolk powder by means of composite enzymatic hydrolysis with multiple proteases can not only increase the yield from about 28% of conventional products in the existing publicly disclosed technology to 60%, and the proportion of protein peptide segments with a relative molecular weight ≤ 2000Da in the finished hydrolyzed egg yolk powder reaches more than 95%, but also significantly improve the flavor and taste of the product, without obvious hydrolysis bitterness and fishy smell. Brief Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] The present invention will be further described below in conjunction with the drawings and embodiments.

[0030] Figure 1 It is a graph of experimental data on the promoting effect of hydrolyzed egg yolk powder concentration on osteoblasts;

[0031] Figure 2 It is a graph of experimental data on the promoting effect of hydrolyzed egg yolk powder on tibia growth;

[0032] Figure 3 It is a graph of experimental data on the influence of hydrolyzed egg yolk powder on tibia bone density and other indicators;

[0033] Figure 4 It is a graph of experimental data on the influence of hydrolyzed egg yolk powder on metabolic indicators; Detailed Embodiments

[0034] The following is combined with Figures 1-4The present invention will be described in detail. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0035] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0036] The main disadvantages of the existing enzymatic hydrolysis process are low enzymatic hydrolysis efficiency, low product yield, relatively high relative molecular weight of the hydrolyzed egg yolk powder in the enzymatic hydrolysis product, and obvious fishy smell of the product. In view of the above technical disadvantages, the present invention proposes a preparation method of hydrolyzed egg yolk powder, and this preparation technology has high repeatability and can realize industrial production.

[0037] Egg yolk protein mainly has a globulin structure. Using a high-pressure enzymatic hydrolysis tank, through high-temperature and high-pressure treatment, the volatile odor substances in the egg yolk protein are evaporated to improve the flavor and taste of the product; at the same time, through high-temperature and high-pressure treatment, the protein structure is opened, so that the protease hydrolysis sites are exposed and are more likely to bind to proteases. Proteases derived from papaya, pineapple, and Bacillus subtilis are selected for combined directional enzymatic hydrolysis to improve the enzymatic hydrolysis efficiency, shorten the enzymatic hydrolysis time, and increase the protein yield; during the high-temperature and high-pressure process, the volatile odor substances in the egg yolk protein are evaporated to improve the flavor and taste of the product. Among them, papain and alkaline protease derived from Bacillus subtilis are endopeptidases, which can more effectively hydrolyze from the inside of the protein with an open spatial structure to obtain more peptide components, and then through the end-cutting action of bromelain and flavor enzyme, the proportion of hydrophobic amino acids at the peptide ends is reduced, and the product flavor is improved.

[0038] Moreover, after high-temperature and high-pressure cooking pretreatment and then preparing hydrolyzed egg yolk powder by means of combined enzymatic hydrolysis with multiple proteases, not only can the yield of about 28% of conventional products in the existing publicly disclosed technology be increased to 60%, and the proportion of protein peptide segments with a relative molecular weight ≤ 2000 Da in the finished hydrolyzed egg yolk powder reaches more than 95%, but also the flavor and taste of the product are significantly improved, and there is no obvious hydrolysis bitterness or fishy smell.

[0039] An industrial preparation process of hydrolyzed egg yolk powder that can promote tibia growth, the specific steps are as follows:

[0040] a. Take 150 kg of defatted egg yolk powder and add it to 1000 kg of water (the mass ratio of defatted egg yolk powder to water is 1 - 3:10 - 20), and adjust the pH of the material liquid to 8.0 - 8.5 with 7.5% sodium hydroxide aqueous solution;

[0041] b. After stirring and mixing evenly, use a high-pressure cooking reactor to raise the temperature to 110 - 120 °C, cook under high temperature and pressure for 3 hours, and continue to keep warm for more than 5 hours;

[0042] c. After the heat preservation is completed, the material liquid is homogenized by a colloid mill;

[0043] d. Add 0.5 - 3.0% compound protease (mainly papain, bromelain, alkaline protease from Bacillus subtilis, flavor enzyme, etc.) to the homogenized material liquid, raise the temperature to 45 - 60 °C, and continuously enzymolyze for 8 hours;

[0044] e. After the enzymolysis is completed, separate by a disc separator;

[0045] f. Raise the temperature of the separated supernatant to 80 °C, add activated carbon and diatomaceous earth, adsorb and keep warm for 1 hour, then add diatomaceous earth, stir and mix evenly, and filter through a plate and frame filter;

[0046] g. Concentrate the sugar content of the filtered supernatant to 20 - 30° by a vacuum low-temperature thin-film evaporator;

[0047] h. Sterilize the concentrated material liquid by UHT instant high-temperature sterilization;

[0048] i. After sterilization, the material liquid is made into a finished product through spray drying and packaging.

[0049] Determination of the basic physical and chemical properties of hydrolyzed egg yolk powder:

[0050] Item Test Result Moisture % 3.14 Ash % 6.69 Protein Content (dry basis) % 82.52 Acid-soluble Protein Content (dry basis) % 72.88 Degree of Hydrolysis % 37.81 Fat % 1.96

[0051] Table 1. Basic physical and chemical indicators

[0052] Molecular Weight, Da Relative Proportion, % ＜180 13.97 180-500 56.61 500-1000 18.39 1000-2000 7.62 2000-3000 1.99 3000-5000 1.10 5000-10000 0.32 ≤1000 88.97 ≤2000 96.57

[0053] Table 2. Relative molecular mass distribution of hydrolyzed egg yolk powder

[0054]

[0055] Table 3. Lysine content in hydrolyzed egg yolk powder

[0056] Analysis of relevant experimental data and research results of hydrolyzed egg yolk powder:

[0057] 1. Experiment 1: Effect of high-temperature and high-pressure cooking on the yield of hydrolyzed egg yolk powder

[0058] Take 150 g of defatted egg yolk powder and add it to 1000 g of water, adjust the pH of the material liquid to 8.0 ± 0.2 with 7.5% sodium hydroxide aqueous solution;

[0059] After stirring and mixing evenly, use an autoclave to raise the temperature to 118 °C, cook under high temperature and pressure for 3 hours, and continue to keep warm for more than 5 hours;

[0060] After the heat preservation is completed, 0.8% compound protease is added to the material liquid, and the temperature is raised to 50 - 55 °C, and enzymatic hydrolysis is continued for 6 hours;

[0061] After the enzymatic hydrolysis is completed, it is separated by a high-speed centrifuge (5000 r / min) for 30 min. The supernatant is taken and the temperature is raised to 80 °C. Activated carbon is added, and after adsorption and heat preservation for 1 hour, filtration is carried out to obtain the filtrate, which is freeze-dried to obtain 96 g of powder. The theoretical yield is calculated to be 64%.

[0062] 2. Experiment 2: Effect of high-temperature and high-pressure cooking duration on the yield of hydrolyzed egg yolk powder

[0063] Take 150 g of defatted egg yolk powder and add it to 1000 g of water. Adjust the pH of the material liquid to 8.0 ± 0.2 with 7.5% sodium hydroxide aqueous solution; after stirring and mixing evenly, use an autoclave to raise the temperature to 118 °C, carry out high-temperature and high-pressure cooking for 6 hours, and continue heat preservation for more than 5 hours; after the heat preservation is completed, 0.8% compound protease is added to the material liquid, and the temperature is raised to 50 - 55 °C, and enzymatic hydrolysis is continued for 6 hours; after the enzymatic hydrolysis is completed, it is separated by a high-speed centrifuge (5000 r / min) for 30 min. The supernatant is taken and the temperature is raised to 80 °C. Activated carbon is added, and after adsorption and heat preservation for 1 hour, filtration is carried out to obtain the filtrate, which is freeze-dried to obtain 97.5 g of powder. The theoretical yield is calculated to be 65%.

[0064] 3. Experiment 3: Promoting effect on osteoblasts

[0065] Experimental method: After subculturing and seeding the MC3T3-1 cells on the plate, incubate overnight to ensure that the cells are completely adherent before use in the experiment.

[0066] Weigh 0.05 g of hydrolyzed egg yolk powder, place it in a 5 mL centrifuge tube, add 1 mL of cell culture medium, and vortex to mix evenly to prepare a sample stock solution with a concentration of 50 mg / mL. Then, use the cell culture medium to sequentially dilute the sample stock solution with a concentration of 5 mg / mL and the final sample working solution with a concentration of 0.5 mg / mL in gradient. Add 100 μL of different sample working solutions to a 96-well plate respectively, and place it in an incubator for static culture for 24 h. The next day, aspirate the original sample working solution, wash it once with PBS, add 100 μL of cell proliferation detection solution containing CCK8, place it in an incubator for 1 h, then measure the absorbance OD value at 450 nm and carry out corresponding calculations. The data is as Figure One shown.

[0067] CCK8 detection principle: Viable cells can metabolize CCK8 into yellow formazan substances. The more viable cells there are, the deeper the yellow color. Conversely, the fewer viable cells there are, the lighter the color. Calculations are carried out through OD value measurement.

[0068] 4. Experiment 4: Experiment on promoting tibia growth

[0069] Experimental method: Twenty-four SD rats were randomly divided into three groups (NCD group (distilled water), BPP group (prepared by the existing enzymatic hydrolysis technology in the market), EYH group (product prepared by the process of Example 1)), with 8 rats in each group. The dosage for the BPP group was 1.0 mg / 100 g, bw, and that for the EYH group was 1.0 mg / 100 g, bw. Another group with sterile water was used as the blank control group. The rats were intragastrically administered with hydrolyzed egg yolk powder at the above dosages for 42 days. Then, the urine of the rats was collected, and the rats were anesthetized and blood was taken from the heart. After standing at room temperature for 2 h, the serum was obtained by centrifugation. Subsequently, the tibia was completely dissected, and the distance between the proximal articular surface and the distal articular surface of the tibia (excluding the joint) was measured with a vernier caliper, which was the tibia length after 42 days of feeding. The average value of three measurements was taken. After measuring the length, the rat tibia was fixed in 4% paraformaldehyde. Four left tibias (close to the average length of the tibias in this group) were selected from each group for micro-CT analysis, and the Scaner software of the Skyscan1276 type Micro CT was used to scan the tibia samples.

[0070] 5. Result analysis:

[0071] 1. Effect on tibia length

[0072] The hydrolyzed egg yolk powder prepared by the technology of this patent significantly promoted the growth of the left tibia of rats (P < 0.05)( Figure 2 A), and the length of the right tibia was also significantly higher than that of the NCD group and the BPP group. Summarizing the lengths of the left and right tibias of the rats, it was found that BPP significantly promoted the elongation of the tibia of rats (P < 0.05), while the promoting effect of EYH on tibia elongation reached an extremely significant level (P < 0.01)( Figure 2 C).

[0073] 2. Effect on indexes such as bone mineral density of rat tibia

[0074] Trabecular bone is the extended part of cortical bone in cancellous bone, that is, trabecular bone is connected to cortical bone and presents an irregular three-dimensional network structure in the bone marrow cavity, like a loofah network or sponge-like, playing a role in supporting hematopoietic tissue. In the porous network structure formed by the connection of trabecular bones, the arrangement of trabecular bones is consistent with the direction of the pressure and tension borne by the bone, so it can bear a large weight, and the bone mass of trabecular bones is closely related to its microstructure. Trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N) and trabecular bone separation (Tb.Sp) are the main indexes for evaluating the spatial morphological structure of trabecular bones. In the case where bone catabolism is greater than bone anabolism, such as in osteoporosis, the values of Tb.Th and Tb.N decrease, while the value of Tb.Sp increases.

[0075] It can be seen from Figure 3 that there were no significant differences in bone volume fraction, trabecular bone-related indexes and bone mineral density among the NCD group, BPP group and EYH group.

[0076] 3. Effects on the metabolic indices of rats

[0077] Bone-specific alkaline phosphatase (BALP) is an extracellular enzyme of osteoblasts. The proliferation, differentiation, and maturation of BALP are closely related to the normal growth and development of bones. BALP participates in the bone formation process, is relatively stable in serum, and is a marker of osteoblast maturation and activity. Clinical studies have shown that the BALP level has a linear relationship with the activities of osteoblasts and pre-osteoblasts. Quantitative determination and dynamic observation of serum BALP can be used as effective parameters for monitoring changes in bone formation. BALP is considered to be one of the most accurate bone formation markers.

[0078] Urinary deoxypyridinoline (DPD) is a bone metabolism marker discovered in recent years that can reflect bone lesions and has good sensitivity and specificity. Hydrolyzed egg yolk powder promotes the growth of rats' bones but does not cause bone lesions.

[0079] It can be seen from Figure 4 that there were no significant differences in the alkaline phosphatase activity in serum and the deoxypyridinoline content in urine among the groups.

[0080] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it, and cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of hydrolyzed egg yolk powder in the preparation of a product that can promote tibia growth, characterized in that, The proportion of protein peptide segments with a relative molecular weight of ≤2000 Da in the hydrolyzed egg yolk powder reaches over 95%, and the lysine content is ≥7.0%; The preparation of the hydrolyzed egg yolk powder includes: Step 1: Add defatted egg yolk powder to water, with the mass ratio of defatted egg yolk powder to water being 1-3:10-20, and adjust the pH of the material liquid to 8.0-8.5 with 7.5% sodium hydroxide aqueous solution; Step 2: After stirring and mixing evenly, use a high-pressure cooking reactor to raise the temperature to 110-120 °C, carry out high-temperature and high-pressure cooking for 3 hours, and continue to keep warm for more than 5 hours; Step 3: After the heat preservation ends, homogenize the material liquid with a colloid mill; Step 4: Add 0.5-3.0% compound protease to the homogenized material liquid. The compound protease includes papain, bromelain, alkaline protease derived from Bacillus subtilis, and flavor enzyme, raise the temperature to 45-60 °C, and continuously carry out enzymatic hydrolysis for 8 hours; Step 5: After the enzymatic hydrolysis ends, separate with a disc separator; Step 6: Heat the separated supernatant to 80 °C, add activated carbon and diatomaceous earth, adsorb and keep warm for 1 hour, then add diatomaceous earth, stir and mix evenly, and filter through a plate and frame filter; Step 7: Concentrate the sugar content of the filtered supernatant to 20-30° with a vacuum low-temperature thin-film evaporator; Step 8: Sterilize the concentrated material liquid by UHT instantaneous high-temperature sterilization; Step 9: After sterilization, spray-dry and package the material liquid to obtain the finished product of hydrolyzed egg yolk powder.

2. An industrial preparation process of hydrolyzed egg yolk powder that can promote tibia growth, characterized in that, The specific steps are as follows: Step 1: Add defatted egg yolk powder to water, and adjust the pH of the material liquid to 8.0-8.5 with 7.5% sodium hydroxide aqueous solution; Step 2: After stirring and mixing evenly, use a high-pressure cooking reactor to raise the temperature to 110-120 °C, carry out high-temperature and high-pressure cooking, and continue to keep warm; Step 3: After the heat preservation ends, homogenize the material liquid with a colloid mill; Step 4: Add compound protease to the homogenized material liquid, raise the temperature to 45-60 °C, and continuously carry out enzymatic hydrolysis for 6-12 hours; Step 5: After the enzymatic hydrolysis ends, separate with a disc separator; Step 6: Add activated carbon and diatomaceous earth to the separated supernatant, and filter through a plate and frame filter after adsorption; Step 7: Concentrate the sugar content of the filtered supernatant to 20-30° with a vacuum low-temperature thin-film evaporator; Step 8: Sterilize the concentrated material liquid by UHT instantaneous high-temperature sterilization; Step 9: After sterilization, spray-dry and package the material liquid to obtain the finished product of hydrolyzed egg yolk powder; In the hydrolyzed egg yolk powder prepared by the above preparation process, the proportion of protein peptide segments with a relative molecular weight of ≤2000 Da reaches over 95%, and the lysine content is ≥7.0%. In Step 1, the mass ratio of defatted egg yolk powder to water is 1-3:10-20. In Step 2, the heat preservation duration should be kept above 5 hours. The compound protease in Step 4 mainly includes papain, bromelain, alkaline protease derived from Bacillus subtilis, and flavor enzyme. The concentration of the compound protease in Step 4 should be 0.5-3.0%. The flavor and taste of the hydrolyzed egg yolk powder product prepared by the above preparation process have been significantly improved.

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