TWI711386B - Preparation method of queen fetal active peptide - Google Patents

Abstract

The present invention relates to a method for preparing a queen fetal active peptide, which includes preparing a queen fetus into a queen fetal reaction solution, and hydrolyzing the queen fetal reaction solution with a first fruit enzyme to heat to obtain a queen fetal hydrolysis reaction solution. The hydrolysis reaction liquid is heated by the second fruit enzyme to hydrolyze to obtain a hydrolysis solution product. The hydrolysis solution product is heated for a period of time to stop the enzyme reaction, and is cooled to a predetermined temperature to obtain a hydrolyzed queen fetal solution, and hydrolyze the queen fetal solution After rapid freeze-drying, the queen fetal active peptide can be obtained as a hydrolyzed product of queen fetal enzyme and health food, which has antioxidant capacity, regulates blood pressure, and is beneficial to human body absorption.

Description

Method for preparing queen fetal active peptide

The present invention relates to a method for preparing a queen fetal active peptide, in particular to a technology for preparing a queen fetal active peptide.

According to the theory, the queen fetus refers to the larva of the queen bee, which is mainly a by-product produced in the process of preparing royal jelly. During the growth of the queen fetus, royal jelly is the main source of nutrition. Because the queen fetus is rich in protein, amino acids, vitamins and minerals, and is believed to have the effects of improving autonomic disorders and menopausal disorders, enhancing human metabolism, improving sleep, anti-aging, and prolonging life, it has been widely used in recent years. Favor and affection of consumers.

According to what is known, in the past, beekeepers took out the fetuses of the queen bees as feed or discarded them during the process of making royal jelly, and some of them were used as ingredients for frying. There are also some related companies who freeze-dried fresh queen bee fetuses, grind them into powder, and then make them into capsules, which can be sold as nutritious foods as shown in reference [1,2]. According to some research institutions, royal jelly has immune regulation, antibacterial effects, and antioxidant capabilities as shown in references [3,4]. However, because there are not too many technical literatures for research and discussion on royal jelly, the physiological activity of royal jelly is speculated. , Queen fetus may have similar health benefits.

Generally speaking, protein is composed of amino acids, and peptides are hydrolyzed products of protein through enzyme action or chemical action. Therefore, the queen fetus is made by hydrolysis technology. The resulting active peptide can also be said to be a small molecule protein. These hydrolysates produced by proteins are polymers composed of two or more amino acids, which play an important role in the regulation of cell physiology and metabolic functions. The structure of peptides is small due to their small molecules, rich in nutrients and easy to absorb, and has obvious physiological regulation functions, such as improving immunity, improving intestinal function, lowering blood pressure, lowering blood lipids, and promoting metabolism, as shown in References [5-7] . Free Radical, also called free radical, is a substance with unpaired electrons, because electrons must be in pairs in order to be in a stable state, so free radicals are very unstable, in order to make themselves stable. , Free radicals will grab the electrons of other molecules or release electrons everywhere, and they will continue to attack or destroy the cells of various organs of the human body, causing diseases as shown in Reference [8]. The role of antioxidants is to neutralize free radicals and convert excess free radicals in the body into less toxic substances by redox methods to prevent the damage caused by free radicals.

Specifically, it is known that the industry in the related art has used the powder obtained after pulverization and drying of the queen fetus as a raw material for health food; or uses a single enzyme to hydrolyze the queen fetus protein. However, health foods made directly from crushed queen fetuses may cause allergic symptoms due to some specific proteins contained in the queen fetus. Therefore, there are many techniques to use proteolytic enzymes to decompose the protein of the queen fetus to obtain low molecular weight. Protein. However, these methods are directly decomposed by the original queen bee fetus and only one enzyme at a time. The product contains incompletely decomposed protein still higher than the decomposed protein, resulting in poor protein decomposition efficiency. It can be seen that the conventional single-enzyme hydrolysis of queen fetal protein does have the above-mentioned defects and is not perfect. Therefore, there is still a need for improvement. In view of this, how to develop a technology for preparing royal fetal active peptides that decompose its macromolecular proteins into small peptides after being treated with different enzymes has become a technology that the industry in the related technical field is eager to solve. Subject.

The technical problem to be solved by the present invention is to improve the deficiency caused by the conventional single-enzyme hydrolysis of queen fetal protein.

The main purpose of the present invention is to provide a method for preparing the active peptides of queen bee fetus, which is mainly to decompose the macromolecular protein into small molecular peptides after the queen bee fetus is treated with different enzymes. After the process of concentration and drying, a product can be prepared. As the hydrolyzed product of queen fetal enzyme or the active peptide of fetal queen of health food, it has many health effects such as anti-oxidation ability, regulating blood pressure and facilitating absorption by the human body. The technical means to achieve the main purpose of the invention include formulating the queen fetus into a reaction solution of the queen fetus, heating the reaction solution of the queen fetus with the first fruit enzyme for hydrolysis to obtain a hydrolysis reaction solution of the queen fetus, and hydrolyzing the reaction solution of the queen fetus Heat the second fruit enzyme for hydrolysis to obtain a hydrolyzed solution product, heat the hydrolyzed solution product for a period of time to stop the enzyme reaction, and cool to a predetermined temperature to obtain a hydrolyzed queen fetal solution, and quickly freeze the hydrolyzed queen fetal solution After drying, the queen fetus active peptide is obtained.

Compared with the prior art, the effect of the present invention is that the re-formulated queen fetal reaction liquid reacts better with enzymes, can shorten the required action time and make the reaction more complete, and through the combined action of the enzymes, the effect is Obviously better, the ratio of decomposed protein is obviously increased, and the content of flavonoids is relatively small.

10: Finished product of active peptide of queen bee fetus

Figure 1 is a schematic diagram of protein analysis of the hydrolyzed queen fetus of the present invention.

Figure 2 is a schematic diagram of the ACE inhibitory activity analysis of the hydrolyzed and unhydrolyzed controls of the present invention.

Figure 3 is a schematic diagram of the appearance of the finished product stored at two different temperatures according to the present invention.

Figure 4 is a schematic diagram of the preparation process of queen fetal peptide according to the present invention.

Figure 5 is a schematic diagram of the preparation process of the queen fetal peptide according to the present invention.

In order to allow your reviewer to further understand the overall technical features of the present invention and the technical means to achieve the purpose of the invention, specific examples and diagrams are used to describe in detail as follows: Please refer to Figures 3 to 5, which is the invention. The specific embodiment of the main purpose includes the following steps:

(a) Steps for remaking the queen fetus: freeze-dry the queen fetus into a powdered queen fetus, and then re-formulate the powdered queen fetus into an aqueous solution of the queen fetus, and use a vibration machine (ie, ultrasonic oscillator and vortex oscillator) The combination) is oscillated, and the queen fetal aqueous solution is uniformly mixed by the oscillating action of the ultrasonic oscillator and the vortex oscillator to obtain the queen fetal reaction liquid.

(b) The first stage hydrolysis step: the queen fetal reaction solution prepared in step (a) is hydrolyzed with the first fruit enzyme at a temperature of 30 to 45° C. for 40 to 380 minutes to obtain the queen fetal hydrolysis reaction solution.

(c) The second stage hydrolysis step: the hydrolysis reaction solution of the queen bee fetus prepared in step (b) is hydrolyzed with the second fruit enzyme at a temperature of 50-78°C for 40-80 minutes to obtain a hydrolyzed solution product; One fruit enzyme is different from the second fruit enzyme.

(d) Heating to stop the reaction step: heating the hydrolyzed solution product of step (c) to 80~120°C and continuing for 15~45 minutes to stop the enzyme reaction to obtain the hydrolyzed queen fetal solution.

(e) Freeze-drying step: When the hydrolyzed queen fetal solution in step (d) is cooled to a predetermined temperature, freeze-drying is quickly performed to obtain the finished product 10 of the queen fetal active peptide.

Based on the foregoing, in a preferred embodiment of the present invention, in step (b), the queen fetal reaction solution is hydrolyzed with the first fruit enzyme at 37°C for 60 minutes. In step (c), the hydrolysis reaction solution of the queen fetus is hydrolyzed with the second fruit enzyme at 72°C for 60 minutes. In step (d), the hydrolyzed solution product is heated to 100° C. for 30 minutes to stop the enzyme reaction.

In a specific embodiment of the present invention, in step (d), the predetermined temperature is 40°C.

In a specific embodiment of the present invention, the concentration of the aqueous solution of queen fetal solution prepared in step (a) is 10-50%, and the concentration of the aqueous solution of queen fetal solution is preferably 25%.

In a specific embodiment of the present invention, the first fruit enzyme used in step (b) is pineapple enzyme, and the pineapple enzyme activity is 8000-12000 (CDU/ml).

In a specific embodiment of the present invention, the second fruit enzyme used in step (b) is papaya enzyme, and the papaya enzyme has an activity of 20,000 to 40,000 (FCCpu/ml).

More specifically, please refer to Figs. 3 and 5, which are a preferred embodiment of the method for preparing the active peptide of queen bee fetus according to the present invention, which includes the following steps:

(a) Step: freeze-dry the queen fetus into a powdered queen fetus, then re-formulate the powdered queen fetus into a queen fetal aqueous solution, and oscillate the queen fetus with an ultrasonic oscillator and a vortex oscillator The aqueous solution is uniformly mixed to obtain a 25% concentration of the queen fetal reaction solution.

(b) Step: The queen fetal reaction solution prepared in step (a) is hydrolyzed with pineapple enzyme with an activity of 10000 (CDU/ml) at 37° C. for 60 minutes to obtain a queen fetal hydrolysis reaction solution.

(c) Step: Hydrolyze the queen fetal hydrolysis reaction solution prepared in step (b) with papaya enzyme with an activity of 30,000 (FCCpu/ml) at 72°C for 60 minutes to obtain a hydrolysis Solution product.

Step (d): Heat the hydrolyzed solution product of step (c) to 100°C and continue for 30 minutes to stop the reaction of the enzyme to obtain a hydrolyzed queen fetal solution.

Step (e): When the hydrolyzed queen fetal solution in step (d) is cooled to 40° C., freeze-drying is performed quickly to obtain the finished product 10 of the queen fetal active peptide.

Specifically, the finished product 10 of the queen fetal active peptide prepared in step (e) has a protein content of 0.79 ± 0.01 mg/g, a peptide content of 28.18 ± 1.12 mg/g, and a total phenol content of 29.29 ±1.12mg of GAE/g, flavonoid content is 0.33±0.02mg of QEAC/g. In addition, the finished product 10 of the queen fetal active peptide can also be used to prepare at least one health food. The health food can be a capsule-type health food; or a queen fetal enzyme hydrolyzed product.

Furthermore, according to the results of pineapple enzyme and papaya enzyme hydrolysis, the compound enzyme hydrolysis formula is adjusted to carry out the optimal process condition mode. The optimal reaction conditions are 25% queen fetal reaction solution, and pineapple enzyme (10000CDU/ml) and papaya Enzyme (30000FCCpu/ml) is used to process the enzyme production process. After reacting at 37°C for 1 hour, the reaction conditions are adjusted to 72°C and then acting for 1 hour. After the reaction, it is quickly heated to 100°C for 30 minutes to stop the enzyme reaction. The protein electrophoresis analysis is used to preliminarily analyze the protein hydrolysis rate and molecular weight, and will test the optimal reaction time, temperature and other conditions. At the same time, the reaction test of different complex enzymes will be tested to optimize the process.

In the present invention, the quality analysis of the samples prepared in the above specific embodiments is performed by freeze-drying the hydrolyzed queen bee fetus and finally using the powder as the final product formulation for quality analysis. Analysis of the protein conversion (hydrolysis) rate reached 95%. As shown in Figure 1, the molecular weight of the hydrolyzed queen fetal protein is less than 30kDa, and the moisture content of the sample after preliminary freeze-drying is 11.5%. And analyze its protein, peptides, total phenols and total flavonoids content (as shown in Table 1 below), from unhydrolyzed And the protein content in the hydrolyzed sample can confirm that the protein conversion (hydrolysis) rate is above 95%.

Table 1 shows the composition analysis of hydrolyzed, unhydrolyzed and royal jelly. The results in Table 1 show that the peptide content and total phenol content of the hydrolyzed queen fetus are higher than those of unhydrolyzed queen fetus and royal jelly, while the flavonoids are lower For unhydrolyzed samples, they may be lost during the manufacturing process. As the amount of peptides increases, positive results are obtained for the goal of product development.

The present invention simultaneously sends the completed queen fetal hydrolyzed peptide product and queen fetus samples to the Taiwan Inspection Company for entrusted analysis of its eight major nutrients. The results are shown in Table 2 below. Table 2 shows the queen fetal enzyme hydrolyzed samples and the queen fetus Analysis of the eight major nutrients.

The results of the sample quality analysis found that the peptide and total phenol content of the product have been greatly increased, which means that the present invention continues to perform product efficacy evaluation and analysis, including the antioxidant evaluation of the queen bee fetal enzyme hydrolysis product and the Angiotension Converting Enzyme (ACE) enzyme Inhibition activity test. In the present invention, the hydrolyzed fetal queen fetus freeze-dried powder is taken and dissolved in a buffer (pH 5.2), the final volume is 10 ml, and the supernatant is centrifuged to obtain the supernatant after being fully shaken to dissolve. The samples are analyzed for antioxidant and Superoxidase respectively Dismutase (SOD) activity, SOD determination kit (Sigma-Aldrich) was used to test its SOD activity. The results are shown in Table 3. It is found from the results that the hydrolyzed queen fetus product has both antioxidant DPPH and reducing power (Reducing Power). It is better than the unhydrolyzed product, and the whole queen bee fetal enzyme hydrolysate has good antioxidant capacity. Table 3 shows the antioxidant activity analysis and comparative analysis of the hydrolyzed queen bee fetal enzyme samples:

Since the hydrolysate of queen bee fetal enzyme is rich in peptides, the present invention aims at inhibiting the activity of vasoconstrictor enzyme (ACE) to explore its blood pressure regulating function. The samples were analyzed at different concentrations for the analysis of their ACE inhibitory activity, and analyzed by high performance liquid chromatography (HPLC). The enzyme hydrolyzed queen fetus was dissolved in 100mM boric acid solution and diluted, and 25μL and 20μL of appropriately diluted samples were taken After ACE solution (50mU/mL) and 100μL HHL solution (15mM) were mixed uniformly, they were placed in a 37℃ water bath for 40 minutes, and 100μL HCl (1M) was added to terminate the enzyme reaction. 1.5 mL of ethyl acetate was added and the mixture was shaken for 30 seconds and ultrasonically for 2 minutes to extract hippuric acid into the organic layer, and centrifuged at 1500×g for 10 minutes. Take 1 mL of the upper layer liquid into a new centrifuge tube, and use a dry bath machine to heat in a dry bath at 90° C. for 30 minutes in an exhaust cabinet to completely evaporate the ethyl acetate. After cooling to room temperature, add 1mL deionized water to re-dissolve, filter through 0.22μm filter membrane, and analyze by high performance liquid chromatography (HPLC) (detector: UV detector (UV/Vis Detertor: 228nm) , Waters ^® TaperSlit, US), chromatography tube: Cosmosil C18-AR-II (Cosmosil, Japan) flow direction to LC grade acetonitrile (Acetonitrile, E. Merck, Germany and 0.08% acetic acid (20: 80, v/ v), the flow rate of 1.0mL/min analysis, the results are shown in Figure 2, the results show that the hydrolyzed queen fetus has higher ACE inhibitory activity than the unhydrolyzed product, and the IC50 of the hydrolyzed product ACE inhibitor The IC50 of the unhydrolyzed product is 0.1%, and the IC50 of the unhydrolyzed product is 5%. Therefore, the hydrolyzed product will be used in the development of health food for blood pressure regulation in the future.

In order to ensure whether the developed product complies with relevant health regulations, the present invention conducts a microbiological test of the product to ensure that it will not cause harm to the human body. Test items include E. coli colony testing and E. coli testing. Refer to the detection method of the Food and Drug Administration of the Ministry of Health and Welfare of Taiwan, weigh 5g of the enzyme hydrolyzed queen bee fetus in a sterile Erlenmeyer flask and add 45mL of sterile 85% physiological laboratory water, after serial dilution, take 100μL of the dilution to solid PCA Coat the plate medium and place it in an incubator at 35°C for 48 hours. Observe the growth of the colony and count the number of colonies to determine the total number of bacteria in the sample. In the determination of E. coli, the present invention uses chromocult coli culture medium Chromocult ^® Coliform Agar (Merck KGaA, Germany) to determine the colony detection and E. coli. The results are shown in Table 4 and Table 5. The total bacterial count and E. coli group test of the samples stored at two different temperatures are in compliance with the "Taiwan Good Food Product Verification and Inspection Project Specifications and Standards", the E. coli group ( MPN/g)<10 ³ , E. coli (MPN/g) is negative. Table 4 is the microbiological analysis table of samples stored at different times:

Since product quality is affected by the environment, in order to test, the present invention places the product at two different temperatures (25, 40°C) and analyzes the color and moisture content of the product when stored for 3 and 6 months. We use an infrared moisture meter (kett, Japan) to measure the moisture content of the sample, and use a colorimeter to analyze the change in the standard color difference of the sample to observe the three items of L*, a*, and b* The data changes, and the results are shown in Table 5. When the samples are stored at room temperature of 25°C and 40°C, their appearance and color should be stable at 25°C and higher temperature of 40°C, and they are quite stable in terms of moisture changes. The finished product of the present invention is 10 The color at 2 temperatures is shown in Figure 3. In terms of moisture content and microbiological testing, the stability of the quality of the finished product 10 is determined and is beneficial to future product packaging design. Table 5 shows the moisture and color changes of samples stored at different times.

According to the conditions obtained by the laboratory of the National Huwei University of Science and Technology, the present invention was originally designed to be used with the existing equipment of the factory to carry out a mass production test in a factory that can process at least 50 kilograms of queen bee tires per batch. However, we It is found that if fresh queen bee fetus is directly used as the enzyme hydrolysis target, it is found that the viscosity of the insect body fluid is too high after homogenization, and its viscosity leads to an enzyme reaction Poor, because the body fluid is not good for the experiment, we re-adjust the process. We must first freeze-dry the queen fetus into a powder, and then use the dried powder to prepare a 25% queen fetus reaction solution, and use pineapples. Enzyme (10000CDU/ml) and papaya enzyme (30000FCCpu/ml) are used to process the enzyme process. The reaction conditions are adjusted to 72°C for 1 hour at 37°C for 1 hour, and the reaction is quickly heated to 100°C (30 Minutes) to stop the enzyme reaction. Then use protein electrophoresis to analyze the protein hydrolysis rate and molecular weight, and analyze the peptide content to confirm the complete reaction of the production process.

After the measurement is completed, the present invention formulates a mass production process as shown in FIG. 4. With the existing technology and equipment of the applicant, Fengzhixiang Company, a batch of 50 kg of raw material for queen bee fetus can be freeze-dried to produce about 20 kg of dry powder. Considering the mass production in the future, in addition to the original process of re-making the queen fetal reaction solution, the first and second stages of hydrolysis and freeze-drying of the queen fetal reaction solution should also be entrusted to Yiguan Chemical Factory to produce products to reduce Pollution hazards, which in turn comply with food hygiene.

After the above specific implementation and description, the present invention mainly uses pineapple enzyme (10000CDU/ml, 37°C) and papaya enzyme (300000FCCpu/ml, 72°C) to hydrolyze the queen fetal protein into small molecule peptides. According to the results, these two edible enzymes hydrolyze their protein into small molecule peptides, and the molecular weight of the hydrolyzed peptides is about 17~31kDa. In order to explore the difference between hydrolyzed and unhydrolyzed queen fetuses, the present invention measures its antioxidant capacity and inhibits angiotensin-converting enzyme (ACE). The results show that hydrolyzed queen fetuses perform better in antioxidant tests. The results of the ACE activity inhibition test found that the ACE inhibition rate of the queen fetus after hydrolysis can reach twice that of the unhydrolyzed queen fetus at the same concentration. Unhydrolyzed hydrolyzable queen tire tread queen ACE inhibitory activity of IC ₅₀ were 0.3mg / ml and 5mg / ml, with minimal hydrolysis of the representative tire queen, you can have the effect of ACE inhibition. Therefore, it is hoped that in the future, we will be able to verify related functions such as anti-fatigue, anti-aging, and anti-cancer, so as to develop the hydrolysate of queen bee fetus into the best health products.

The foregoing is only a feasible embodiment of the present invention, and is not intended to limit the scope of the present invention. Any equivalent implementation of other changes based on the content, features and spirit of the following claims are all Should be included in the scope of the patent of the present invention.

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10‧‧‧Queen bee fetal active peptide product

Claims (6)

A preparation method of a queen fetal active peptide, which comprises: (a) the step of remaking the queen fetus: freeze-drying and crushing the queen fetus to prepare a powdered queen fetus, and then re-compound the powdered queen fetus with water to form a queen fetus Aqueous solution, and uniformly mix the queen fetal aqueous solution with a shaking machine to obtain a queen fetal reaction liquid; (b) the first stage hydrolysis step: the queen fetal reaction liquid prepared in step (a) is used with a first fruit enzyme Hydrolyze at 37°C for 60 minutes to obtain a queen fetal hydrolysis reaction solution; (c) the second stage hydrolysis step: use the queen fetal hydrolysis reaction solution prepared in step (b) with a second fruit enzyme in 72 Hydrolysis is carried out at a temperature of ℃ for 60 minutes to obtain a hydrolyzed solution product; wherein, the first fruit enzyme is different from the second fruit enzyme; (d) heating to terminate the reaction step: the hydrolyzed solution product of step (c) Heat to 100°C and continue for 30 minutes to stop the enzyme reaction to obtain a hydrolyzed queen fetal solution; and (e) freeze-drying step: When the hydrolyzed queen fetal solution in step (d) is cooled to a temperature of 40°C, it will quickly Freeze-drying is performed to obtain the queen fetal active peptide; wherein the first fruit enzyme is pineapple enzyme; the second fruit enzyme is papaya enzyme; the protein content of the queen fetal active peptide is 0.79±0.01 mg/g, The peptide content is 28.18±1.12mg/g, the total phenol content is 29.29±1.12mg of GAE/g, and the flavonoid content is 0.33±0.02mg of QEAC/g. The method according to claim 1, wherein, in step (a), the oscillating device includes an ultrasonic oscillator and a vortex oscillator, and the ultrasonic oscillator and the vortex oscillator are oscillated to make The aqueous solution of the queen fetus is uniformly mixed to obtain the reaction solution of the queen fetus. The method according to claim 1, wherein the concentration of the aqueous solution of the queen fetus prepared in step (a) is 10-50%. The method according to claim 3, wherein the concentration of the aqueous solution of the queen fetus prepared in step (a) is 25%. The method according to claim 1, wherein the pineapple enzyme activity is 8000 to 12000 (CDU/ml). The method according to claim 1, wherein the papaya enzyme activity is 20,000 to 40,000 (FCCpu/ml).

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