A production process of original ecology royal jelly

Abstract

The application provides a production process of original royal jelly, which comprises the following steps: adding slow-release pheromone condensates into an artificial royal platform by using original nectar plants and water sources, adding organic acid substances into supplementary artificial bee feed, and treating collected royal jelly by immobilized enzyme, so that the productivity and immunity of worker bees are improved, and the original, high-quality, high-oxidation-resistance royal jelly product is obtained.

Description

Technical Field

[0001] This invention belongs to the field of honey technology, specifically relating to a production process for natural royal jelly. Background Technology

[0002] Royal jelly is a secretion from the pharyngeal glands of young worker bees that raise larvae in the hive. It enters the liver and spleen meridians; its main functions are to nourish and strengthen the body, benefit the liver, and invigorate the spleen. It is used to treat weakness after illness, malnutrition in children, frailty in the elderly, infectious hepatitis, hypertension, rheumatoid arthritis, and duodenal ulcers. Royal jelly is a secretion produced by the hypopharyngeal and mandibular glands of worker bees. It has vasodilatory and antihypertensive activities, induces a decrease in serum cholesterol levels, and possesses antibacterial, anti-allergic, anti-inflammatory, immunomodulatory, and antioxidant properties.

[0003] Bee pheromones are secreted by specialized glands in individual bees and spread through contact or air. They are chemical substances with specific functions, mutually promoting and regulating the activities of the bee colony. Previous studies have shown that injecting pheromones can increase worker bee activity and improve the health and survival rate of larvae and pupae within the hive. The larval pheromones released by queen bee larvae contain chemicals that induce worker bees to nurture them, thereby increasing the yield and quality of royal jelly. However, because most pheromone molecules are highly volatile and easily affected by environmental factors such as temperature, sunlight, and wind and rain, pheromone waste and reduced effectiveness can occur. Furthermore, the biological cycle of pheromone release needs to be considered. How to stably release pheromones to provide a lasting positive feedback loop for bees is the focus of this invention.

[0004] Artificial bee feed mainly includes sugar feed and pollen substitutes. Sugar feed is generally used to replace honey in the hive, providing the necessary carbohydrates for the normal growth and reproduction of the bee colony. The main sugar feeds include white sugar syrup, etc. Bees obtain their nutrition from the bee bread they consume; therefore, the bee bread has a significant impact on the royal jelly produced by bees. Although an appropriate amount of protein can maximize the development of worker bees' hypopharyngeal glands, thereby increasing their immune function, dietary protein does not significantly affect the effective components in royal jelly. Therefore, selecting a suitable artificial feed component to increase the effective components in royal jelly is one of the key directions of this invention.

[0005] Royal jelly possesses various nutritional and health benefits, with its antioxidant capacity being the most prominent. Freshly collected royal jelly contains abundant water-soluble proteins, but due to their large molecular size, their antioxidant activity is poor. Furthermore, royal jelly acid is a unique unsaturated fatty acid with a pungent and irritating taste. To improve its antioxidant activity and taste, it is typically processed using methods such as ultra-high pressure hydrolysis and enzymatic hydrolysis. Ultra-high pressure hydrolysis can quickly, efficiently, and without pollution, yielding a product with high nutritional content and good taste; however, it requires high-pressure equipment, resulting in high costs and relatively complex operation, limiting its widespread applicability. Enzymatic hydrolysis can reduce the pungent taste of royal jelly, but because the enzyme components are difficult to separate from the royal jelly product, it leads to the loss of nutritional components. Therefore, this invention employs an immobilized enzyme method for enzymatic hydrolysis. By optimizing the immobilization process of the enzyme, a royal jelly product with a sweet taste, rich nutrition, and high antioxidant properties is obtained. Summary of the Invention

[0006] This invention discloses a production process for raw royal jelly to solve any of the above-mentioned and potential problems in the prior art.

[0007] To solve the above-mentioned technical problems, the preparation method of the present invention is as follows:

[0008] One-day-old larvae are transferred into artificial queen cells in the hive frame. Pheromones gel is placed in the artificial queen cells. Artificial feed is added to the outside of the hive partition. The royal jelly production temperature is above 15℃, and there is no continuous cold wave. The royal jelly is gently scraped along the inner wall of the cell base with a royal jelly collection pen or scraper and scraped into a royal jelly bottle. The royal jelly collection time is 60 minutes. After collection, it is immediately placed in a refrigerator at 2℃ for later use. After the royal jelly of the whole frame has been collected, the beeswax inside and around the unaccepting cells is cut off with a knife. A small amount of residual royal jelly is dipped from the accepting cells and applied to the unaccepting cells with a royal jelly collection pen. Then, the larvae are transferred and put back into the production colony.

[0009] Immobilized enzyme treatment: Take the above royal jelly, add sucrose to adjust the pH to 5-7, then add immobilized enzyme at a solid-liquid ratio of 1:20, and enzymatically hydrolyze by vortexing at 37℃ for 3 hours with a vortexing frequency of 50Hz. Filter out the immobilized enzyme to obtain the final product.

[0010] Preparation of pheromone gel: Pheromones and ethanol are mixed at a volume ratio of 1:2-2.5. Then, the ethanol solution of pheromones is added to a 2-3% sodium alginate solution, wherein the volume ratio of the ethanol solution of pheromones to the sodium alginate solution is 1:2-2.5. A 1-4% ester solution is added dropwise to the mixture. The resulting mixture is subjected to ultrasonic treatment at a frequency of 80-120 kHz to obtain a pheromone-containing emulsion. The sodium alginate emulsion containing pheromones is added dropwise to a 6% (w / w) calcium chloride aqueous solution, and the gel solidifies to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres.

[0011] The pheromone is a mixture of methyl linoleate, ethyl linolenate, and tetradecyl myristate in equal proportions;

[0012] The ester is a 2-5% (w / w) solution of 4-nitrophenyl lauryl ester.

[0013] The pheromone gel particles have a particle size of 2-12 mm, and the amount of gel added to each artificial king is 0.3-0.8 mg per cubic centimeter.

[0014] Preparation of supplemental artificial feed: Mix 18-24 parts soybean meal, 28-35 parts corn flour, 8-10 parts corn gluten meal, 40-50 parts white sugar, 1-3 parts organic acid, and 0.5-0.8 parts dicalcium phosphate evenly.

[0015] The organic acid is N-succinimide myristic acid.

[0016] Preparation of immobilized enzyme: Take 8-10 parts of mesoporous silica porous material, add 1.5-2 parts of 5% (w / w) papain sodium chloride solution, and add 0.5-0.8 parts of adhesive dropwise. Place it on a magnetic stirrer and react at 200 r / min for 6 h. After the reaction is complete, store the immobilized enzyme in a 4℃ refrigerator for later use.

[0017] The adhesive is a 30% N-(5-hydroxynicotinamide)-L-glutamic acid solution.

[0018] The advantages and beneficial effects of this invention are as follows:

[0019] 1. This invention provides a production process for natural royal jelly. By utilizing natural nectar-producing plants and water sources, adding slow-release pheromone globules to artificial queen cells, adding organic acid substances to supplemental artificial bee feed, and treating the collected royal jelly with immobilized enzymes, the production capacity and immunity of worker bees are improved. This process can produce natural, high-quality royal jelly products with high antioxidant properties.

[0020] 2. This invention improves worker bees' acceptance of artificial queen cells through the slow release of pheromone globules, which exhibit good stability and persistence, thereby promoting royal jelly production. The artificial feed prepared by this invention, through scientific formulation, maximizes the development of worker bees' hypopharyngeal glands, increasing yield while also promoting the synthesis of endogenous fatty acids, thus increasing the content of effective components in royal jelly. The enzymatic hydrolysis treatment with immobilized enzymes can easily and effectively remove the pungent taste from royal jelly and improve its antioxidant properties, resulting in a high-quality, highly antioxidant, pristine royal jelly product.

[0021] 3. In the pheromone gel prepared by this invention, 4-nitrophenyl laurate helps the pheromone gel mix into an ordered layered stack during formation, and is stabilized by the interaction between the ester structure and alkyl chain, which are similar to those of pheromones. This layered stacking structure makes the pheromone gel a storage-capable sustained-release pheromone gel. Compared with pure pheromones or simply mixed pheromones, the pheromones in this gel can achieve more effective sustained release, thus exhibiting better control of the release rate, good hydrolytic stability, and higher activity.

[0022] 4. Adding the prepared pheromone gel to the artificial queen cell can mimic larval secretions, thus increasing worker bees' acceptance of the queen cell, improving the quality of queen rearing, and promoting royal jelly production. Furthermore, providing an appropriate amount of pheromone can attract more worker bees to protect the queen cell, maintain temperature, and secrete royal jelly to feed the queen bees, ensuring the quality of the royal jelly.

[0023] 5. Although an appropriate amount of protein can maximize the development of worker bees' hypopharyngeal glands and thus increase yield, feeding protein does not significantly affect the effective components of royal jelly. Therefore, this invention selects to add the organic acid N-succinimide myristic acid to regulate the artificial feed. On the one hand, the organic acid can be converted into 10-HAD more quickly in the bee's body, which can effectively increase the content of 10-HDAA, 3-HDAA and sebacic acid in royal jelly, and the conversion rate is fast and long-lasting.

[0024] 6. N-Succinimidylmyristic acid is a fatty acid derivative, so its metabolic pathway has the advantages of better absorption and higher activity compared to other organic acids. The structure of N-succinimidylmyristic acid may make it more stable in the metabolism of bees. A stable metabolic pathway can ensure that it remains active in the body for a long time, thus continuously having a positive impact on the physiological functions of worker bees, promoting the synthesis of endogenous fatty acids in worker bees. The mandibular glands of worker bees can directly utilize the 14-carbon saturated fatty acids in N-succinimidylmyristic acid, or synthesize it with short-chain molecules, significantly increasing the activity of amylase, trypsin, superoxide dismutase, lysozyme, and phenol oxidase in worker bees, significantly reducing the malondialdehyde content in worker bee bodies, and improving the digestive, antioxidant, and immune capabilities of worker bees.

[0025] 7. Cyclic oligosaccharide compounds are commonly used to enhance enzyme immobilization. While they offer good immobilization, this process can cause conformational changes in the enzyme, leading to a loss of enzyme activity. Therefore, N-(5-hydroxynicotinyl)-L-glutamic acid is chosen not only to enhance enzyme immobilization but also to help maintain the enzyme's three-dimensional structure. This ensures the enzyme remains stable in the immobilized environment, preventing deformation or aggregation and maximizing enzyme activity.

[0026] 8. The structural characteristics of N-(5-hydroxynicotinamide)-L-glutamic acid, combined with the structure of porous materials, form a relatively loose and uneven surface, exposing more binding sites to proteases and creating conditions for enzyme adsorption. N-(5-hydroxynicotinamide)-L-glutamic acid makes the surface structure of the immobilized enzyme more compact and rough. Because N-(5-hydroxynicotinamide)-L-glutamic acid has abundant binding sites, it increases the loading capacity of porous materials. The hydroxyl and nitrogen ions and multiple carboxyl groups on the N-(5-hydroxynicotinamide)-L-glutamic acid ring can catalyze enzyme activity, thereby improving the efficiency of enzyme catalysis.

[0027] 9. N-(5-hydroxynicotinyl)-L-glutamate influences the shape of mesoporous silica to a certain extent, making it predominantly brush-like. This brush-like structure provides excellent fixation for papain and can synergistically catalyze pectinase, facilitating the inactivation of microorganisms and harmful enzymes without the addition of preservatives. It also alleviates the pungent and astringent taste of royal jelly, maintaining a pleasant flavor. The fixed enzyme can also degrade amino acids in royal jelly, producing growth factors and polypeptides. Royal jelly treated with the fixed enzyme significantly improves its in vitro antioxidant activity and has a good regulatory effect on human metabolism and immune function. Furthermore, this process enzymatically hydrolyzes royal jelly's main protein 1, reducing allergic reactions in susceptible individuals. Detailed Implementation

[0028] The invention will be further described in detail below with reference to the embodiments. All the following embodiments are based on the following conditions: the nectar source is an abundant and continuous nectar and pollen source within a 3km radius of the beehive; the water source is mineral-rich stream water; the specifications of the royal jelly collection frame are the same as the brood frame: the top bar and side bars are both 13mm; four 13mm wide and 8mm thick base plates are horizontally installed inside the frame; royal jelly production can begin when the number of bees in the royal jelly-producing colony reaches 8 or more frames; the hive is divided into two areas using a queen excluder: a brood rearing area and a royal jelly production area. The brood rearing area contains the queen bee and 3 combs: old pupae, empty combs, and egg combs for bee colony reproduction; the royal jelly production area contains, in order from both sides, honey and pollen combs, new pupae combs, larval combs, and royal jelly frames; the royal jelly production temperature is above 15℃, and the process is carried out under the condition of no continuous cold waves.

[0029] Example 1

[0030] (1) Preparation of pheromone gel: Pheromones methyl linoleate, ethyl linolenate, and tetradecyl myristate are mixed in equal proportions and then mixed with ethanol at a volume ratio of 1:2.2. The ethanol solution of pheromone is then added to a 2.5% sodium alginate solution, wherein the volume ratio of the ethanol solution of pheromone to the sodium alginate solution is 1:2.2. 3% of a 4% 4-nitrophenyl laurate solution is added dropwise to the mixture. The resulting mixture is subjected to ultrasonic treatment at a frequency of 100 kHz to obtain a pheromone-containing emulsion. The sodium alginate emulsion containing pheromone is added dropwise to a 6% calcium chloride aqueous solution, and the gel solidifies to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres.

[0031] (2) Preparation of supplementary artificial feed: Mix 21 parts soybean meal, 32 parts corn flour, 9 parts corn gluten meal, 45 parts white sugar, 2 parts N-succinimide myristic acid, and 0.6 parts dicalcium phosphate evenly to obtain the feed.

[0032] (3) Transfer the one-day-old larvae into the work platform of the pulp frame, and add 0.5 mg / cm 3 Place the pheromone gel in the artificial queen cell, supplement the artificial feed on the outside of the hive partition, and gently scrape along the inner wall of the cell base with a pheromone collection pen or scraper to remove the royal jelly. Scrape the royal jelly into a jelly bottle and collect the royal jelly. The collection time is 60 minutes. After taking it out, immediately place it in a refrigerator at 2°C for later use. After the royal jelly of the whole frame has been collected, cut off the beeswax inside and around the unaccepted cells with a knife. Use a pheromone collection pen to dip a small amount of residual royal jelly from the accepting cells and apply it to the unaccepted cells. Then transplant the larvae and put them back into the production colony.

[0033] Example 2

[0034] (1) Preparation of pheromone gel: Pheromones methyl linoleate, ethyl linolenate, and tetradecyl myristate are mixed in equal proportions. Pheromones and ethanol are mixed at a volume ratio of 1:2. Then, the ethanol solution of pheromones is added to a 3% sodium alginate solution. The volume ratio of the ethanol solution of pheromones to the sodium alginate solution is 1:2. 1% of a 5% 4-nitrophenyl laurate solution is added dropwise to the mixture. The resulting mixture is subjected to ultrasonic treatment at a frequency of 120 kHz to obtain a pheromone-containing emulsion. The sodium alginate emulsion containing pheromones is added dropwise to a 6% calcium chloride aqueous solution. The gel solidifies to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres.

[0035] (2) Preparation of supplementary artificial feed: Mix 18 parts soybean meal, 35 parts corn flour, 8 parts corn gluten meal, 50 parts white sugar, 3 parts N-succinimide myristic acid, and 0.5 parts dicalcium phosphate evenly.

[0036] (3) Transfer the one-day-old larvae into the work platform of the pulp frame, and add 0.3 mg / cm 3 Place the pheromone gel in the artificial queen cell, supplement the artificial feed on the outside of the hive partition, and gently scrape along the inner wall of the cell base with a pheromone collection pen or scraper to remove the royal jelly. Scrape the royal jelly into a jelly bottle and collect the royal jelly. The collection time is 60 minutes. After taking it out, immediately place it in a refrigerator at 2°C for later use. After the royal jelly of the whole frame has been collected, cut off the beeswax inside and around the unaccepted cells with a knife. Use a pheromone collection pen to dip a small amount of residual royal jelly from the accepting cells and apply it to the unaccepted cells. Then transplant the larvae and put them back into the production colony.

[0037] Example 3

[0038] (1) Preparation of pheromone gel: Pheromones methyl linoleate, ethyl linolenate, and tetradecyl myristate are mixed in equal proportions. Pheromones and ethanol are mixed at a volume ratio of 1:2.5. Then, the ethanol solution of pheromones is added to a 2% sodium alginate solution. The volume ratio of the ethanol solution of pheromones to the sodium alginate solution is 1:2.5. 4% of a 2% 4-nitrophenyl laurate solution is added dropwise to the mixture. The resulting mixture is subjected to ultrasonic treatment at a frequency of 80 kHz to obtain a pheromone-containing emulsion. The sodium alginate emulsion containing pheromones is added dropwise to a 6% calcium chloride aqueous solution. The gel solidifies to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres.

[0039] (2) Preparation of supplementary artificial feed: Mix 24 parts soybean meal, 28 parts corn flour, 10 parts corn gluten meal, 40 parts white sugar, 1 part N-succinimide myristic acid, and 0.8 parts dicalcium phosphate evenly to obtain the feed.

[0040] (3) Transfer the one-day-old larvae into the work platform of the pulp frame, and add 0.8 mg / cm 3 Place the pheromone gel in the artificial queen cell, supplement the artificial feed on the outside of the hive partition, and gently scrape along the inner wall of the cell base with a pheromone collection pen or scraper to remove the royal jelly. Scrape the royal jelly into a jelly bottle and collect the royal jelly. The collection time is 60 minutes. After taking it out, immediately place it in a refrigerator at 2°C for later use. After the royal jelly of the whole frame has been collected, cut off the beeswax inside and around the unaccepted cells with a knife. Use a pheromone collection pen to dip a small amount of residual royal jelly from the accepting cells and apply it to the unaccepted cells. Then transplant the larvae and put them back into the production colony.

[0041] Comparative Example 1

[0042] The difference between this comparative example and Example 1 is that in this comparative example, pheromones are directly mixed and injected into the artificial penis. Specifically, the process involves mixing pheromones of methyl linoleate, ethyl linoleate, and tetradecyl myristate in equal proportions and injecting them into each artificial penis at a concentration of 0.5 mg / cm³. 3 Simply inject pheromones; the rest is the same as in Example 1.

[0043] Comparative Example 2

[0044] The difference between this comparative example and Example 1 is that the pheromones in this comparative example are made into pheromone capsules, and the specific process is as follows:

[0045] Using 25 parts of sodium octenyl succinate starch, emulsified modified starch-809, gelatin, and β-cyclodextrin as wall materials, they were mixed in a mass ratio of 15:5:3:2. 100 parts of purified water were added, and the mixture was stirred at 8000 rpm for 10 minutes to dissolve. Then, 12 parts of the mixed pheromone solution were added to a mixture of pheromone methyl linoleate, ethyl linolenate, and tetradecyl myristate in equal proportions. The mixture was stirred at 10000 rpm for 2 minutes and then homogenized under high pressure at 20 MPa for 2 minutes to obtain pheromone capsules. The pheromone concentration was 0.5 mg / cm³. 3 The pheromone capsules were placed in an artificial kingpin, and the rest was the same as in Example 1.

[0046] Comparative Example 3

[0047] The difference between this comparative example and Example 1 is that the 4-nitrophenyl laurate solution is a mannitol dioctanoate solution, while the rest of the implementation methods are the same as in Example 1.

[0048] Comparative Example 4

[0049] The difference between this comparative example and Example 1 lies in the amount of 4-nitrophenyl laurate solution added. Specifically, the pheromone gel preparation is as follows: pheromone methyl linoleate, ethyl linolenate, and tetradecyl myristate are mixed in equal proportions and then mixed with ethanol at a volume ratio of 1:2.2. The pheromone ethanol solution is then added to a 2.5% sodium alginate solution, with a volume ratio of 1:2.2. 10% of a 4% (w / w) 4-nitrophenyl laurate solution is added dropwise to the mixture. The resulting mixture is then ultrasonically treated at a frequency of 100 kHz to obtain a pheromone-containing emulsion. This pheromone-containing sodium alginate emulsion is then added dropwise to a 6% (w / w) calcium chloride aqueous solution, causing gel solidification to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres. The rest of the process is the same as in Example 1.

[0050] Comparative Example 5

[0051] The difference between this comparative example and Example 1 lies in the amount of 4-nitrophenyl laurate solution added. Specifically, the pheromone gel preparation is as follows: pheromone methyl linoleate, ethyl linolenate, and tetradecyl myristate are mixed in equal proportions and then mixed with ethanol at a volume ratio of 1:2.2. The pheromone ethanol solution is then added to a 2.5% sodium alginate solution, with a volume ratio of 1:2.2. 0.5% of a 4% (w / w) 4-nitrophenyl laurate solution is added dropwise to the mixture. The resulting mixture is then ultrasonically treated at a frequency of 100 kHz to obtain a pheromone-containing emulsion. This pheromone-containing sodium alginate emulsion is then added dropwise to a 6% (w / w) calcium chloride aqueous solution, causing gel solidification to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres. The rest of the process is the same as in Example 1.

[0052] Experiment 1: Determination of Wangtai Acceptance Rate

[0053] Determination of pheromone acceptance rate: Eight production frames were prepared for each group, with 16 artificial pheromones in each production frame, and 128 artificial pheromones in each group. The pheromone substances of Examples 1-3 and Comparative Examples 1-5 were placed into the artificial pheromones. After 3 days, the number of artificial pheromones and the number of accepted artificial pheromones were counted. The number of accepted artificial pheromones divided by the number of artificial pheromones was the pheromone acceptance rate.

[0054] The test results of Examples 1-3 and Comparative Examples 1-5 are shown in Table 1 below:

[0055] Table 1. Acceptance Rate of Wangtai

[0056] Group Wangtai's acceptance rate / % Example 1 96.09 Example 2 94.53 Example 3 93.75 Comparative Example 1 86.72 Comparative Example 2 87.50 Comparative Example 3 90.63 Comparative Example 4 92.19 Comparative Example 5 89.06

[0057] Comparative Example 6

[0058] The difference between this comparative example and Example 1 is that no organic acid components were added to the artificial feed in this comparative example. The artificial feed was prepared by mixing 21 parts soybean meal, 32 parts corn flour, 9 parts corn gluten meal, 45 parts white sugar, and 0.6 parts dicalcium phosphate evenly. The rest is the same as in Example 1.

[0059] Comparative Example 7

[0060] The difference between this comparative example and Example 1 is that the N-succinimide myristic acid in this comparative example is citric acid, while the rest is the same as in Example 1.

[0061] Comparative Example 8

[0062] The difference between this comparative example and Example 1 lies in the amount of N-succinimide myristic acid added in the artificial feed. Specifically, the artificial feed was prepared by mixing 21 parts soybean meal, 32 parts corn flour, 9 parts corn gluten meal, 45 parts white sugar, 5 parts N-succinimide myristic acid, and 0.6 parts dicalcium phosphate evenly. The rest was the same as in Example 1.

[0063] Comparative Example 9

[0064] The difference between this comparative example and Example 1 lies in the amount of N-succinimide myristic acid added in the artificial feed. Specifically, the artificial feed was prepared by mixing 21 parts soybean meal, 32 parts corn flour, 9 parts corn gluten meal, 45 parts white sugar, 0.5 parts N-succinimide myristic acid, and 0.6 parts dicalcium phosphate evenly. The rest was the same as in Example 1.

[0065] Example 4

[0066] This embodiment follows the steps described in Embodiment 1:

[0067] (4) Preparation of immobilized enzyme: Take 9 parts of mesoporous silica porous material, add 1.8 parts of 5% papain sodium chloride solution, and add 0.7 parts of 30% N-(5-hydroxynicotinyl)-L-glutamic acid solution. Place it on a magnetic stirrer and react at 200 r / min for 6 h. After the reaction, store the immobilized enzyme in a 4℃ refrigerator for later use.

[0068] (5) Immobilized enzyme treatment: Take the above royal jelly, add sucrose to adjust the pH to 6, add immobilized enzyme at a solid-liquid ratio of 1:20, and enzymatically hydrolyze by vortexing at 37℃ for 3 hours with a vortexing frequency of 50Hz. Filter out the immobilized enzyme to obtain the final product.

[0069] Example 5

[0070] This embodiment follows the steps described in Embodiment 1:

[0071] (4) Preparation of immobilized enzyme: Take 8 parts of mesoporous silica porous material, add 2 parts of 5% papain sodium chloride solution, and add 0.8 parts of 30% N-(5-hydroxynicotinyl)-L-glutamic acid solution. Place it on a magnetic stirrer and react at 200 r / min for 6 h. After the reaction is completed, store the immobilized enzyme in a 4℃ refrigerator for later use.

[0072] (5) Immobilized enzyme treatment: Take the above royal jelly, add sucrose to adjust the pH to 5, add immobilized enzyme at a solid-liquid ratio of 1:20, and enzymatically hydrolyze by vortexing at 37℃ for 3 hours with a vortexing frequency of 50Hz. Filter out the immobilized enzyme to obtain the final product.

[0073] Example 6

[0074] This embodiment follows the steps described in Embodiment 1:

[0075] (4) Preparation of immobilized enzyme: Take 10 parts of mesoporous silica porous material, add 1.5 parts of 5% papain sodium chloride solution, and add 0.5 parts of 30% N-(5-hydroxynicotinyl)-L-glutamic acid solution. Place it on a magnetic stirrer and react at 200 r / min for 6 h. After the reaction is completed, store the immobilized enzyme in a 4℃ refrigerator for later use.

[0076] (5) Immobilized enzyme treatment: Take the above royal jelly, add sucrose to adjust the pH to 7, add immobilized enzyme at a solid-liquid ratio of 1:20, and enzymatically hydrolyze by vortexing at 37℃ for 3 hours with a vortexing frequency of 50Hz. Filter out the immobilized enzyme to obtain the final product.

[0077] Comparative Example 10

[0078] The difference between this comparative example and Example 4 is that the N-(5-hydroxynicotinyl)-L-glutamic acid solution is a β-cyclodextrin solution; otherwise, it is the same as Example 4.

[0079] Comparative Example 11

[0080] The difference between this comparative example and Example 4 lies in the amount of β-cyclodextrin solution added. Specifically, the immobilized enzyme was prepared as follows: 9 parts of mesoporous silica porous material were taken, and 1.8 parts of a 5% (w / w) papain sodium chloride solution were added to it. Then, 1.2 parts of a 30% (w / w) N-(5-hydroxynicotinyl)-L-glutamic acid solution were added dropwise. The mixture was placed on a magnetic stirrer and reacted at 200 r / min for 6 h. After the reaction, the immobilized enzyme was stored in a 4°C refrigerator for later use. The rest was the same as in Example 4.

[0081] Comparative Example 12

[0082] The difference between this comparative example and Example 4 lies in the amount of β-cyclodextrin solution added. Specifically, the immobilized enzyme was prepared as follows: 9 parts of mesoporous silica porous material were taken, and 1.8 parts of a 5% (w / w) papain sodium chloride solution were added to it. 0.2 parts of a 30% (w / w) N-(5-hydroxynicotinyl)-L-glutamic acid solution were added dropwise. The mixture was placed on a magnetic stirrer and reacted at 200 r / min for 6 h. After the reaction, the immobilized enzyme was stored at 4°C for later use. The rest was the same as in Example 4.

[0083] Experiment 2: Determination of Royal Jelly Components

[0084] Each batch of royal jelly samples was stored at -20°C to determine the 10-HDA content. Five samples were randomly measured, and the average value was taken. The 10-HDA content was determined using high-performance liquid chromatography with internal standard method.

[0085] Table 2 10-HAD Test Results

[0086] Group 10-HAD / % Example 4 2.53 Example 5 2.48 Example 6 2.45 Comparative Example 6 1.99 Comparative Example 7 2.14 Comparative Example 8 2.36 Comparative Example 9 2.23 Comparative Example 10 2.16 Comparative Example 11 2.38 Comparative Example 12 2.27

[0087] Experiment 3: Determination of the antioxidant capacity of royal jelly:

[0088] (1) DPPH free radical scavenging rate: Prepare a 79 mg / L DPPH-ethanol solution, dissolve the bee product sample in anhydrous ethanol to obtain a sample solution with a mass concentration of 1000 ug / mL, take 0.5 mL of the sample solution, add 5.0 mL of DPPH-ethanol solution and mix well, react at 37℃ for 1 h, use anhydrous ethanol as blank, measure the color at a wavelength of 517 nm, and calculate the DPPH free radical scavenging rate. DPPH free radical scavenging rate = (blank absorbance - sample absorbance) / blank absorbance x 100.

[0089] (2) ABTS free radical scavenging rate: 25 mL of 7.4 mol / L LABTS solution and 25 mL of 2.6 mol / L potassium persulfate solution were measured and mixed in equal volumes. The mixture was reacted at room temperature in the dark for 12-16 h. Then, it was diluted 40-50 times with anhydrous ethanol and placed at room temperature in the dark for 30 min to prepare the ABTS working solution. The bee product sample was dissolved in anhydrous ethanol to prepare a sample solution with a concentration of 20 mg / mL. 20 L of the sample solution (280 W LABTS working solution) was applied to a 96-well microplate, and the absorbance value A1 was measured at 734 nm. The absorbance value A2 was obtained by replacing the sample solution with deionized water. Vitamin C was used as a positive control. The ABTS free radical scavenging rate was calculated as follows: Scavenging rate (%) = (2-A1) / A2 x 100:

[0090] (3) Superoxide anion free radical scavenging rate: Take 4.5 mL of 50 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer into a test tube, then take 2.0 mL of distilled water into a test tube, and then take 1.0 mL of bee product sample solution (microcapsules dissolved in ethanol, concentration 20 mg / mL) into the test tube. After mixing thoroughly, place it in a 25℃ water bath for 20 min. Then take 0.5 mL of 3.0 mol / L pyrogallol solution into the test tube, shake it quickly, zero the tube with distilled water, and measure the absorbance A1 at a wavelength of 325 nm using an ultraviolet spectrophotometer. The absorbance value A2 is obtained by replacing the microcapsule sample solution with deionized water. Vitamin C is used as a positive control. The superoxide anion free radical scavenging rate is calculated as follows: Scavenging rate (%) = (A2-A1) / A2x100.

[0091] Table 3 Antioxidant Activity Assay

[0092] Group DPPH free radical scavenging rate (%) ABTS free radical scavenging rate (%) Superoxide anion radical scavenging rate (%) Example 4 93.17 94.8 91.52 Example 5 92.65 93.87 90.75 Example 6 92.86 92.56 90.16 Comparative Example 6 65.78 66.84 69.61 Comparative Example 7 67.84 69.47 70.94 Comparative Example 8 82.75 84.79 86.78 Comparative Example 9 73.42 80.93 79.62 Comparative Example 10 62.71 64.82 65.47 Comparative Example 11 85.73 82.76 86.94 Comparative Example 12 76.49 75.82 76.83

Claims

1. A production process for natural royal jelly, characterized in that, The production process specifically includes the following steps: One-day-old larvae are transferred into artificial queen cells in the hive frame. Pheromones gel is placed in the artificial queen cells. Artificial feed is added to the outside of the hive partition. The royal jelly production temperature is above 15℃, and there is no continuous cold wave. The royal jelly is gently scraped along the inner wall of the cell base with a royal jelly collection pen or scraper and scraped into a royal jelly bottle. The royal jelly collection time is 60 minutes. After collection, it is immediately placed in a refrigerator at 2℃ for later use. After the royal jelly of the whole frame has been collected, the beeswax inside and around the unaccepting cells is cut off with a knife. A small amount of residual royal jelly is dipped from the accepting cells and applied to the unaccepting cells with a royal jelly collection pen. Then, the larvae are transferred and put back into the production colony. Immobilized enzyme treatment: Take the above royal jelly, add sucrose to adjust the pH to 5-7, then add immobilized enzyme at a solid-liquid ratio of 1:20, and enzymatically hydrolyze by vortexing at 37℃ for 3 hours with a vortexing frequency of 50Hz. Filter out the immobilized enzyme to obtain the final product. Preparation of pheromone gel: Pheromones and ethanol are mixed at a volume ratio of 1:2-2.

5. Then, the ethanol solution of pheromones is added to a 2-3% sodium alginate solution, wherein the volume ratio of the ethanol solution of pheromones to the sodium alginate solution is 1:2-2.

5. A 1-4% ester solution is added dropwise to the mixture. The resulting mixture is subjected to ultrasonic treatment at a frequency of 80-120 kHz to obtain a pheromone-containing emulsion. The sodium alginate emulsion containing pheromones is added dropwise to a 6% (w / w) calcium chloride aqueous solution, and the gel solidifies to form gel spheres. The spheres are filtered out, washed, and dried to obtain pheromone gel spheres. The pheromone is a mixture of methyl linoleate, ethyl linolenate, and tetradecyl myristate in equal proportions; The ester solution is a 2-5% (w / w) solution of 4-nitrophenyl lauryl ester. Preparation of supplemental artificial feed: Mix 18-24 parts soybean meal, 28-35 parts corn flour, 8-10 parts corn gluten meal, 40-50 parts white sugar, 1-3 parts organic acid, and 0.5-0.8 parts dicalcium phosphate evenly. The organic acid is N-succinimide myristic acid; Preparation of immobilized enzyme: Take 8-10 parts of mesoporous silica porous material, add 1.5-2 parts of 5% (w / w) papain sodium chloride solution, and add 0.5-0.8 parts of adhesive. Place it on a magnetic stirrer and react at 200 r / min for 6 h. After the reaction, store the immobilized enzyme in a 4℃ refrigerator for later use. The adhesive is a 30% N-(5-hydroxynicotinamide)-L-glutamic acid solution.

2. The production process of raw royal jelly according to claim 1, characterized in that: The pheromone gel particles have a particle size of 2-12 mm, and the amount of gel added to each artificial king is 0.3-0.8 mg per cubic centimeter.

3. The application of royal jelly prepared by the production process of claim 1 or 2 above in health products, characterized in that: The health supplement is packaged in 5g portions.