Preparation process of royal jelly fermented bee product

Abstract

The invention discloses a preparation process of a royal jelly fermented bee product, which comprises the following steps: fermenting bee food to obtain a bee food product with high nutritional ingredients, enhancing the absorption of worker bees and the yield of royal jelly, and adding substances for increasing the tolerance of the bees into the bee food to improve the metabolism of the bees to pesticide residues, thereby reducing the pesticide residues in the royal jelly and improving the yield of the royal jelly. And finally, the collected royal jelly is subjected to modified protease treatment, so that the nutrition and health-care functions of the royal jelly are improved, the taste is improved, and the obtained bee product has the advantages of high oxidation resistance, immunity enhancement, liver protection and the like.

Description

Technical Field

[0001] The invention relates to the technical field of royal jelly, and in particular to a preparation process of royal jelly fermented bee products. Background Art

[0002] Royal jelly is the secretion of the pharyngeal glands of young worker bees that cultivate larvae in the beehive. It belongs to the liver and spleen meridians. It is mainly used to nourish and strengthen the liver and spleen. It can treat weakness after illness, malnutrition in children, physical weakness in the elderly, infectious hepatitis, hypertension, rheumatoid arthritis, and duodenal ulcers. Royal jelly is a high-protein food. Long-term consumption of royal jelly can bring many health benefits, such as enhancing immunity, improving sleep quality, delaying aging, antibacterial and anti-inflammatory, and protecting the liver.

[0003] Honey is made from plant essence extracted by bees through a purely natural biological production process. It has a strong and special natural fragrance and contains a variety of nutrients such as amino acids, nucleic acids, fructose, vitamins A, C, E, selenium, zinc, manganese, magnesium and other trace elements, as well as more than 140 active ingredients. It is a good food that people often use for daily consumption, longevity, recovery from illness, and beauty.

[0004] Bee food is the food of honey bees and the main food source of young bees. It plays an important role in the reproduction and survival of bee colonies. Natural bee food is sealed in the beehive by bees. Artificial collection will destroy the structure of the beehive and affect the living habits of the bee colony. The yield of natural bee food is low, and it only supplies nutrition for the larvae. Artificial forced collection will affect the reproduction and survival of the entire bee colony. The composition of natural bee food is complex, and it contains impurities such as bee eggs that are not easy to remove, so it has high requirements for production and processing. Therefore, the raw material of artificial fermented bee food is pollen, which has no restrictions on the source and can be mass-produced. And the nutritional content of bee food can be guaranteed after fermentation. Controlling the strains and oxygen in the fermentation process to regulate the nutritional content of bee food is the research focus of this invention.

[0005] Royal jelly has a variety of physiological and pharmacological effects and is widely used in health foods, foods and daily chemicals. Since 2001, there have been problems such as abuse of pesticides and pesticides in the field. The problem of pesticide residues in bee products represented by honey and royal jelly has attracted worldwide attention. The pesticide residue limit has been upgraded to a conventional quality requirement for exporting bee products, and has also become a green barrier to the export trade of bee products. Although after years of efforts, my country still has not completely solved the problem of pesticide residues. In China, reports of banned drugs being detected in bee products occur from time to time. In order to further reduce the residues of pesticides in bee products and improve the quality of bee products, the present invention starts with bee food, and by fermentation, bee food is prepared so that bees increase the degradation of pesticide residues, thereby reducing the pesticide residues in royal jelly, to ensure the quality of bee products.

[0006] Most bee products on the market are made by mixing honey, royal jelly, etc., which have extremely high nutritional value, hypoglycemic properties, high antioxidant properties and other functions. However, bee products made by simple direct mixing still contain allergenic protein components. Different bee product raw materials have their own unique biologically active components. Their mixing may cause the interaction of certain components and affect their biological activity; therefore, it is particularly important to use some technical means to transform royal jelly. Enzymatic hydrolysis technology is an effective means of modifying protein materials. The products obtained by enzymatic hydrolysis often have better physical and chemical properties, nutritional properties and biological activity than raw protein, and can better meet the needs of related industries. The present invention further improves the utilization efficiency and functional properties of royal jelly protein by modified protease. Summary of the invention

[0007] In order to solve the above technical problems, the specific preparation process of the present invention is as follows:

[0008] Add artificially fermented bee food on the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, take out the royal jelly with a jelly pen, scrape it into a jelly bottle, collect the royal jelly, add 1-2 parts of water and 0.03 parts of modified protease to 10-20 parts of royal jelly, stir and disperse at a speed of 300r / min for 10min, the temperature is 40-45°C, enzymolysis is performed for 2-4h, 10% ammonium sulfate solution by mass fraction is added for salting out and alcohol precipitation, centrifuge at a speed of 5000rpm for 15min to remove excess liquid to obtain pure royal jelly, wait until it cools to room temperature, and store it at 4°C; mix the royal jelly after enzymolysis with honey in a ratio of 1:10 to obtain the bee product;

[0009] The artificial fermentation of bee bread comprises: adding 0.1-0.3% of lactic acid bacteria strains into a culture dish containing 50 parts of sterile water, adding 10 parts of brown sugar and culturing at 37°C for 12 hours to obtain lactic acid bacteria culture solution, weighing 500 parts of pollen, grinding and sieving, pouring into a fermentation tank, adding sterile water with a solid-liquid ratio of 13:100, fully mixing, and then high-pressure sterilization for 30 minutes; after cooling, adding 10% of lactic acid bacteria culture solution, and adding 1-3% of fermentation promoter, setting the temperature of the fermentation tank to 33°C, the stirring speed to 445RPM, stopping the fermentation when the pH change on the display screen of the fermentation tank is <0.02 for 2 consecutive days, and after the fermentation is completed, centrifuging and drying the fermented product to obtain the bee bread.

[0010] The fermentation promoting substance is sucrose dodecanoate.

[0011] The artificially fermented bee bread further comprises mixing, stirring and dispersing the dried bee bread with a tolerance solution having a mass fraction of 20-30% in a ratio of 1:0.002-0.005.

[0012] The tolerable substance is kaempferol glucuronide solution.

[0013] The modified protease comprises: firstly dispersing and dissolving 8-15 parts of a modifier in 100-120 parts of a hydrochloric acid solution with a mass fraction of 37%, placing the solution in a 60°C constant temperature water bath and stirring the solution at a constant temperature of 30 minutes at a rotation speed of 400-500RPM, and dropping a NaOH solution to adjust the pH value to 6.8-7.2; dropping 0.2-0.4% of Esperase 8.0L, stirring the solution at room temperature for 2 hours at a rotation speed of 100-200RPM, and after the reaction is completed, performing 6 cycles of ultrafiltration on the solution using an ultrafilter with a molecular weight cutoff of 50kDa, and after the ultrafiltration is completed, adjusting the pH value to 4.8 using a hydrochloric acid solution, and storing the solution at 0°C for future use.

[0014] The modifier is propylsulfonamide.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention discloses a preparation process of royal jelly fermented bee products. By fermenting bee bread, a bee bread product with high nutritional content is obtained, the absorption of worker bees and the production of royal jelly are enhanced, and the tolerance of bees to pesticides and other substances is added to the bee bread, thereby reducing pesticide residues in the royal jelly. Finally, the collected royal jelly is treated with modified protease to improve the nutritional and health functions of the royal jelly and improve the taste. The obtained bee product has multiple advantages such as high antioxidant activity, enhanced immunity, and liver protection.

[0017] 2. After fermentation, the total sugar content, total protein content, and amino acid content in the bee bread increased. This is because after the liquid fermentation is stirred, the contact area between the lactic acid bacteria and the pollen becomes larger, which is conducive to the full fermentation of the pollen. From the perspective of large-scale production, liquid fermentation lactic acid bacteria reproduce quickly and can improve production efficiency; on the other hand, the total sugar, total protein and amino acid content of bee bread produced by liquid fermentation are higher, which ensures the quality of artificially fermented bee bread. After artificial fermentation, the bee bread is rich in various amino acids, and these amino acids are very helpful in enhancing the immunity of worker bees. The reason is that the ammonia molecules in the lactic acid bacteria culture solution produce various amino acids through a series of reactions.

[0018] 3. The structure of sucrose dodecanoate contains both hydrophilic sucrose parts and lipophilic dodecanoic acid chains. This hydrophilic-lipophilic balance helps to form a stable emulsified system and improve the solubility of the substrate. Sucrose dodecanoate is used as a fermentation promoter. It can reduce the surface tension of the liquid, increase the contact area between lactic acid bacteria and pollen in liquid fermentation, and promote the transfer and reaction of substances. By improving the microenvironmental conditions, the metabolic activity of lactic acid bacteria is promoted, thereby accelerating the production of proteins and amino acids. The presence of sucrose dodecanoate may make the amino acids and other organic substances produced during the fermentation process easier to be absorbed and utilized by worker bees, thereby improving the nutritional value of bee food.

[0019] 4. Kaempferol glucuronide is a derivative of flavonoids. It is more effective than simple kaempferol flavonoid compounds in regulating bee metabolism and can regulate bee metabolism through multiple pathways and targets.

[0020] 5. Therefore, by feeding a certain amount of kaempferol glucuronide, the average survival time of worker bees after long-term ingestion of imidacloprid can be extended. Kaempferol glucuronide regulates the tolerance of Chinese honey bees to pesticides by inducing up-regulation of key P450s genes and increases the degradation and digestion ability of bees to pesticide residues. Kaempferol glucuronide can significantly improve the tolerance of Chinese honey bees to pesticide residues, enhance the metabolism of pesticides, and ensure the yield and quality of royal jelly.

[0021] 6. Kaempferol glucuronide can significantly increase the activity of detoxification enzymes in honey bees, and participate in the detoxification metabolism of pesticides in honey bees. The advance treatment of kaempferol glucuronide can provide protection for subsequent pesticide exposure by upregulating the expression of key P450s genes, and improve the degradation of pesticide residues in honey bees. This effect enables honey bees to metabolize more effectively, thereby reducing pesticide residues in royal jelly.

[0022] 7. By modifying the amidation of proteases with propylsulfonamide, specific residues of proteases are selectively modified, especially free carboxyl and amino groups, so that the active center of the enzyme can be enhanced without affecting other important structures. Through modification, propylsulfonamide can improve the stability of proteins in high temperature, extreme pH or organic solvent environments, thereby reducing the risk of denaturation and maintaining the activity of the enzyme. By introducing hydrophilic or hydrophobic groups, propylsulfonamide can improve the solubility and fluidity of proteins, thereby enhancing their physiological activity and biological functions. Thus, high enzyme activity and functional modification can be achieved at the same time.

[0023] 8. The application of modified protease in enzymatic hydrolysis of royal jelly has significant benefits and advantages. First, the proteolysis of royal jelly to produce bioactive peptides can effectively improve its nutritional and health functions and increase the content of 10-hydroxy-2-decenoic acid in royal jelly. These bioactive peptides have multiple physiological functions such as antioxidant peptides and antihypertensive peptides. In addition, the enzymatic modification process is gentle and will not destroy the original functional properties of the protein, which allows the final product to maintain the original nutritional components and physiological activity of royal jelly.

[0024] 9. After enzymatic hydrolysis, royal jelly has rich amino acid types and high content, and the relative molecular weight of the enzymatic hydrolysis product has significantly decreased, the viscosity has decreased, and it has good fluidity. In addition, enzymatic hydrolysis also changes the color of royal jelly, significantly improves permeability, and significantly increases the content of 10-hydroxy-2-decenoic acid in royal jelly. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the embodiments.

[0026] All raw materials used in the embodiments of the present invention are from Jiangxi Fengfujia Technology Co., Ltd., wherein the raw materials or chemical reagents, unless otherwise specified, are obtained through conventional commercial channels.

[0027] Example 1

[0028] Artificial fermentation of bee bread: adding 0.2% lactic acid bacteria strains into a culture dish containing 50 parts of sterile water, adding 10 parts of brown sugar, and culturing at 37°C for 12 hours to obtain lactic acid bacteria culture solution, weighing 500 parts of pollen, grinding and sieving, pouring into a fermentation tank, adding sterile water with a solid-liquid ratio of 13:100, fully mixing, and then high-pressure sterilization for 30 minutes; after cooling, accessing 10% lactic acid bacteria culture solution, and adding 2% sucrose dodecanoate, setting the fermentation tank temperature to 33°C, the stirring speed to 445RPM, and stopping the fermentation when the pH change on the fermentation tank display screen is <0.02 for 2 consecutive days, and after the fermentation is completed, the fermented product is centrifuged and dried to obtain bee bread;

[0029] Example 2

[0030] Artificial fermentation of bee bread: adding 0.1% lactic acid bacteria strains to a culture dish containing 50 parts of sterile water, adding 10 parts of brown sugar, and culturing at 37°C for 12 hours to obtain lactic acid bacteria culture solution, weighing 500 parts of pollen, grinding and sieving, pouring into a fermentation tank, adding sterile water with a solid-liquid ratio of 13:100, fully mixing, and then high-pressure sterilization for 30 minutes; after cooling, accessing 10% lactic acid bacteria culture solution, and adding 3% sucrose dodecanoate, setting the fermentation tank temperature to 33°C, the stirring speed to 445RPM, and stopping the fermentation when the pH change on the fermentation tank display screen is <0.02 for 2 consecutive days, and after the fermentation is completed, the fermented product is centrifuged and dried to obtain bee bread;

[0031] Example 3

[0032] Artificial fermentation of bee bread: adding 0.3% lactic acid bacteria strains into a culture dish containing 50 parts of sterile water, adding 10 parts of brown sugar, and culturing at 37°C for 12 hours to obtain lactic acid bacteria culture solution, weighing 500 parts of pollen, grinding and sieving, pouring into a fermentation tank, adding sterile water with a solid-liquid ratio of 13:100, fully mixing, and then high-pressure sterilization for 30 minutes; after cooling, accessing 10% lactic acid bacteria culture solution, and adding 1% sucrose dodecanoate, setting the fermentation tank temperature to 33°C, the stirring speed to 445RPM, and stopping the fermentation when the pH change on the fermentation tank display screen is <0.02 for 2 consecutive days, and after the fermentation is completed, the fermented product is centrifuged and dried to obtain bee bread;

[0033] Comparative Example 1

[0034] In this comparative example, the bee bread is not fermented. The specific process is: 500 parts of pollen are weighed, ground and sieved, and then poured into a fermentation tank, and sterile water with a solid-liquid ratio of 13:100 is added, mixed thoroughly, and then high-pressure sterilization is performed for 30 minutes.

[0035] Comparative Example 2

[0036] In this comparative example, sucrose dodecanoate was not added, and the rest was the same as in Example 1.

[0037] Comparative Example 3

[0038] In this comparative example, sucrose dodecanoate is sucrose, and the rest is the same as in Example 1.

[0039] Comparative Example 4

[0040] The dosage of sucrose dodecanoate in this comparative example is different, specifically: 0.2% of lactic acid bacteria strains are added to a culture dish containing 50 parts of sterile water, and 10 parts of brown sugar are added and cultured at 37°C for 12 hours to obtain a lactic acid bacteria culture solution, 500 parts of pollen are weighed, ground and sieved, and poured into a fermentation tank, and sterile water with a solid-liquid ratio of 13:100 is added and fully mixed, and then high-pressure sterilization is performed for 30 minutes; after cooling, 10% of the lactic acid bacteria culture solution is added, and 5% of sucrose dodecanoate is added, the fermentation tank temperature is set to 33°C, the stirring speed is set to 445RPM, and the fermentation is stopped when the pH change on the display screen of the fermentation tank is <0.02 for 2 consecutive days. After the fermentation is completed, the fermented product is centrifuged and dried to obtain bee bread;

[0041] Comparative Example 5

[0042] The dosage of sucrose dodecanoate in this comparative example is different, specifically: 0.2% of lactic acid bacteria strains are added to a culture dish containing 50 parts of sterile water, and 10 parts of brown sugar are added and cultured at 37°C for 12 hours to obtain a lactic acid bacteria culture solution, 500 parts of pollen are weighed, ground and sieved, and then poured into a fermentation tank, and sterile water with a solid-liquid ratio of 13:100 is added and fully mixed, and then high-pressure sterilization is performed for 30 minutes; after cooling, 10% of the lactic acid bacteria culture solution is added, and 0.5% of sucrose dodecanoate is added, the fermentation tank temperature is set to 33°C, the stirring speed is set to 445RPM, and the fermentation is stopped when the pH change on the display screen of the fermentation tank is <0.02 for 2 consecutive days, and after the fermentation is completed, the fermented product is centrifuged and dried to obtain bee bread;

[0043] Experiment 1: Determination of content in bee food

[0044] Determination of total sugar content: The total sugar content in bee bread was determined by anthrone colorimetry. Add 0.2g of bee bread prepared in Example 1-3 and Comparative Example 1-4 to a test tube, add 5mL of distilled water, shake evenly, cover the tube mouth and boil in a boiling water bath for 10min, cool the running water to room temperature, filter, and prepare the filtrate into 25mL of aqueous solution. Then take 0.1mL of the above solution, then add 0.9mL of distilled water to dilute, measure the absorbance value of the solution, and repeat the operation 3 times to measure the content.

[0045] Total protein content: The content of the bee breads prepared in Examples 1-3 and Comparative Examples 1-4 was determined by Kjeldahl nitrogen determination.

[0046] Total amino acid content: weigh about 0.20 g of the bee bread samples prepared in Examples 1-3 and Comparative Examples 1-4 into a hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid into the hydrolysis tube, add 2 to 3 drops of phenol, shake to fully dissolve the sample, and then fill with high-purity nitrogen; seal the tube while filling with ammonia, place the sealed hydrolysis tube in a constant temperature drying oven at (110 ± 1) ° C, hydrolyze for 22 hours, and then take out and cool.

[0047] Open the hydrolysis tube, transfer all the hydrolyzate to a 50ml volumetric flask, dilute with deionized water, and rinse the hydrolysis tube several times. After filtering the hydrolyzate, draw 1ml of the filtrate into a 10ml tube, concentrate at about 50℃, dissolve the residue with 1-2ml water, evaporate to dryness, dissolve with 1.0ml 0.02moL / L dilute hydrochloric acid, filter and provide for instrument measurement.

[0048] Chromatographic conditions: Chromatographic column: chromatographic column for hydrolyzed amino acids: detection wavelength: 570nm, of which the detection wavelength for proline is 440nm; analytical column temperature: 57°C; reaction column temperature: 135°C; injection volume: 20μl; elution solution flow rate: 0.4ml / min; post-column derivatization reagent flow rate: 0.35ml / min. The measurement results are shown in Table 1.

[0049] Table 1 Determination of bee bread content

[0050] Group Total sugar content (%) Total protein content (%) Total amino acid content (%) Example 1 48.5 32.4 15.1 Example 2 48.3 31.6 14.9 Example 3 48.1 31.5 14.6 Comparative Example 1 16.3 22.3 11.2 Comparative Example 2 35.9 20.5 11.8 Comparative Example 3 38.5 24.3 12.9 Comparative Example 4 46.8 29.7 14.2 Comparative Example 5 42.8 26.3 13.6

[0051] The following implementation modes are all carried out on the basis of Example 1.

[0052] Example 4

[0053] 0.4% of a 25% kaempferol glucuronide solution was added dropwise to the fermented bee bread of Example 1, and the mixture was stirred evenly to obtain the final artificially fermented bee bread.

[0054] Modified protease: First, 12 parts of propylsulfonamide are dispersed and dissolved in 110 parts of a 37% hydrochloric acid solution by mass, and placed in a 60°C constant temperature water bath and stirred at a constant temperature for 30 minutes at a speed of 450RPM, and NaOH solution is added dropwise to adjust the pH to 7.0; 0.3% protease Esperase 8.0L is added dropwise, and then stirred at room temperature for 2 hours at a speed of 150RPM. After the reaction is completed, the above solution is ultrafiltered for 6 cycles using an ultrafilter with a molecular weight cutoff of 50kDa. After the ultrafiltration is completed, the pH value is adjusted to 4.8 with a hydrochloric acid solution and stored at 0°C for later use.

[0055] Artificially fermented bee feed is added to the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, the royal jelly is taken out with a jelly pen, scraped into a jelly bottle, and the royal jelly is collected; 1.5 parts of water and 0.03 parts of modified protease are added to 15 parts of royal jelly, and the mixture is stirred and dispersed at a speed of 300 r / min for 10 minutes at a temperature of 42°C, and enzymolysis is performed for 3 hours; 10% ammonium sulfate solution by mass is added for salting out and alcohol precipitation, and the mixture is centrifuged at a speed of 5000 rpm for 15 minutes to remove excess liquid to obtain pure royal jelly, which is cooled to room temperature and stored at 4°C; the enzymatically hydrolyzed royal jelly and honey are mixed in a ratio of 1:10 to obtain the bee product.

[0056] Example 5

[0057] 0.5% of a 20% kaempferol glucuronide solution was added dropwise to the fermented bee bread of Example 1, and the mixture was stirred evenly to obtain the final artificially fermented bee bread.

[0058] Modified protease: First, 15 parts of propylsulfonamide are dispersed and dissolved in 100 parts of a 37% hydrochloric acid solution by mass, and placed in a 60°C constant temperature water bath and stirred at a constant temperature of 30 minutes at a speed of 500RPM, and NaOH solution is added dropwise to adjust the pH to 6.8; 0.4% protease Esperase 8.0L is added dropwise, and then stirred at room temperature for 2 hours at a speed of 100RPM. After the reaction is completed, the above solution is subjected to 6 cycles of ultrafiltration using an ultrafilter with a molecular weight cutoff of 50kDa. After the ultrafiltration is completed, the pH value is adjusted to 4.8 with a hydrochloric acid solution and stored at 0°C for later use.

[0059] Artificially fermented bee feed is added to the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, the royal jelly is taken out with a jelly pen, scraped into a jelly bottle, and the royal jelly is collected; 1 part of water and 0.03 parts of modified protease are added to 20 parts of royal jelly, and the mixture is stirred and dispersed at a speed of 300 r / min for 10 minutes, the temperature is 45°C, and enzymolysis is performed for 2 hours; 10% ammonium sulfate solution by mass is added for salting out and alcohol precipitation, and the mixture is centrifuged at a speed of 5000 rpm for 15 minutes to remove excess liquid to obtain pure royal jelly, which is cooled to room temperature and stored at 4°C; the enzymatically hydrolyzed royal jelly and honey are mixed in a ratio of 1:10 to obtain the bee product.

[0060] Example 6

[0061] 0.2% of a 30% kaempferol glucuronide solution was added dropwise to the fermented bee bread of Example 1, and the mixture was stirred evenly to obtain the final artificially fermented bee bread.

[0062] Modified protease: First, 8 parts of propylsulfonamide are dispersed and dissolved in 120 parts of a 37% hydrochloric acid solution by mass, and placed in a 60°C constant temperature water bath and stirred at a constant temperature for 30 minutes at a speed of 400RPM, and NaOH solution is added dropwise to adjust the pH to 7.2; 0.2% protease Esperase 8.0L is added dropwise, and then stirred at room temperature for 2 hours at a speed of 200RPM. After the reaction is completed, the above solution is subjected to 6 cycles of ultrafiltration using an ultrafilter with a molecular weight cutoff of 50kDa. After the ultrafiltration is completed, the pH value is adjusted to 4.8 with a hydrochloric acid solution and stored at 0°C for later use.

[0063] Artificially fermented bee feed is added to the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, the royal jelly is taken out with a jelly pen, scraped into a jelly bottle, and the royal jelly is collected; 2 parts of water and 0.03 parts of modified protease are added to 10 parts of royal jelly, and the mixture is stirred and dispersed at a speed of 300r / min for 10 minutes at a temperature of 40°C, and enzymolysis is performed for 4 hours; 10% ammonium sulfate solution by mass is added for salting out and alcohol precipitation, and the mixture is centrifuged at a speed of 5000rpm for 15 minutes to remove excess liquid to obtain pure royal jelly, which is cooled to room temperature and stored at 4°C; the enzymatically hydrolyzed royal jelly and honey are mixed in a ratio of 1:10 to obtain the bee product.

[0064] Comparative Example 6

[0065] In this comparative example, no kaempferol glucuronide solution was added to the bee bread, and the remaining implementation methods were the same as those in Example 4.

[0066] Comparative Example 7

[0067] In this comparative example, the kaempferol glucuronide solution is a quercetin solution, and the rest of the implementation manner is the same as Example 4.

[0068] Comparative Example 8

[0069] In this comparative example, the amount of kaempferol glucuronide solution added is different, specifically: 0.4% of a 40% kaempferol glucuronide solution by mass is added to the fermented bee bread in the above Example 1, and the mixture is stirred evenly to obtain the final artificially fermented bee bread; the rest of the implementation methods are the same as those in Example 4.

[0070] Comparative Example 9

[0071] In this comparative example, the amount of kaempferol glucuronide solution added is different, specifically: 0.4% of a 10% kaempferol glucuronide solution by mass is added to the fermented bee bread in the above Example 1, and the mixture is stirred evenly to obtain the final artificially fermented bee bread; the rest of the implementation methods are the same as those in Example 4.

[0072] Comparative Example 10

[0073] This comparative example does not modify the protease, and the specific process is:

[0074] 0.4% of a 25% kaempferol glucuronide solution was added dropwise to the fermented bee bread of Example 1, and the mixture was stirred evenly to obtain the final artificially fermented bee bread.

[0075] Artificially fermented bee feed is added to the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, the royal jelly is taken out with a jelly pen, scraped into a jelly bottle, and the royal jelly is collected; 1.5 parts of water and 0.03 parts of protease Esperase 8.0L are added to 15 parts of royal jelly, and the mixture is stirred and dispersed at a speed of 300r / min for 10 minutes at a temperature of 42°C, and enzymolysis is performed for 3 hours; a 10% ammonium sulfate solution by mass is added for salting out and alcohol precipitation, and the mixture is centrifuged at a speed of 5000rpm for 15 minutes to remove excess liquid to obtain pure royal jelly, which is cooled to room temperature and stored at 4°C; the enzymatically hydrolyzed royal jelly and honey are mixed in a ratio of 1:10 to obtain the bee product.

[0076] Comparative Example 11

[0077] In this comparative example, propylsulfonamide is ethylenediamine, and the rest is the same as in Example 4.

[0078] Comparative Example 12

[0079] The amount of propylsulfonamide added in this comparative example is different. The specific modified protease is: first, 20 parts of propylsulfonamide are dispersed and dissolved in 110 parts of a 37% hydrochloric acid solution by mass, and placed in a constant temperature water bath at 60°C and stirred at constant temperature for 30 minutes at a speed of 450RPM, and a NaOH solution is added dropwise to adjust the pH to 7.0; 0.3% protease Esperase 8.0L is added dropwise, and then stirred at room temperature for 2 hours at a speed of 150RPM. After the reaction is completed, the above solution is subjected to 6 cycles of ultrafiltration using an ultrafilter with a molecular weight cutoff of 50kDa. After the ultrafiltration is completed, the pH value is adjusted to 4.8 with a hydrochloric acid solution and stored at 0°C for use; the rest is the same as Example 4.

[0080] Comparative Example 13

[0081] The amount of propylsulfonamide added in this comparative example is different. The specific modified protease is: first, 5 parts of propylsulfonamide are dispersed and dissolved in 110 parts of a 37% hydrochloric acid solution by mass, and placed in a constant temperature water bath at 60°C and stirred at constant temperature for 30 minutes at a speed of 450RPM, and a NaOH solution is added dropwise to adjust the pH to 7.0; 0.3% protease Esperase 8.0L is added dropwise, and then stirred at room temperature for 2 hours at a speed of 150RPM. After the reaction is completed, the above solution is subjected to 6 cycles of ultrafiltration using an ultrafilter with a molecular weight cutoff of 50kDa. After the ultrafiltration is completed, the pH value is adjusted to 4.8 with a hydrochloric acid solution and stored at 0°C for use; the rest is the same as Example 4.

[0082] Experiment 2: Determination of pesticide residues in royal jelly

[0083] After feeding the bee feeds of Examples 4-6 and Comparative Examples 6-9, the obtained royal jelly was tested for pesticide residues, and the detection method was used to determine the pesticide residues in accordance with the national food safety standard GB23200.98-2016. The results are shown in Table 2.

[0084] Table 2

[0085]

[0086] Test 3: Determination of 10-hydroxy-2-decenoic acid content in bee products:

[0087] (1) Preparation of 10-hydroxy-2-decenoic acid reference solution: Weigh 1.000 mg of 10-hydroxy-2-decenoic acid reference solution into a 100 mL volumetric flask and dilute to volume with anhydrous ethanol to obtain a 0.01 mg / mL standard solution. Filter through a 0.45 μm microporous membrane and set aside.

[0088] (2) Preparation of 10-hydroxy-2-decenoic acid test solution: Accurately weigh 1.000 g of the bee products of Examples 4-6 and Comparative Examples 10-13, dilute to 10 mL with anhydrous ethanol, sonicate for 30 min at room temperature, remove, centrifuge at 6000 r / min for 10 min, take the supernatant, and dilute to prepare a sample solution with a mass concentration of 1 mg / mL, filter through a 0.45 μm microporous filter membrane, and set aside.

[0089] (3) Preparation of 10-HDA test solution: Nucleosil C 18 (4.6mm×250mm, 5μm) chromatographic column, column temperature of 30°C, acetonitrile-water (volume ratio of 27:73) as the mobile phase, isocratic elution, flow rate of 1.0mL / min, injection volume of 20μL, detection wavelength of 210nm.

[0090] The bee products prepared in Examples 4-6 and Comparative Examples 10-13 were respectively taken, accurately weighed, and the contents were measured and calculated. The results are shown in Table 3 below.

[0091] Table 310-Hydroxy-2-decenoic acid content determination results

[0092] Sample Group 10-Hydroxy-2-decenoic acid content (%) Example 4 0.1634 Example 5 0.1586 Example 6 0.1594 Comparative Example 10 0.1274 Comparative Example 11 0.1338 Comparative Example 12 0.1504 Comparative Example 13 0.1437

Claims

1. A process for preparing royal jelly fermented bee products, characterized in that: Artificially fermented bee food is added to the outside of the beehive partition, the jelly production temperature is above 15°C, there is no continuous cold wave, the royal jelly is taken out with a jelly pen, scraped into a jelly bottle, and the royal jelly is collected; 1-2 parts of water and 0.03 parts of modified protease are added to 10-20 parts of royal jelly, and the mixture is stirred and dispersed at a speed of 300r / min for 10 minutes at a temperature of 40-45°C, and enzymolysis is performed for 2-4 hours; 10% ammonium sulfate solution by mass is added for salting out and alcohol precipitation, and the mixture is centrifuged at a speed of 5000rpm for 15 minutes to remove excess liquid to obtain pure royal jelly, which is cooled to room temperature and stored at 4°C; the enzymatically hydrolyzed royal jelly and honey are mixed in a ratio of 1:10 to obtain the bee product.

2. The process for preparing royal jelly fermented bee products according to claim 1, characterized in that: The artificial fermentation of bee bread comprises: adding 0.1-0.3% of lactic acid bacteria strains into a culture dish containing 50 parts of sterile water, adding 10 parts of brown sugar and culturing at 37°C for 12 hours to obtain lactic acid bacteria culture solution, weighing 500 parts of pollen, grinding and sieving, pouring into a fermentation tank, adding sterile water with a solid-liquid ratio of 13:100, fully mixing, and then high-pressure sterilization for 30 minutes; after cooling, accessing 10% of lactic acid bacteria culture solution, adding 1-3% of fermentation promoter, setting the temperature of the fermentation tank to 33°C, the stirring speed to 445RPM, stopping the fermentation when the pH change on the display screen of the fermentation tank is <0.02 for 2 consecutive days, and after the fermentation is completed, centrifuging and drying the fermented product to obtain the bee bread.

3. The process for preparing royal jelly fermented bee products according to claim 2, characterized in that: The fermentation promoting substance is sucrose dodecanoate.

4. The process for preparing royal jelly fermented bee products according to claim 2, characterized in that: The artificially fermented bee bread also includes mixing, stirring and dispersing the dried bee bread with 20-30% by mass of a tolerance solution in a ratio of 1:0.002-0.

005.

5. The process for preparing royal jelly fermented bee products according to claim 4, characterized in that: The tolerable substance is kaempferol glucuronide solution.

6. The process for preparing royal jelly fermented bee products according to claim 1, characterized in that: The modified protease comprises: firstly dispersing and dissolving 8-15 parts of a modifier in 100-120 parts of a hydrochloric acid solution with a mass fraction of 37%, placing the solution in a 60°C constant temperature water bath and stirring the solution at a constant temperature of 30 minutes at a rotation speed of 400-500RPM, and dropping a NaOH solution to adjust the pH to 6.8-7.2; dropping 0.2-0.4% of Esperase 8.0L, stirring the solution at room temperature for 2 hours at a rotation speed of 100-200RPM, and after the reaction is completed, performing 6 cycles of ultrafiltration on the solution using an ultrafilter with a molecular weight cutoff of 50 kDa, and after the ultrafiltration is completed, adjusting the pH to 4.8 using a hydrochloric acid solution, and storing the solution at 0°C for later use.

7. The process for preparing royal jelly fermented bee products according to claim 6, characterized in that: The modifier is propylsulfonamide.

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