Method for increasing content of 10-hydroxy-2-decenoic acid in royal jelly and application thereof

Abstract

The invention provides a method for increasing the content of 10-hydroxy-2-decenoic acid (10-HDA) in royal jelly, the content of 10-HDA in royal jelly can be increased by adding a stearic acid and reduced coenzyme II tetrasodium (NADPH) composition in a certain proportion into pollen feed for continuous feeding, and the method has the characteristics of simplicity and convenience in operation, short period, good effect and the like, and is suitable for large-scale production. And a new thought can be provided for high-quality royal jelly production.

Description

Technical Field

[0001] The invention belongs to the field of bee nutrition, and in particular relates to a method for increasing the content of 10-hydroxy-2-decenoic acid (10-HDA) in royal jelly. The method aims to improve the nutritional value and biological activity of royal jelly by adding specific substances. Background Art

[0002] Royal jelly is a milky white or pale yellow, creamy substance secreted by the mandibular and hypopharyngeal glands of worker bees. It is the primary food source for honeybee larvae and queens. It contains a rich variety of nutrients, including proteins, carbohydrates, lipids, vitamins, minerals, enzymes, and other bioactive substances. Royal jelly is reported to possess antibacterial, antioxidant, anti-aging, anti-inflammatory, and anti-tumor properties.

[0003] 10-Hydroxy-2-decenoic acid (10-HDA) is an unsaturated fatty acid unique to royal jelly, also known as royal jelly acid. 10-HDA is a medium-chain fatty acid with antioxidant, anti-tumor, and antibacterial properties. The 10-HDA content is a key indicator for measuring royal jelly quality and distinguishing its authenticity, and has become a core evaluation indicator in the national standard for royal jelly (GB 9697-2008). The national standard (GB 9697-2008) sets clear quality grading requirements for the 10-hydroxy-2-decenoic acid (10-HDA) content in royal jelly. Premium royal jelly must contain 10-HDA content of ≥1.8%, while qualified royal jelly must contain a minimum of ≥1.4%. The market price difference between premium and qualified royal jelly is significant, making increasing the 10-HDA content in royal jelly extremely economically valuable.

[0004] Studies have shown that the biosynthesis of 10-HDA begins with the synthesis of fatty acids ( Figure 1). First, under the catalysis of fatty acid synthase (FAS), acetyl-coenzyme A (Acetyl-CoA) and malonyl-CoA serve as starting substrates, and after condensation, reduction, dehydration, and re-reduction, a long-chain saturated fatty acid, stearic acid (C18), is generated. Since the molecular structure of 10-hydroxy-2-decenoic acid (10-HDA) contains a 10-carbon fatty acid chain, stearic acid needs to gradually shorten its carbon chain through the fatty acid β-oxidation pathway. The β-oxidation pathway is a highly conserved and efficient metabolic process. Its main steps include continuous dehydrogenation, hydration, re-dehydrogenation, and sulfhydration reactions, gradually removing carbon atom units from stearic acid until the carbon chain length is shortened to 10 carbon atoms. Furthermore, under the catalysis of cytochrome P450 monooxygenase, the terminal carbon (ω position) of the 10-carbon chain intermediate metabolite undergoes hydroxylation. In this step, the cytochrome P450 enzyme system, through its unique catalytic mechanism, precisely introduces the hydroxyl group into the 10th position of the carbon chain, thereby forming the biologically active 10-HDA.

[0005] Hu Fuliang et al. investigated the biochemical synthesis pathway of 10-HDA by adding stearic acid to bee feed and found that adding 1%, 5%, and 8% stearic acid significantly increased the 10-HDA content in royal jelly. However, adding stearic acid only increases the amount of stearic acid stored in the bees, providing a substrate for 10-HDA biosynthesis. However, after reaching a certain concentration, it is difficult to further increase the 10-HDA content in royal jelly. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for increasing the 10-HDA content in royal jelly in response to the problem that the content of 10-HDA in royal jelly is difficult to further increase in the prior art.

[0007] The present invention has discovered that by simultaneously adding stearic acid and tetrasodium coenzyme II (NADPH) to the feed, stearic acid provides sufficient substrate for the biosynthesis of 10-HDA. Furthermore, NADPH regulates lipid metabolism pathways, efficiently forming hydroxyl groups and significantly increasing the 10-HDA content in royal jelly. Based on this, the present invention proposes the following solution:

[0008] The first object of the present invention is to provide an application of stearic acid and NADPH in increasing the 10-hydroxy-2-decenoic acid content of royal jelly produced by honeybees.

[0009] The second object of the present invention is to provide the use of stearic acid and NADPH in the preparation of pollen feed additives.

[0010] The third object of the present invention is to provide a bee feed additive comprising an effective amount of stearic acid and NADPH.

[0011] Preferably, the weight ratio of stearic acid to NADPH in the composition is 40,000-120,000:1-5, preferably 80,000:2.5.

[0012] Specifically, in one embodiment of the present invention, the stearic acid addition level in the stearic acid and NADPH composition is 80 g / kg of feed, and the NADPH addition level is 2.5 mg / kg of feed.

[0013] The basic feed for bees can be a conventional feed for producing royal jelly, such as a feed containing pollen and honey. In one embodiment of the present invention, the basic feed contains or consists of air-dried rapeseed pollen and honey, preferably mixed in a weight ratio of 1:1.

[0014] The fourth object of the present invention is to provide the bee feed additive or the use of the feed additive in increasing the content of 10-hydroxy-2-decenoic acid in royal jelly produced by bees.

[0015] The last object of the present invention is to provide a method for increasing the content of 10-hydroxy-2-decenoic acid in royal jelly, which is achieved by the following steps: feeding bees with the above-mentioned bee feed containing stearic acid and NADPH composition; preferably, feeding for 9 days.

[0016] The present invention effectively increases the content of 10-HDA in royal jelly by simultaneously adding stearic acid and NADPH to the feed, thereby improving the quality of royal jelly and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the biochemical synthesis pathway of 10-HDA in Italian worker bees.

[0018] Figure 2 Figure 3. Effects of exogenous addition of stearic acid and NADPH combination on the expression of genes related to 10-HDA synthesis in the mandibular gland of Italian worker bees. Figure A shows the relative expression of the fatty acid synthase (FASN) gene in the mandibular gland of worker bees; Figure B shows the relative expression of the acyl-CoA oxidase 1 (ACOX1) gene in the mandibular gland of worker bees; Figure C shows the relative expression of the cytochrome P450 family member (CYP6AQ1) gene in the mandibular gland of worker bees; and Figure D shows the relative expression of the cytochrome P450 family member (CYP6AS5) gene in the mandibular gland of worker bees. DETAILED DESCRIPTION

[0019] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0020] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0021] Example 1 Bee Feed

[0022] The bee feed of the present invention comprises a basic feed and an additive, wherein:

[0023] Basic feed: air-dried rapeseed pollen and honey, mixed in a weight ratio of 1:1

[0024] Additives: Stearic acid and NADPH composition

[0025] Bee feed was prepared by mixing the feed additive with the base feed at a ratio of 8% by weight. (Because the weight percentage of NADPH can vary by several orders of magnitude, NADPH was ignored when calculating the weight ratio of the pollen feed additive to the bee feed, the same applies below.)

[0026] Example 2 Grouping and feeding of bee colonies

[0027] The present invention randomly divides the raised Italian honey bee colonies into four groups. The first group is fed with a single pollen diet, as a control group (3 groups per group); the second group adds 80g / kg of stearic acid to the pollen diet, as a stearic acid experimental group (3 groups per group); the third group adds 2.5mg / kg of NADPH coenzyme to the pollen diet, as a NADPH experimental group (3 groups per group); the fourth group adds 80g / kg of stearic acid and 2.5mg / kg of NADPH coenzyme to the pollen diet, as a stearic acid-NADPH composition experimental group (3 groups per group). The experimental time of the control group and the experimental group is 14 days (to ensure the scientificity and controllability of the experimental conditions, all experimental group bees are given priority depollening treatment 5 days before the start of the experiment, pollen supply is stopped and no other nutrients are provided. On this basis, pollen feeding and depollening treatment are carried out synchronously within 5 days after the start of the experiment). During the depollination period, each bee colony was equipped with a depollinator. After installation, both the control and experimental groups underwent depollination for 5 days to prevent interference from exogenous pollen during the experimental period. Air-dried rapeseed pollen was ground into a powder using a grinder. This powder was then mixed with honey in a specific ratio (1:1) and kneaded into a pelleted diet. This pelleted diet was then placed on the queen excluder for worker bees to consume. During the feeding process, the daily feed intake of the worker bees was accurately measured using an electronic balance, and the average feed intake was calculated (Table 1). Specific procedures: When the beehive was opened daily, the total weight of the pollen pellets and the attached coverslips was weighed using an electronic balance. This was used to calculate the actual feed intake of the worker bees, thus ensuring data accuracy. The pollen pellets were replaced every three days (500g / 3d). During this period, the supply was dynamically adjusted based on the remaining pollen to optimize feeding conditions.

[0028] Table 1 Average feed intake of experimental bee colonies

[0029]

[0030] Note: Different letters in the same column indicate significant differences (P<0.05), while the same letters indicate no significant differences (P>0.05), the same below.

[0031] As shown in Table 1, the combination of stearic acid and NADPH in the feed can significantly increase the average feed intake of the bee colony compared with the group only adding stearic acid or the group only adding NADPH (P<0.05); but compared with the control group, the combined addition of stearic acid and NADPH had no significant effect on the average feed intake of the bee colony.

[0032] Example 3 Detection of Royal Jelly Yield

[0033] After 3 to 4 days of the test period, a double row of pulp strips was used to make a queen bee base. Each base was coated with a small amount of royal jelly for moistening before use. At the beginning of the experiment, one-day-old larvae were transplanted into the base, and the pulp frame was placed in the super box for feeding. After 72 hours of cultivation, the royal jelly was removed from the base using the clamp worm method, and collected in a 10mL centrifuge tube for weighing, and the weighing results were recorded (Table 2). Subsequently, the collected royal jelly was transferred to a -20°C refrigerator for storage for subsequent detection of the 10-HDA content in the royal jelly.

[0034] Table 2 Comparison of royal jelly production produced by experimental bee colonies

[0035]

[0036] The royal jelly samples from all groups were thick and milky in appearance, with a slightly yellowish color. As shown in Table 2, the total amount of royal jelly produced by the four groups was approximately 250g, and there was no significant difference in royal jelly production among the four groups.

[0037] Example 4 Detection of 10-HDA Content in Royal Jelly

[0038] The 10-HDA content in royal jelly was determined by liquid chromatography. The specific procedure was as follows: After thawing the royal jelly sample at room temperature, 0.5 g of the mixed sample was weighed and placed in a small beaker. 1 mL of hydrochloric acid (0.03 mol / L) and 2 mL of double-distilled water were added and shaken to mix thoroughly. Then, 10 mL of internal standard solution (methyl parahydroxybenzoate solution) was added and the volume was adjusted to 50 mL with anhydrous ethanol. After shaking, the sample was ultrasonicated for 15 minutes and then centrifuged for 10 minutes at 3000 rpm. The supernatant was then placed on a LabAlliance liquid chromatograph for analysis. Chromatographic conditions: Avantor column; diode array detector; detection wavelength 210 nm; mobile phase methanol + 0.2% phosphoric acid solution (methanol:0.2% phosphoric acid solution = 55:45); flow rate 1 mL / min; column temperature 25°C. The test results are shown in Table 3.

[0039] Table 3 10-HDA content in royal jelly (%)

[0040]

[0041] As shown in Table 3, the 10-HDA content in the stearic acid + NADPH combination treatment group was significantly higher than that in the control group, the stearic acid alone group, and the NADPH alone group (P < 0.05). In contrast, the 10-HDA content in royal jelly of bee colonies supplemented with either stearic acid or NADPH alone was not significantly different from that in the control group (P > 0.05). These results indicate that the simultaneous addition of stearic acid and NADPH to the diet is more effective in increasing the 10-HDA content in royal jelly than the stearic acid or NADPH alone groups.

[0042] Example 5: Expression of genes related to 10-HDA synthesis in the mandibular gland of worker bees

[0043] 1. Bee Sample Grouping and Processing

[0044] After nine days of the experimental period, the mandibular glands of honeybees from the experimental colonies were collected from the apiary for quantitative analysis. The procedure was as follows: After live bees were collected, they were immediately placed in a -80°C ultra-low temperature freezer to maintain tissue integrity and metabolic stability. Before sampling, dissecting forceps and insect pins were rigorously disinfected with alcohol and wiped with sterile water to remove residual alcohol, ensuring a sterile environment. During sample processing, frozen bee samples were removed from centrifuge tubes and fixed to wax trays using insect pins. Under a microscope, dissecting forceps were used to precisely incise the epidermal tissue between the two pairs of compound eyes to expose the mandibular glands. The mandibular tissue containing the mandibular glands was then carefully dissected. The dissected mandibular tissue was placed on a pre-prepared glass slide, and 20 μL of filter-sterilized phosphate-buffered saline (PBS) was added to maintain tissue moisture and biological activity. Subsequently, the mandibular tissue was fixed with dissecting forceps, and the junction between the mandibular gland and the palate was precisely separated to obtain the intact mandibular gland. Ten bees were collected from each group, totaling 20 mandibular gland samples; three biological replicates were set for each treatment group.

[0045] 2. Traditional method of total RNA extraction

[0046] (1) The centrifuge tubes, pipette tips, and steel balls used in the extraction process must be sterilized under high pressure (120°C, 40 min) and dried before use.

[0047] (2) Place the maxillary gland tissue in a 1.5 ml centrifuge tube, add 0.5 ml RNAex (AG Aikerui), add small steel balls, and break the homogenate on a tissue homogenizer (180 s, 60 Hz). Let it stand at room temperature for 5 minutes to facilitate complete tissue lysis.

[0048] (3) Add chloroform (1 / 5 the volume of RNAex) to the lysate and mix thoroughly. After standing at room temperature for 5 minutes, centrifuge at 12,000 g for 15 minutes at 4°C.

[0049] (4) Carefully remove the centrifuge tube. The homogenate will now be separated into three layers: the supernatant (containing RNA), the intermediate protein layer, and the lower organic phase. Transfer the supernatant to another new centrifuge tube (do not remove the intermediate protein layer).

[0050] (5) Add 1 / 2 the volume of RNAex isopropanol to the supernatant, mix thoroughly, and let stand at room temperature for 10 minutes.

[0051] (6) Centrifuge at 12,000 g for 10 min at 4°C. Discard the supernatant, taking care not to touch the RNA pellet.

[0052] (7) Add an equal volume of 75% ethanol (750 ml ethanol + 250 ml DEPC water) to the centrifuge tube to wash the RNA precipitate and the tube wall. Centrifuge at 7,500 g for 5 min at 4°C. Carefully discard the supernatant without touching the precipitate.

[0053] (8) Add 50 μL of RNase-free water to the centrifuge tube to dissolve the RNA. Store the dissolved RNA at -80°C.

[0054] 3. Reverse Transcription Reaction

[0055] According to the instructions of the AG Acryl Reverse Transcription Kit, gDNA is removed. The gDNA reaction system is as follows:

[0056] Table 4 gDNA reaction system

[0057]

[0058] After centrifugation, the mixture was placed in a PCR instrument. The reaction conditions were as shown in Table 5.

[0059] Table 5 PCR reaction conditions

[0060]

[0061] 4. Real-time Fluorescence Quantitative PCR

[0062] The primer sequences are shown in Table 6, and the reaction system is shown in Table 7.

[0063] Table 6 Primer information

[0064]

[0065] Table 7 qPCR reaction system

[0066]

[0067] Reaction conditions: first step at 95°C for 30 s; second step at 95°C for 5 s; third step at 60°C for 30 s; 40 cycles of the second and third steps.

[0068] The experimental results are as follows Figure 2 shown

[0069] Compared with the control group, the combined addition of stearic acid and NADPH to the diet significantly increased the relative expression levels of fatty acid synthase (FASN), acyl-CoA oxidase 1 (ACOX1), and cytochrome P450 family members (CYP6AQ1 and CYP6AS5) genes in the mandibular gland of worker bees.

[0070] In summary, the combined exogenous addition of stearic acid and NADPH can significantly enhance the expression of genes related to 10-HDA synthesis in the mandibular gland of worker bees, thereby promoting the secretion of 10-HDA and ultimately leading to an increase in the 10-HDA content in royal jelly.

[0071] Those skilled in the art will appreciate that, although limited by space, the present invention utilizes 80 g / kg of stearic acid and 2.5 mg / kg of NADPH coenzyme added to honeybee feed for experiments, the present invention fully demonstrates the role of NADPH in promoting the efficient conversion of stearic acid to 10-HDA. Those skilled in the art will foresee that the presence of NADPH and stearic acid will enhance 10-HDA synthesis. The specific amounts of stearic acid and NADPH added can be appropriately adjusted by those skilled in the art based on the ranges and research methods disclosed herein.

Claims

1. Application of a stearic acid and NADPH composition in increasing the 10-hydroxy-2-decenoic acid content of royal jelly produced by honeybees.

2. Application of stearic acid and NADPH composition in the preparation of bee feed additives.

3. A bee feed additive comprising an effective amount of stearic acid and NADPH.

4. The feed additive according to claim 3, wherein The weight ratio of stearic acid to NADPH in the composition is 40,000-120,000:1-5, preferably 80,000:2.

5.

5. A bee feed containing the feed additive according to claims 3 to 5.

6. The feed according to claim 5, characterized in that The basic ingredients of the feed contain air-dried rapeseed pollen and honey in a weight ratio of 1:

1.

7. Use of the feed additive according to claims 3-5 in increasing the 10-hydroxy-2-decenoic acid content of royal jelly produced by honeybees.

8. A method for increasing the content of 10-hydroxy-2-decenoic acid in royal jelly, which is achieved by the following steps: feeding honeybees with the feed additive according to claim 3 or 4 or the bee feed according to claim 5 or 6.

9. The method according to claim 8, wherein The stearic acid addition level was 80 g / kg of feed, and the NADPH addition level was 2.5 mg / kg of feed.

10. The method according to claim 8, wherein The bees are fed for 9 days.

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