A method for evaluating quality of royal jelly based on bacteriostatic activity

Abstract

The application discloses a royal jelly quality evaluation method based on bacteriostatic activity, and belongs to the technical field of bioengineering and food quality evaluation. The method evaluates the quality of royal jelly by determining the bacteriostatic activity of the royal jelly. Specifically, the royal jelly to be measured is diluted to 10% volume concentration, then the bacteriostatic activity of the diluted royal jelly on indicator bacteria is determined by adopting a filter paper method, and the quality grade of the royal jelly to be measured is determined according to the bacteriostatic ring diameter. The quality of the royal jelly is evaluated by determining the bacteriostatic activity of the royal jelly, the influence of water content, 10-HDA and freshness on the quality of the royal jelly is considered in principle, the method has the characteristics of simple operation and intuitive evaluation, and is easy to popularize and apply, thereby laying an important foundation for the quality detection technology of royal jelly.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering technology and food quality evaluation technology, and in particular to a method for evaluating the quality of royal jelly based on antibacterial activity. Background Technology

[0002] Royal jelly, also known as bee jelly, is a milky white or pale yellow jelly-like substance secreted by the well-developed hypopharyngeal and mandibular glands of worker bees. Royal jelly has a pH of 3.6–4.2 and contains 60–70% water, 11–23% sugars, 9–18% protein, 4–8% esters, and trace amounts of vitamins and minerals. It possesses various physiological functions, including anti-aging, antibacterial, anti-fatigue, anti-inflammatory, antioxidant, anti-tumor, anti-diabetic, and anti-mutagenic effects.

[0003] The national standard GB 9697-2008 for royal jelly classifies it into superior and qualified grades based on the content of moisture and 10-hydroxy-2-decenoic acid (10-HDA). Superior grade royal jelly has a moisture content ≤ 67.5 g / 100g and a 10-HDA content ≥ 1.8 g / 100g; qualified grade royal jelly has a moisture content ≤ 69.0 g / 100g and a 10-HDA content ≥ 1.4 g / 100g. However, in practical applications, it has been found that evaluating royal jelly quality solely based on these two indicators is biased. For example, moisture content is easily controlled artificially, while 10-HDA is relatively stable and shows almost no change even under unsuitable storage conditions. In fact, it has been found that the physiological activity of fresh royal jelly is much higher than that of stored royal jelly, and the longer the storage time, the lower the physiological activity. This clearly demonstrates that royal jelly is highly perishable, and its quality is easily affected by storage conditions. Improper storage can significantly alter the physical properties, chemical composition, and physiological efficacy of royal jelly. Current national standards are ineffective in evaluating royal jelly quality, necessitating a simple and easily applicable method for its evaluation.

[0004] To effectively evaluate the quality of royal jelly and establish grading and pricing standards, as well as storage methods for sales, distribution, and consumption, researchers have conducted numerous experiments to identify valuable quality evaluation indicators. Considering the close relationship between the "freshness" of royal jelly and its quality, researchers screened various potential "freshness" evaluation indicators, attempting to combine "freshness" indicators with national standard physicochemical indicators to jointly evaluate royal jelly quality. Reported "freshness" indicators include enzymes (superoxide dismutase, glucose oxidase), color, furosine, furfural, free amino acids, 57KU protein, and ATP and its degradation products. However, due to various constraints, no single "freshness" evaluation indicator for royal jelly is currently universally recognized.

[0005] The quality of royal jelly is closely related to its physical properties, chemical composition, and physiological effects, making it difficult to find a single evaluation index. At the same time, there are currently no reports on evaluating the quality of royal jelly based on its antibacterial activity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for evaluating the quality of royal jelly based on antibacterial activity, in order to solve the problems existing in the prior art. This method takes into account the influence of moisture, 10-HDA and freshness on the quality of royal jelly in principle, and has the characteristics of simple operation and intuitive evaluation, which makes it easy to promote and apply, and lays an important foundation for the quality detection technology of royal jelly.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a method for evaluating the quality of royal jelly by measuring its antibacterial activity.

[0009] Furthermore, the royal jelly to be tested was first diluted to a volume concentration of 10%, and then the antibacterial activity of the diluted royal jelly against indicator bacteria was determined by the filter paper disc method. The quality grade of the royal jelly to be tested was determined based on the diameter of the inhibition zone.

[0010] Furthermore, the indicator bacterium is *Cytobacillus oceanisediminis* CO29, classified as *Cytobacillus oceanisediminis*, with accession number CGMCC NO.22874. This strain has been deposited at the China Center for Microbial Culture Collection, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 13, 2021.

[0011] Furthermore, the quality grade of the royal jelly to be tested includes two grades: qualified and unqualified.

[0012] If the diameter of the inhibition zone is greater than or equal to 7.2 mm, the royal jelly to be tested is qualified; if the diameter of the inhibition zone is less than 7.2 mm, the royal jelly to be tested is unqualified.

[0013] Furthermore, the filter paper method is as follows: after the filter paper is soaked in diluted royal jelly, it is placed in an indicator bacteria plate and incubated at a constant temperature of 37°C for 18-20 hours. Then, the diameter of the inhibition zone of the indicator bacteria is measured to evaluate the quality grade of the royal jelly.

[0014] This invention also provides the application of a strain in evaluating the quality of royal jelly, comprising the following steps:

[0015] (1) Culture of the strain: Add the strain to the liquid culture medium and culture it until it enters the logarithmic phase. Then, spread the bacterial solution on a solid culture medium plate to obtain the indicator bacteria plate.

[0016] (2) First, soak the filter paper in the royal jelly to be tested diluted to a volume concentration of 10%, then place it in an indicator bacteria plate and incubate at a constant temperature of 37°C for 18-20 hours. Then measure the diameter of the inhibition zone of the indicator bacteria and judge the quality grade of the royal jelly to be tested based on the results.

[0017] The strain is *Cytobacillus oceanisediminis* CO29, classified as *Cytobacillus oceanisediminis*, with accession number CGMCC NO.22874. This strain has been deposited at the China Center for Microbial Culture Collection, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 13, 2021.

[0018] The present invention discloses the following technical effects:

[0019] This invention screened nine strains, including Gram-positive bacteria, Gram-negative bacteria, yeast, and filamentous fungi, and found that *Bacillus subtilis*, a marine sediment bacterium, is highly sensitive to royal jelly, easy to cultivate, grows rapidly, and is non-toxic and harmless. It can be used as an indicator strain for evaluating the antibacterial activity of royal jelly. Furthermore, it was determined that using a 10% aqueous solution of royal jelly as the test sample can improve experimental repeatability and reduce error. Subsequent experiments on the antibacterial activity of royal jelly under different storage conditions and on the changes in antibacterial activity of royal jelly from different origins or nectar sources clarified that the standard for a qualified royal jelly product is an inhibition zone greater than or equal to 7.2 mm. Therefore, this invention provides a new method for evaluating the quality of royal jelly based on its antibacterial activity.

[0020] This invention evaluates the quality of royal jelly by measuring its antibacterial activity. In principle, it takes into account the effects of moisture, 10-HDA, and freshness on the quality of royal jelly. It is also characterized by simple operation and intuitive evaluation, making it easy to promote and apply, and laying an important foundation for the quality testing technology of royal jelly. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results show the inhibitory effects of royal jelly on various tested bacteria;

[0023] Figure 2 To determine the antibacterial effect of royal jelly under different storage conditions: A: -18℃, B: 4℃, C: 25℃, D: room temperature, not protected from light, E: 37℃;

[0024] Figure 3 The antibacterial effect of different royal jelly samples after storage at 4℃ in the dark for 60 days was investigated. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the reagents used are commercially available reagents.

[0031] The marine sediment Bacillus used in the following examples is the marine sediment Bacillus CO29 disclosed in the inventor's previous patent application (CN113621541A), classified as Cytobacillus Oceanisediminis, with accession number CGMCC NO.22874. This strain has been deposited at the China Culture Collection Center for Microbial Cultures, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 13, 2021. The other strains are commercially available.

[0032] Antibacterial activity is one of the important physiological activities of royal jelly and also the easiest to measure. Reported antibacterial active substances in royal jelly include 10-HDA, various proteins, and polypeptides. 10-HDA is a quality differentiation indicator in the national standard for royal jelly, but it cannot reflect the freshness of the royal jelly. Moisture content is also a quality differentiation indicator in the national standard for royal jelly; the moisture content increases with prolonged storage, determining the mass of antibacterial substances per unit weight of royal jelly. Changes in the content of protein and polypeptide antibacterial active substances can, to some extent, reflect the freshness of royal jelly. Therefore, antibacterial activity is a comprehensive quality evaluation indicator for royal jelly that takes into account 10-HDA, moisture content, and protein and polypeptide antibacterial active substances. Based on this, the inventors conducted research on evaluating royal jelly quality based on antibacterial activity.

[0033] Example 1

[0034] Screening of indicator bacteria

[0035] The key to antibacterial experiments lies in the selection of indicator bacteria. Good indicator bacteria should be highly sensitive to royal jelly, easy to culture, grow rapidly, and be non-toxic and harmless. Therefore, we selected the following strains for antibacterial experiments to identify suitable indicator strains for royal jelly antibacterial activity: Gram-positive bacteria: *Cytobacillus oceanisediminiss*, *Bacillus subtilis*, *Staphylococcus vitulinus*; Gram-negative bacteria: *Escherichia coli*, *Acinetobacter pittii*, *Chryseobacterium indologenes*; yeast: *Yarrowia lipolytica*; filamentous fungi: *Aspergillus niger*, *Aspergillus oryzae*.

[0036] Culture medium:

[0037] Bacteria: Beef extract peptone medium: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, natural pH. If preparing a solid medium, add 2% agar. Autoclave at 121°C for 20 min.

[0038] Yeast: YEPD medium: yeast extract 10 g / L, glucose 20 g / L, tryptone 20 g / L, pH adjusted to 6.0 with H2SO4. If preparing a solid medium, add 2% agar. Sterilize the prepared medium by steaming at 115°C for 20 min.

[0039] Filamentous fungi: PDB medium: 200 g / L potato, 20 g / L glucose, natural pH. To prepare a solid medium, add 2% agar to the PDB medium to obtain PDA medium. Autoclave the prepared medium at 121°C for 20 minutes.

[0040] Indicator culture plate preparation: Appropriate types of liquid culture media are used for the activation of corresponding strains, while solid culture media are used for the preparation of indicator culture plates. For bacteria and yeasts, after activation, the strains are cultured to the logarithmic growth phase, shaken well, and 100 μL of the bacterial suspension is evenly spread onto the surface of a solid culture plate. For filamentous fungi, after activation, the strains are cultured for 3 days, and 100 μL (1×10⁻⁶) of the suspension is taken and spread evenly onto the surface of a solid culture plate. 6 A conidial suspension (number of spores per mL) was evenly spread on the surface of a solid culture medium plate.

[0041] Preparation of filter paper discs: Ordinary laboratory filter paper was punched into discs with a diameter of 6 mm using a puncher, placed in a test tube, sealed, and sterilized by autoclaving at 121°C for 20 min.

[0042] Antibacterial test: Sterile filter paper was picked up with sterile forceps and immersed in a 90% aqueous solution of fresh royal jelly (sample A). Upon removal, the filter paper was held briefly at the edge of the container to remove excess solution. The filter paper was then placed on an indicator bacterial plate and pressed firmly with forceps to ensure close contact with the culture medium. A sterile water-soaked filter paper was used as a blank control. Bacteria were incubated at 37℃ for 18–20 h, yeast at 30℃ for 24 h, and filamentous fungi at 30℃ for 72 h. The diameter of the inhibition zone around the indicator bacterial colonies was observed and measured (five replicates were performed for each royal jelly sample; the highest and lowest values ​​were discarded, and the average value was taken as the measured value) to evaluate the in vitro antibacterial activity of royal jelly. Results are shown below. Figure 1 .

[0043] Depend on Figure 1It was found that fresh royal jelly (sample A) had varying degrees of inhibitory effect on bacteria, but no inhibitory effect on *Yersinia lipolyticis*, *Aspergillus oryzae*, and *Aspergillus niger*. Fresh royal jelly showed stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The sensitivity of various bacterial strains to royal jelly was ranked as follows: *Bacillus subtilis* > *Bacillus subtilis* > *Staphylococcus aureus* > *Acinetobacter p. p.* > *Escherichia coli* > *Flavobacterium indosum*. Considering the high sensitivity of *Bacillus subtilis* to royal jelly, and the fact that this strain is easy to culture, grows rapidly, and has no pathogenicity or other toxic effects, this strain was selected as an indicator bacterium to evaluate the antibacterial activity of royal jelly.

[0044] Example 2

[0045] Determining the dilution ratio of royal jelly

[0046] As shown in Table 1, when using the filter paper method to detect the antibacterial activity of royal jelly, a higher concentration (90%) of royal jelly leads to greater errors due to its higher viscosity. To improve experimental repeatability and reduce errors, we investigated the antibacterial activity of royal jelly at different dilutions against different bacteria.

[0047] Almost all experimental procedures were the same as in Example 1, except that during the antibacterial experiment, sterile filter paper discs were picked up with sterile tweezers and immersed in 10%, 30%, 50%, 70%, and 90% fresh royal jelly aqueous solutions (sample A) for subsequent filter paper disc antibacterial experiments. The results are shown in Table 1.

[0048] Table 1. Inhibitory effects of royal jelly on various test bacteria.

[0049]

[0050]

[0051] Note: "-" in the attached table indicates no antibacterial effect.

[0052] Table 1 shows that the antibacterial activity of fresh royal jelly (sample A) gradually increased with increasing concentration. A 90% concentration of royal jelly aqueous solution inhibited the growth of all six tested bacteria to varying degrees. However, when the royal jelly concentration decreased to 10%, it only inhibited the growth of *Bacillus subtilis*, while showing no significant inhibitory effect on the other strains. This indicates that *Bacillus subtilis* is highly sensitive to the antibacterial substances in royal jelly; even a 10% concentration of fresh royal jelly significantly inhibits its growth. The significantly reduced viscosity of 10% fresh royal jelly greatly improved experimental repeatability, proving that this method is entirely feasible for determining the antibacterial activity of bacterial strains.

[0053] Example 3

[0054] Changes in the antibacterial activity of royal jelly under different storage conditions

[0055] It is generally believed that the quality of royal jelly is highly correlated with its freshness, and inappropriate storage methods can lead to a significant decline in its quality. To investigate the relationship between the freshness of royal jelly and its antibacterial activity, we studied the antibacterial effect of royal jelly (sample A) on Bacillus subtilis in marine sediments after storage at -18℃, 4℃, 25℃, room temperature (without light), and 37℃ for 7, 14, 21, 28, 35, 42, and 60 days. Considering that the antibacterial activity of a 10% concentration of royal jelly aqueous solution is relatively low and may further decrease with storage, thus failing to reflect changes in freshness, this experiment also prepared royal jelly aqueous solutions of different concentrations for antibacterial studies. Other experimental procedures were basically the same as in Example 1, and the experimental results are as follows: Figure 2 As shown in AE.

[0056] Depend on Figure 2 The results from the AE analysis show that, overall, the antibacterial activity of fresh royal jelly samples (-18℃) is higher than that of samples stored at 4℃, which in turn is higher than that of samples stored at 25℃, which are stored at room temperature without light protection, which is higher than that of samples stored at 37℃. At each storage temperature, the higher the sample concentration, the stronger the antibacterial activity. For samples stored at -18℃, regardless of concentration, the antibacterial activity did not change significantly over different storage times. For samples stored at 4℃, the antibacterial activity at a 10% concentration decreased with prolonged storage time, while the antibacterial activity of other concentrations did not change significantly with storage time. This indicates that within 60 days, the antibacterial activity of royal jelly stored at 4℃ decreases, but not significantly; this decrease is only noticeable at a 10% concentration. This also demonstrates the rationality of using a 10% royal jelly concentration to study antibacterial activity, as it significantly improves the sensitivity of the method evaluation. Samples stored at 25℃, at room temperature without light protection, and at 37℃, except for the 90% concentration sample, all showed a decrease in antibacterial activity with prolonged storage time. This indicates that the antibacterial activity of royal jelly can reflect its freshness.

[0057] Marconi et al. (J. Agric. Food Chem 2002, 50, 2825-2829) believe that royal jelly can retain its nutritional components after 10 months of storage at 4℃. Wu Liming (Research on Freshness Indicators and Evaluation Methods of Royal Jelly, Zhejiang University, 2008) found that royal jelly can retain its nutritional components after 3 months of storage at 4℃. Wu et al. (LWT-Food Science and Technology 2015, 63, 504-510) believe that royal jelly can maintain its freshness after 2 months of storage at 4℃. Therefore, if we conservatively assume that royal jelly can maintain its freshness after 2 months of storage at 4℃, the diameter of the inhibition zone of a 10% concentration royal jelly solution is 7.8±0.3 mm. Therefore, we initially define products with an inhibition zone diameter greater than 7.5 mm as qualified, and those with an inhibition zone diameter less than 7.5 mm as unqualified.

[0058] Example 4

[0059] Changes in antibacterial activity of royal jelly from different origins or nectar sources stored at 4℃ for 60 days

[0060] The origin or nectar source of royal jelly can alter its composition, leading to variations in its antibacterial activity. To ensure the universality of this invention, we randomly selected seven royal jelly samples (sample information is shown in Table 2) and studied the changes in their antibacterial activity over 60 days of storage at 4°C. The results are shown in Table 2. Figure 3 .

[0061] Table 2 Information on Royal Jelly Samples

[0062]

[0063] Depend on Figure 3 It can be seen that royal jelly from different origins and nectar sources, after being diluted to a 10% concentration and stored in the dark at 4°C for 60 days, showed that the smallest inhibition zone was 7.5 mm and the largest was 8.1 mm. This indicates that the antibacterial activity of different samples varied slightly. Considering the standard deviation during the measurement and the diversity of samples, the definition of the inhibition zone of qualified products in Example 3 was conservatively revised. Products with an inhibition zone diameter greater than or equal to 7.2 mm were defined as qualified products, while products with an inhibition zone diameter less than 7.2 mm were defined as unqualified products.

[0064] Based on the above examples, we believe that royal jelly stored at -18℃ for 60 days, 4℃ for 60 days, 25℃ for 21 days, at room temperature without light for 14 days, or at 37℃ for 7 days can all be considered as qualified products; while other storage conditions will lead to the appearance of unqualified royal jelly. These results are consistent with the currently accepted practical experience.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for evaluating the quality of royal jelly, characterized in that, First, the royal jelly to be tested was diluted to a volume concentration of 10%. Then, the antibacterial activity of the diluted royal jelly against indicator bacteria was determined by the filter paper disc method. The quality grade of the royal jelly to be tested was determined based on the diameter of the inhibition zone. The indicator bacterium is *Bacillus subtilis* (a marine sediment bacterium). Cytobacillus oceanized iminiss (), its accession number is CGMCC NO.22874; The quality grades of the royal jelly to be tested include two levels: qualified and unqualified. If the diameter of the inhibition zone is greater than or equal to 7.2 mm, the royal jelly to be tested is qualified; if the diameter of the inhibition zone is less than 7.2 mm, the royal jelly to be tested is unqualified.

2. The method according to claim 1, characterized in that, The filter paper method is as follows: after soaking the filter paper in diluted royal jelly, it is placed in an indicator bacteria plate and incubated at 37°C for 18-20 hours, and then the diameter of the inhibition zone of the indicator bacteria is measured.

3. The application of a strain in evaluating the quality of royal jelly, characterized in that, Includes the following steps: (1) Culture of the strain: Add the strain to the liquid culture medium and culture it until it enters the logarithmic phase. Then, spread the bacterial solution on a solid culture medium plate to obtain the indicator bacteria plate. (2) First, soak the filter paper in the royal jelly to be tested diluted to a volume concentration of 10%, then place it in an indicator bacteria plate and incubate at a constant temperature of 37°C for 18-20 hours. Then measure the diameter of the inhibition zone of the indicator bacteria and judge the quality grade of the royal jelly to be tested based on the results. The strain is *Bacillus subtilis* (Marine sediment bacillus). Cytobacillus oceanized iminiss (), its accession number is CGMCC NO.22874; The quality grades of the royal jelly to be tested include two levels: qualified and unqualified. If the diameter of the inhibition zone is greater than or equal to 7.2 mm, the royal jelly to be tested is qualified; if the diameter of the inhibition zone is less than 7.2 mm, the royal jelly to be tested is unqualified.

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