Freshness Of Royal Jelly Research Articles

Impact of Different Storage Temperature on the Enzymatic Activity of Apis mellifera Royal Jelly

Royal Jelly is a nutrient secretion of nurse bees and a high interest functional food in human nutrition. Very little information is available on its chemical composition integrity and enzymatic activity during shelf life and assessment of new freshness markers are desirable for its conservation. In this study, the activity of glucose oxidase, five proteases and two antioxidant enzymes in refrigerated and frozen Royal Jelly for different storage times was preliminary investigated. Refrigeration determined a significantly reduction in glucose oxidase and carboxypeptidase A-like activity in Royal Jelly after one year of storage while no differences were recorded in the activity of these enzymes in frozen samples. After one year of storage glucose oxidase and carboxypeptidase A-like activity resulted higher in frozen samples frozen than in refrigerate ones. Results obtained suggest that the activities of these enzymes may be good markers of Royal Jelly freshness within 1 year at refrigeration condition. Freezing could be a valid alternative storage method to ensure a higher preservation of glucose oxidase and carboxypeptidase A-like activities for at least 1 year. Further investigation to determine the timing of glucose oxidase inactivation/degradation under refrigerated conditions and the enzymatic activity trend under prolonged frozen conditions are desirable.

Development of a Freshness Assay for Royal Jelly Based on the Temperature- and Time-Dependent Changes of Antimicrobial Effectiveness and Proteome Dynamics of Royal Jelly Proteins.

Although the antimicrobial, nutritional, and health-promoting properties of royal jelly (RJ) have been widely confirmed, the effects of storage temperature and time on RJ quality remain to be further explored. Herein, the antimicrobial and proteomic dynamics of RJ stored under different conditions were comprehensively investigated to identify consistent and sensitive markers of RJ degradation. We confirmed the negative correlation between antimicrobial properties and increased the storage temperature and duration in RJ. Using surface plasmon resonance, we showed the protein degradation-induced conformation changes in RJ, which reflected the overall variation in RJ proteins caused by the storage conditions. Further proteomic and western blotting analyses demonstrated the sensitivity and reliability of major RJ protein 4 (MRJP4) as a measure of temperature- and time-dependent RJ changes. Based on these results, we developed a colloidal gold immunoassay strip for MRJP4 detection, providing a reliable, simple, and rapid method for the evaluation of RJ freshness.

Rheological and microstructural characterization of royal jelly at different temperatures

AbstractIn this study, effect of temperature (4, 10, 25, and 35°C) on microstructural and rheological properties of royal jelly (RJ) was investigated. Our results indicated that pH and proximate composition of RJ was insignificantly affected by temperature, but °Brix values increased. Microstructural images showed that RJ particles were homogeneous at 10 and 25°C, but swelled at 35°C. RJ samples showed shear thinning behavior with yield stress. The increasing temperature caused the increase of yield stress due to swelling of particles. All samples were characterized as weak gel‐like behavior because G′ > G″ at up to 100 rad/s. Three interval thixotropic test (3ITT) showed that RJ samples could partially recover after high shear deformation, but increasing temperature decreased recovery percentage of samples. This study revealed that the temperature of RJ should never exceed to 10°C to obtain better recovery during the processing.Practical applicationsApicultural products such as royal jelly (RJ) have become very important nutritional products in recent times due to bioactive properties and are increasingly attracted by consumers. Bioactive properties of RJ can highly be deteriorated by process temperature and storage conditions. RJ has creamy and milk‐like structure and contains high amounts of proteins and carbohydrates. Therefore, the rheological characterization of RJ can provide significant information about structure, freshness, end product quality for consumers, as well as process equipment design and energy consumption for manufacturers. The freshness of RJ is determined with the analysis of 10‐HDA, furosine, superoxide dismutase and 57 kDa proteins. This study revealed that the rheological characterization of RJ at different temperatures may be helpful to estimate freshness and process conditions easier than analytical methods.

Transgenic silkworms secrete the recombinant glycosylated MRJP1 protein of Chinese honeybee, Apis cerana cerana.

Major royal jelly protein-1 (MRJP1) is the most abundant glycoprotein of royal jelly (RJ) and is considered a potential component of functional foods. In this study, we used silkworm transgenic technology to obtain five transgenic silkworm lineages expressing the exogenous recombinant Chinese honeybee, Apis cerana cerana, protein-1 (rAccMRJP1) under the control of a fibroin light chain (Fib-L) promoter in the posterior silk glands. The protein was successfully secreted into cocoons; specifically, the highest rAccMRJP1 protein content was 0.78% of the dried cocoons. Our results confirmed that the protein band of the exogenous rAccMRJP1 protein expressed in the transgenic silkworm lineages was a glycosylated protein. Therefore, this rAccMRJP1 protein could be used as an alternative standard protein sample to measure the freshness of RJ. Moreover, we also found that the overall trend between the expression of the endogenous and exogenous genes was that the expression level of the endogenous Fib-L gene declined as the expression of the exogenous rAccMRJP1 gene increased in the transgenic silkworm lineages. Thus, by employing genome editing technology to reduce silk protein expression levels, a silkworm bioreactor expression system could be developed as a highly successful system for producing various valuable heterologous proteins, potentially broadening the applications of the silkworm.

Freshness determination of royal jelly by analyzing decomposition products of adenosine triphosphate

Royal jelly (RJ), widely used in food products, is highly perishable. To ensure its quality, it is necessary to identify an indicator of RJ freshness. In this study, we measured the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), hypoxanthine (Hx), adenosine (Ao), and adenine (Ai) in RJ stored at different temperatures and for different storage periods. An F-value, defined as ratio between the sum of Ao, Ai, HxR, and Hx and the sum of ATP, ADP, AMP, IMP, HxR, Hx, Ao, and Ai, was evaluated as an indicator of RJ freshness. When the F-value of an RJ sample was evaluated, the equivalent biochemical age could be determined based on linear equations. An F-value of 20%, with an equivalent biochemical age of 60.69 days when stored at 4 °C or 6.82 days when stored at 30 °C, was the calculated RJ freshness threshold value in this study. This novel indicator could contribute to the establishment of standardized criteria for the identification of RJ freshness and provide more rigorous standards for harvesting, packaging, and storage procedures of RJ.

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody.

Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.

Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly

Nucleotides, nucleosides and nucleobases play a greater role in the physiological activity of organisms which are highly present in royal jelly (RJ). The objective of the present study is to develop a HPLC method to simultaneous determine nucleotides, nucleosides and nucleobases in RJ and access them in fresh and commercial RJ samples. The LOD and LOQ were 12.2–99.6μg/L and 40.8–289.4μg/L, respectively with nearly 100.9% recoveries. Except uric acid, all other compounds were found in RJ samples. Significant difference in the average content of compounds in fresh (2682.93mg/kg) and commercial samples (3152.78mg/kg) were observed. AMP, adenosine and adenine were found predominant in all the samples. Significant higher levels of ATP, ADP and AMP was seen in fresh RJ samples, and IMP, uridine, guanosine, and thymidine was seen in commercial RJ samples. The investigated compounds can be used as indexes for assessment RJ freshness and quality.

Assessment of royal jelly freshness by HILIC LC–MS determination of furosine

Royal jelly (RJ) is a high added value product, which can be spoiled and may lose its health-promoting properties according to the storage temperature and duration. Furosine, a compound generated at the early stages of the Maillard reaction, is a good marker of the freshness of RJ which can be used to detect heat treatments or prolonged storage of RJ samples. The determination of furosine content of RJ has been developed and validated by LC–MS. This new analytical method has the advantages of being sensitive and rapid. Moreover it avoids the time-consuming and cost-intensive SPE purification step of the usual HPLC-UV method.The influence of the storage temperature was studied on 55 French RJs. Ten of them were additionally analyzed just after harvest which exhibited furosine contents around 20.1mg/100g of protein. The furosine content remained stable when stored 9 months at −18°C, whereas a slight increase was observed for a storage temperature of +2/+5°C, with values increasing up to 61.3mg/100g of protein. The analysis of 27 commercial RJs, after 9 months of storage at +2/+5°C, displayed higher furosine contents up to 219.9mg/100g of protein with a mean at 97.1mg/100g of protein. These results underline that furosine is a suitable marker for assessing the quality of storage conditions and freshness of RJ.

Using proteomics platform to develop a potential immunoassay method of royal jelly freshness

So far royal jelly (RJ) has been widely used as a kind of popular and traditional food for health promotion. However, the quality of RJ is vulnerable to improper storage conditions. In order to prohibit the low quality RJ products entering the market and consequently affect the health of humans, it is necessary to define the quality parameters and establish corresponding detection methods for the freshness of RJ. In this research, we applied two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry to research major royal jelly proteins (MRJPs) changes under different storage conditions after 6-month storage, looking for a stable and reliable protein marker which was also feasible to detect the freshness of RJ. Further research with the help of Western blotting analysis (WB) confirmed that, under room temperature, MRJP5 began to hydrolyze within 30 days and would completely degrade within 75 days, indicating that MRJP5 can be adapted as a freshness marker for RJ products. Moreover, the assessment results of the freshness of 12 commercial RJ products with WB showed that MRJP5 was present in twelve RJ samples at different abundance levels which further confirmed the detection of MRJP5 could be a feasible method to assess the quality and freshness of commercial RJ products.

Fast Determination of Royal Jelly Freshness by a Chromogenic Reaction

Royal jelly is one of the most important products of honeybees. Given its role in development of bee brood into fertile individuals of the royal caste it is also used in health products for human consumption. Royal jelly spoils and loses its health-promoting properties depending on storage duration and conditions. To ensure product quality before selling, it is therefore necessary to assess royal jelly freshness. Many indexes of freshness have been suggested, but they all lack reliability or require complex and time-consuming analyses. Here we describe a method to detect royal jelly freshness based on a chromogenic reaction between royal jelly and HCl. We demonstrate that analyses based on color parameters allow for the discrimination of royal jelly samples based on the duration of their storage. Color parameters of royal jelly stored at -18 and 4 °C for 28 d remained comparable to that of fresh samples, which supports the reliability of the method. The method of freshness determination described is practical, cheap, and fast and can thus be used in real-time when trading royal jelly. The method developed can be used to assess royal jelly freshness. It is practical, cheap, and fast and can thus be used in real-time when trading royal jelly.

Proteomics Analysis of Major Royal Jelly Protein Changes under Different Storage Conditions

Protein changes in fresh royal jelly (RJ) were compared when stored at -20, 4 degrees C, and room temperature (RT) for 12 months. Protein was partially identified using combinations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), gel filtration chromatography, nanoLC MS/MS, and a protein engine identification tool applied to the honeybee genome. Significantly more protein spots were found in fresh (85 spots) and -20 degrees C (81 spots) stored RJ than in samples stored at 4 degrees C (73 spots) and at RT (70 spots) for 1 year. Most identified spots, 56, 57, 51, 46, corresponding to RJ sample of the fresh, -20 degrees C, 4 degrees C, and RT, were assigned to major royal jelly proteins (MRJPs). Marked differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. The quantity of MRJP1 decreased significantly following the temperature trend in all images, but MRJP 2 and -3 did not increase or decrease following the temperature trend, thus, suggesting that MRJP 1-3 are sensitive to temperature. However, MRJP4, 5, glucose oxidase (GOD), peroxiredoxin (PRDX), and glutathione S-transferase (GST) S1 were clearly absent in all images in samples held at RT for 1 year. This indicates that they are the proteins most sensitive to storage temperature and protein markers for freshness of RJ. Combining chromatography and nanoLC MS/MS results, we tentatively conclude that MRJP5 is a reliable freshness marker and that the best way to maintain quality of RJ is under freezing conditions.

Furosine: a Suitable Marker for Assessing the Freshness of Royal Jelly

Fifteen commercial samples of royal jelly, consisting of 10 imported samples, and 5 samples of known origin obtained freshly harvested from beekeepers, were analyzed for protein, lysine, and furosine content. In addition, a commercial sample of royal jelly, at the beginning of its commercial shelf life, was stored for 10 months both at 4 degrees C and at room temperature in order to assess the development of the Maillard reaction (furosine) and relative nutritional damage (blocked lysine). The commercial royal jelly products contained different amounts of furosine, ranging from 37.1 to 113.3 mg/100 g protein, evidence of different storage times and conditions. The average furosine content of the royal jelly samples of known origin and harvesting was significantly lower than that of the imported samples (41.7 versus 73.6 mg/100 g protein, respectively). With regard to shelf life, furosine content increased significantly from 72.0 mg/100 g protein to 500.8 mg/100 g protein after 10 months of storage at room temperature, while it increased to a much lower level (100.5 mg/100 g protein) when the royal jelly was stored at 4 degrees C. However, nutritional damage, expressed as blocked lysine (calculated indirectly from the furosine content), was minor or negligible, 11.9 and 2.3% of total lysine, in samples stored at room temperature and at 4 degrees C, respectively. Lysine was determined by an innovative procedure based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that furosine is a suitable index for assessing the quality and freshness of royal jelly.

Inhibition of specific degradation of 57-kDa protein in royal jelly during storage by ethylenediaminetetraacetic acid.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M., Biosci. Biotechnol. Biochem., 65, 277-284 (2001).). Here, we investigated the effects of various proteinase inhibitors on proteinase activity in RJ and on the specific degradation of 57-kDa protein during storage. Ethylenediaminetetraacetic acid (EDTA), but not other inhibitors, inhibited the proteinase activity in RJ, and dose-dependently suppressed storage-dependent degradation of 57-kDa protein. These results suggest that EDTA inhibits a specific proteinase activity in RJ, thereby suppressing the degradation of 57-kDa protein during storage at high temperature.

Fifty-seven-kDa Protein in Royal Jelly Enhances Proliferation of Primary Cultured Rat Hepatocytes and Increases Albumin Production in the Absence of Serum

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001) Biosci. Biotechnol. Biochem. 65, 277–284.). Here, we investigated the physiological effects of 57-kDa protein on primary cultured rat hepatocytes in the absence of serum. The 57-kDa protein and RJ significantly stimulated hepatocyte DNA synthesis, whereas bovine serum albumin and RJ stored at 40°C for 7 days, which lacks 57-kDa protein, did not. The mitogenic activity of 57-kDa protein was lost after treatment with trypsin. These results indicate that 57-kDa protein acts as a mitogen. The stimulatory effect of 57-kDa protein was dose-dependent and was more potent at lower than at higher cell densities. The 57-kDa protein also prolonged the cell proliferation of primary cultured rat hepatocytes, with an enhancement of albumin production compared to untreated cells. Therefore, 57-kDa protein is likely to promote liver regeneration and may have a cytoprotective action on hepatocytes.

Antifatigue effect of fresh royal jelly in mice.

We investigated the antifatigue effect of royal jelly (RJ), which had been stored at -20 degrees C from immediately after collection, in male Std ddY mice. The mice were accustomed to swimming in an adjustable-current swimming pool, then subjected to forced swimming five times during 2 wk, and the total swimming period until exhaustion was measured. They were separated into three groups with equal swimming capacity, which were administered RJ, RJ stored at 40 degrees C for 7 d (40-7d RJ), or the control solution including casein, cornstarch, and soybean oil before swimming. All mice were forced to swim for 15 min once; then the maximum swimming time to fatigue was measured after a rest period. The swimming endurance of the RJ group significantly increased compared with those of the other groups. The mice in the RJ group showed significantly decreased accumulation of serum lactate and serum ammonia and decreased depletion of muscle glycogen after swimming compared with the other groups, whereas there was no significant difference between the 40-7d RJ group and the control group in these parameters after swimming. A quantitative analysis of constituents in RJ showed that 5 7-kDa protein, which we previously identified as a possible freshness marker of RJ, was specifically degraded in RJ stored at 40 degrees C for 7 d, whereas the contents of various vitamins, 10-hydroxy-2-decenoic acid, and other fatty acids in RJ were unchanged. These findings suggest that RJ can ameliorate the physical fatigue after exercise, and this antifatigue effect of RJ in mice seems to be associated with the freshness of RJ, possibly with the content of 5 7-kDa protein.