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ELSEVIER0308-8146(95)00031-3

Food Chemistry 54 (1995) 195-200Copyright 0 1995 Elsevier Science LimitedPrinted in Great Britain. All rights reserved

030%8146/951$9.50

Changes in protein components and storagestability of Royal Jelly under various conditions

Chinshuh Chen* & Soe-Yen ChenDepartment of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung. Taiwan 402

(Received 16 September 1994; revised version received and accepted 5 January 1995)

The changes in quality of Royal Jelly (RJ) stored at -20C 4C and room tem-peratures under dark and light conditions for up to 7 months were investigated.The results showed that colour, viscosity, fractions of water-soluble protein(WSP) and simple sugars of RJ changed significantly during storage at roomtemperature, but not at -20C. The viscosity and browning intensity increasedwith storage time, and were enhanced by the ambient temperature. Two majorfractions in WSP of RJ were obtained by gel filtration, while four to five differ-ent protein components were found when subjected to SDS-PAGE. The relativeamounts and molecular weight distributions of these WSP components variedduring storage. The results indicated that the quality deterioration of RJ duringstorage was due to the Maillard browning reaction.

INTRODUCTIONNatural food products that have undergone the leastprocessing are now popular. Among them, royal jelly(RJ) is one of the most attractive and lasting products.It has been a commercial product, especially in dieteticsand cosmetics, in many countries for more than 30years. RJ is a secretion from the hypopharyngeal andmandibular glands of worker bees (&is mellifera L.). Itis a yellowish, creamy and acidic material with aslightly pungent taste. In the society of honey-bees, RJis fed temporarily (less than 3 days) to the brood ofworkers and drones, but it is the only food of thequeen bee throughout her life (Townsend & Lucas,1940; Lercker et al., 1981). The queen bee is wellknown for her reproductive ability, large body size andlong life span. RJ is thus generally regarded as themajor reason for the significant morphological andfunctional differences between queen and worker bees(Karaali et al., 1988). Commercially, RJ can be pro-duced in significant quantities using the Doolittlequeen-rearing technique (Lensky, 1971).

The composition of RJ varies with seasonal andregional conditions. The average moisture content ofRJ is 60-700/o, crude protein 12-15%, lipid 3-6%, totalsugars lO-16% and pH 3.54.5 (Takenaka, 1982; Howeet al., 1985). The storage conditions of RJ depend uponthe types of product. Raw or natural RJ, which is soldand consumed as it is, is stored at -20C or lower, while* To whom correspondence should be addressed Two freshly harvested RJ samples (RJ-I and RJ-2) were

195

the lyophilised type, either alone or in combination withother ingredients, is generally in capsulated or tabletforms and is stored at cool or room temperature. How-ever, RJ spoils or deteriorates and loses its commercialvalue eventually when it is stored improperly. It was re-ported that there was a gradual deterioration of proteincomponents (Okada et al., 1979) and an increase in itsviscosity and titratable acidity (Takenaka et al., 1986;Yatsunami, 1988). These changes might be due to theMaillard reaction or lipid oxidation. Proteins of freshRJ consist of water-soluble and water-insoluble frac-tions. Depending upon the extraction conditions used,the water-soluble protein (WSP) was the major fractionand made up 4689% of the total protein (Tomoda etal., 1977; Takenaka & Echigo, 1983); consequently, thechange of WSP fraction might play an important role inthe quality deterioration of RJ. However, there are veryfew data available on the changes in the composition ofRJ during storage, especially in protein components.

In this study, the changes in quality of raw RJ, par-ticularly in WSP, during a storage period of sevenmonths both in the refrigerator and at room tempera-ture were investigated. Finally, the feasibility of apply-ing results obtained to detecting quality changes of RJduring storage was evaluated.

MATERIALS AND METHODSPreparation of royal jelly samples


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196 Chinshuh Chen, Soe-Yen Chensupplied directly from two different beekeepers in middleand southern Taiwan during June 1993. Bees (&is mel-&era L.) were fed with nectar and pollen from two fruittrees, longan (Nephelium longana) and lychee (Litchi chi-newis). Each RJ sample was dispensed into 28 sterile,air-tight glass bottles (50ml). These bottles were thenrandomly divided into four groups; seven bottles foreach group were stored at -20C 4C, and at room tem-perature in bright and dark places of laboratory, respec-tively. The light source was a fluorescent lamp (280 lux)and samples were illuminated for 15 h a day. The aver-age room temperature was about 24 f 3C and all bot-tled samples were protected from light by wrapping withaluminium foil, except those at room temperature in thebright place. Sampling was performed monthly and eachbottle was sampled once only, in order to avoid thepotential repeated freeze-thaw damage. Cold or frozensamples, were allowed to equilibrate to room tempera-ture and then stirred with a glass rod to homogeneitybefore analysis. All analyses were done in duplicate.Measurement of viscosityThe viscosity was measured using a Brookfield digitalviscometer (Model DV-II, Brookfield Engineering Lab.)equipped with a small sample adapter (12 ml) and a no.28 spindle. The RJ sample was previously equilibratedto 30.0 f O.lC. The measurement was taken three timeswith the speed of spindle being set at 100 rpm.Measurement of browning intensityOne gram of sample was diluted with distilled water toa final volume of 100 ml (Solution A). Ten millilitres ofthe Solution A was further diluted with an equalamount of distilled water and measured for absorbanceat 420 nm using a spectrophotometer (Hitachi, U-2000). In addition, 50 ml of the above Solution A waswithdrawn and mixed with 2.5 ml 10% trichloroacetic(TCA) solution and stirred for 5 min. The filtrate wasthen collected through a membrane filter (0.45 pm) tomeasure the absorbance at 420 nm.Extraction of WSPTwo types of WSP, WSP-1 and WSP-2, were preparedfrom RJ. For the preparation of WSP-1, 5 ml of distilledwater were added to 2.5 g sample; the supematant aftercentrifugation (5000 x g,30 min) was designated WSP-1. The extraction of WSP-2 was done as described byTakenaka and Echigo (1983) with modifications. Fifteenmillilitres of 0.01 M phosphate buffer (pH 7.2) was addedto 2.0 g of RJ samples and centrifuged (5000 X g, 30min) at 4C. The supematant was withdrawn and satu-rated with 90% ammonium sulphate in an ice bath. Theprecipitate was collected after centrifugation (7000 X g,50 min, 4C) and redissolved with 10 ml of 0.01 M phos-phate buffer (pH 7.2), followed by dialysing against dis-tilled water in a cold room for about 2 days. The dialysedsolution obtained, as WSP-2, was then lyophilised.

Gel filtration chromatographyThe proteins in the WSP-2 were separated by gel filtra-tion. The separation was carried out on a Sephacryl S-200 (Pharmacia, LKB) column (1.6 cm D X 90 cm H).The flow rate through the column was 15 ml/h using0.01 M phosphate buffer, pH 7.2, as eluant. The relativeamount of proteins in the eluted fractions was detectedcontinuously with a UV-detector (ISCO, UA-6) at 280nm. The area under a peak in the chromatogram wasapproximated by cutting the area out of the graphpaper and weighing it (Robyt & White, 1987). Theratio of area of leading peak (peak 1) to that of thesecond peak (peak 2) was defined as R, value.SDS-PAGE analysisThe SDS-PAGE analysis of WSP-2 was performedaccording to Cooper (1977). 7.5% polyacrylamide gelwas cast and run at 120 V using a Dual Mini SlabKit (Atto, AE-6450). Protein bands were stained withCoomassie brilliant blue. The molecular weight mark-ers were supplied the electrophoresis calibration kit forlow-molecular-weight protein (Pharmacia LKB).Sugar analysisSucrose, glucose and fructose were determined byHPLC (Ball, 1990). One gram of RJ sample was mixedwith 5 ml of 80% (v/v) aqueous ethanol at 4C for 30min. The supernatant after centrifugation (7000 X g,30min) was filtered through a membrane filter (0.45 pm)before analysis. The column was a pre-packed column(4 mm id. X 250 mm L), packing material wasLichrosorb NH*. Aqueous acetonitrile solution (ace-tonitrile/water = 87 : 13, v/v) was used as eluant, theflow rate was 1.0 ml/min.Total nitrogen determinationThe Kjeldahl method (AOAC, 1980) for the determina-tion of organic nitrogen was used to determine the totalnitrogen contents in the WSP-1 and RJ. One gram ofRJ sample or aliquots of WSP-1 solution were mixedwith 15 ml cont. sulphuric acid, and placed in a diges-tion tube, digested in a microwave digestion system(Maxidigest MX-350) for 15 min, followed by distillingand titrating with a Kjeltec System (Tecator, model1026). The nitrogen content (%N, w/w) was then calcu-lated, and the ratio of total nitrogen content for WSP-1solution to that of RJ sample was defined as R, value.

RESULTS AND DISCUSSIONChanges in the viscosity of RJ during storageThe viscosity of fresh RJ-2 was about 2060~~ and itremained almost unchanged up to 7 months when thesample was stored at -20C (Table 1). However, the


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Changes in protein components of Roy al Jelly during storage 197Table 1. Changes in viscosity of royal jelly (RJ-2) stored underdifferent conditions

Viscosity at storage period (month)Storage conditions ~ 0 1 3 5 7-20C 2060 2070 2100 2020 23004C 2060 2350 2790 3300 3630Room temperature andBright Place 2060 Eh E E EDark Place 2060 E E E EUnit in centipoise (cp).The actual viscosities exceeded the upper limit (5000 cp) ofthe viscometer used under the specified condition as describedin the Materials and Methods section.

viscosity increased linearly with time as storage temper-ature increased to 4C. Furthermore, a remarkablechange was observed for those samples stored at roomtemperature, the increase of viscosity was so sharp thatit failed to be measured by the instrument. The viscos-ity was thus a sensitive parameter in tracing the storagestability of RJ.Browning reaction of RJ during storageThe yellowish white colour of freshly harvested RJbecame darkened gradually as storage time increased.Figure 1 shows how the browning reaction occurred in

1 . 2 0

1 . 0 0

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P%1oo0.60E5 0.60

i3 0.400.20

0.00 0

RJ - 1- 4 + * - A- A

.RJ-2

1 2 3 4 5 5 7 5Storage Time( Month )

Fig. 1. Changes in browning intensity of royal jelly duringstorage. Samples from two origins (RJ-1 and RJ-2) werestored under different conditions; (0) -20C; (0) 4C; (A)room temperature and bright place; (a room temperature anddark place. Samples stored at room temperature and brightplace were also measured with a pretreatment of TCA (+).

fresh RJs when stored under different conditions. Thebrown intensities increased rapidly and reached max-ima within 2 months, when RJ were stored at roomtemperature in the presence or absence of light. On theother hand, the extent of the browning reactionincreased to a much lower level when RJ were at 4Cand was almost negligible at -20C. Apparently, thebrowning reaction was stimulated by the higher tem-peratures, and little by the presence of light. Similarresults were also observed in colour difference values(AE) of RJ during storage at room temperature (Take-naka et al., 1986).If portions of RJ samples stored at room temperaturefor different periods were further treated with TCA inorder to remove the proteins, the TCA treatmentresulted in a drastic decrease of absorbance to nearlyzero, despite the storage time. This indicated that brownpigments were either tightly absorbed in the proteinfractions or associated with molecules such as glycopro-teins. Takenaka and Echigo (1983) reported that boththe water-soluble and water-insoluble proteins in a freshRJ were actually glycoproteins; the carbohydrate moi-eties were composed of glucose and mannose.Changes of WSP-1 in RJ during storageThe changes of WSP-I in RJ during storage were inves-tigated. The ratio of WSP-1 to total nitrogen content,R,, indicates the extent of hydrolysis of proteins in RJ.R, values were between 0.5 and 0.6 for freshlyharvested RJs (Fig. 2). Townsend and Lucas (1940) andTomoda et al. (1977) found almost equal amounts of

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0.200 1 2 5 4 5 6 7 6Storage lme( Month )

Fig. 2. The ratios (R,) of water-soluble nitrogen to totalnitrogen of two royal jelly samples stored under differentconditions. For an explanation of the symbols, see Fig. 1.


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198 Chinshuh Chen, Soe- Yen Chenwater-soluble and water-insoluble proteins in RJ. How-ever, up to 89% of water-soluble nitrogen in the totalnitrogen content of RJ was also reported (Takenaka &Echigo, 1983). The R, values decreased during the first2-3 months, and reached constant values dependingupon the storage temperature. The total amount of freeamino acids of RJ stored at room temperature was alsofound to be decreased with time (Takenaka et al.,

0.5

0.4

Ec 0.38c\l8 0.2

0.1

Peak 1 Peak 21 1

0 RJ-2

0.00 20 30 40 50

Fraction number( 3ml/tube )Fig. 3. Representative gel filtration chromatograms of water-soluble protein (WSP-2) from royal jelly during storage.WSP-2 was extracted from royal jelly, RJ-2, which has beenstored at -20C for O-2 months. Numbers O-2 indicate thestorage period from 0 to 2 months, respectively.

250.RJ-1

zoo -

cl 1 2 3 4 5 6 7 IIStorage Tims( Month )

Fig. 4. The peak area ratios (R,) of peak 1 and peak 2 in thegel filtration chromatograms of water-soluble proteins (WSP-2) of two royal jelly samples stored under different conditions.For an explanation of the symbols, see Fig. 1.

-79.1-653.9-56.9-49.2

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Fig. 5. SDS-PAGE patterns of water-soluble protein (WSP-2) of royal jelly (RJ-2) stored under different conditions. (a), -20C;(b), 4%; (c), room temperature and dark place; (d), room temperature and bright place. Lanes l-7 refer to the storage periodfrom 0 to 6 months, respectively; lane 8, marker proteins.


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Changes in protein components of Royal Jelly during storage 1991986). This indicated that proteins in RJ hydrolysedsoon after harvesting, especially at room temperature,and this was accompanied by the browning colour. Onthe other hand, the decrease in WSP-1 also impliedthat in increase of water-insoluble protein componentsoccurred. In addition, a rapid increase in viscosity dur-ing the storage of RJ at room temperature wasobserved as above and also by Takenaka et al. (1986).It was thus suggested that some hydrolysates of WSPfractions in RJ aggregated or polymerised again duringstorage and led to the increase in both the amount ofwater-insoluble protein and viscosity.Fractionation of WSP-2 in RJTwo peaks, peaks 1 and 2, were obtained when WSP-2was fractionated by the gel filtration chromatography(Fig. 3) as also shown by Tomoda et a/.( 1977). Duringthe storage period, the peak area of the peak 1, whichelutes first and has greater molecular weight increased,while that of peak 2 decreased. In addition, smallerfragments with lower molecular weights were probablyproduced as the shape of peak 2 tended to spread outgradually. If the ratio of areas of peak 1 to peak 2, R,,was calculated, it was found that the R, valuesincreased significantly within 2 months; this tendencyalso depended upon the storage temperature (Fig. 4).Higher temperatures had greater effects on them, whilethe effect of light was little. This indicated that bothpolymerisation and hydrolysis reactions occurredsimultaneously in RJ during storage. It was probablethat the Maillard browning reaction resulted in poly-merisation of components in RJ as is also shown inFig. 2, and the decrease in area but spreading in shapeof peak 2 was due to the hydrolysis of protein compo-nents in RJ by the proteolytic reaction. Protease hasbeen previously detected in RJ (Chiu, 1992).The changes in the components of WSP-2 in RJ werealso shown by the results of SDS-PAGE. Four distinctand a faint bands were found for a fresh RJ, withmolecular weights ranging from 50 000 to 88 000 (Fig.5). Depending upon the extraction methods of WSP,Tomoda et al. (1977) reported at least five protein frac-tions in the WSP of a fresh RJ, based on solubility in aphosphate buffer containing 0.1 M NaCl. Takenakaand Echigo (1983) obtained five proteins from the WSPextracted by 0.1 M phosphate buffer, and precipitatedwith 60% ammonium sulphate. Instead, Pate1 ez al.(1960) showed four bands in a water-extracted proteinusing paper electrophoresis. The changes of thesebands were indiscernible in a frozen RJ during storage;however, the changes became significant as storagetemperature increased. For example, the bands withmolecular weight greater than 65 000 from the samplestored at room temperature disappeared gradually,resulting in the appearance of new bands with lowermolecular weights (20 000-40 000). In other words, theresults obtained from the SDS-PAGE probably onlyreflected changes of peak 2 in gel filtration chromatog-raphy; i.e. some components in the RJ disintegrated

into smaller fragments during storage. The polymerisedproducts (peak 1) were not detected or differentiated byelectrophoresis under the experimental conditions used.Simple sugars in RJThe major sugars found in the RJ were glucose, fol-lowed by fructose and sucrose. Depending upon theage and beekeeping foods, glucose constituted 50-70%of the total sugars (Tourn et al., 1980; Lercker, 1986).Both glucose and fructose increased rapidly within 2months and then remained constant or decreasedslightly, while sucrose decreased as storage time in-creased (Fig. 6). In addition, the hydrolysis of sucrosewas enhanced at higher temperatures; however, thelight had little effect on it. These results indicated thatthe initial increase of glucose and fructose probablycame from the hydrolysis of sucrose, and some of thesereducing sugars might react with the hydrolysates ofprotein components eventually due to the Maillardbrowning reaction as described previously.

3.00

s.001 1-m

Fig. 6. Changes in sugar contents of royal jelly (RJ-2) storedunder different conditions. For an explanation of the symbols,see Fig. 1.


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200 Chinshuh Chen, Soe- Yen ChenThese results clearly indicated that the browningreaction of RJ during storage was due to the Maillardbrowning reaction. This type of reaction was sensitive

to the ambient temperature. Higher storage tempera-tures stimulated the reaction rates; however, less orlittle stimulating effect was found in the presence of light.The actual contribution of light to the browning reac-tion of RJ stored at room temperature was probablymasked by the effect of the higher temperature used inthis study. In addition, the changing patterns of param-eters such as brown intensity, R,, and R, during storagewere similar between two RJ samples. The results alsoshowed that measurements of colour, viscosity, andWSP fractions basically reflected the quality changes ofraw RJ during storage. It was therefore possible to cor-relate quality loss of RJ products with the changes ofthese parameters, and allow us to detect the qualityloss during storage or freshness of raw RJ products byusing these methods.

ACKNOWLEDGEMENTSThe authors are sincerely grateful to the NationalScience Council, Republic of China, for support of thiswork (project No. NSC 82-409-BOO.5111).

REFERENCESAOAC (1980). Oficial Methods of Analysis of the Associationof OfJial Analytical Chemists (13th edn), ed. Horwitz, W.Association of the Official Analytical Chemists, Washing-ton, DC, USA, pp. 14-15.Ball, G. F. M. (1990). The application of HPLC to the deter-mination of low molecular weight sugars and polyhydricalcohols in food: a review. Food Chem., 35, 117-52.Chiu, S. M. (1992). Effects of temperature and illuminationon the storage stability of royal jelly. MS thesis, NationalChung-Hsing University, Taiwan.

Cooper, T. G. (1977). Electrophoresis. In The Tools of Bio-chemistry, ed. Cooper, T. G. John Wiley & Sons, NewYork, USA, pp. 194-233.Howe, S. R., Dimick, P. S. & Benton, A. W. (1985). Compo-sition of freshly harvested and commercial royal jelly. J.Apic. Res., 24, 52-6 1.Karaali, A., Meydanoglu, F. & Eke, D. (1988). Studies oncomposition, freeze-drying and storage of Turkish royalielly. J. Apic. Res., 27, 182-S.L&sky, Y. -( 1971). Rearing honeybee larvae in queen rightcolonies. J. Aoic. Res.. 10. 99-101.Lercker, G. (1986). Carbohydrate determination of royal jellyby high resolution gas chromatography (HRGC). FoodChem., 19,255-64.Lercker, G., Capella, P., Conte, L. S. & Ruini, F. (1981).Components of royal jelly: I. Identification of the organicacids. Lipids, 16, 912-19.Okada, I., Sakai, T., Matsuka, M. & Furusawa, T. (1979).Changes in the electrophoretic patterns of royal jellyproteins caused by heating and storage. Chem. Abstr., 91,Article 34385~.Patel, N. G., Haydak, M. H. & Gochnauer, T. A. (1960).Electrophoretic components of the proteins in honeybeelarval food. Nature (London). 186. 633-4Robyt, J. F. & White: B. J. (1987). Analyzing and reportingexperimental data. In Biochemical Techniques - Theoryand Practice, eds Robyt, J. F. & White, B. J. Brooks/ColePublishing Co., CA, USA, pp. l-20.Takenaka, T. (1982). Chemical compositions of royal jelly.Honeybee Sci., 3, 69-74.Takenaka, T. & Echigo, T. (1983). Proteins and peptides inroyal jelly. Nippon Nogeikagaku Kaishi, 57, 1203-g.Takenaka, T., Yatsunami, K. & Echigo, T. (1986). Changesin quality of royal jelly during storage. Nippon ShokuhinKogyo Gakkaishi, 33, 1-7.Tomoda, G., Matsuyama, J. & Matsuka, M. (1977). Studieson protein in royal jelly. 2. Fractionation on water-solubleprotein by DEAE-cellulose chromatography, gel filtrationand disc electrophoresis. J. Apic. Res., 16, 125-30.Tourn, M. L., Lombard, A., Belliardo, F. & BulTa, M. (1980).Quantitative analysis of carbohydrates and organic acids inhoneydew, honey and royal jelly by enzymic methods. J.Aoic. Res., 19, 144-6.Townsend, G. F. & Lucas, C. C. (1940). The chemical natureof roval iellv. Biochem. J.. 34. 1155-62.Yatsunamc K (1988). Changesin quality of royal jelly. Hon-eybee Sci., 9, 67-7 1.

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