Ihc-platform.net

Journal of ApiProduct and ApiMedical Science 1(1): 1-6 (2009) ORIGINAL RESEARCH ARTICLE

Quality and standardisation of Royal Jelly

Anna Gloria Sabatini1*, Gian Luigi Marcazzan1, Maria Fiorenza Caboni2,
Stefan Bogdanov3, Ligia Bicudo de Almeida-Muradian 4
1 CRA- Istituto Nazionale di Apicoltura, Bologna, Italy. 2 Dipartimento di Scienze degli Alimenti, Università di Bologna, Italy. 3 Swiss Bee Research Centre Agroscope, Liebefeld Poseux, Berne, Switzerland. 4 Departamento de Alimentos e Nutrição Experimental, Faculdade de Ciências Farmacêuticas da Universidade de São Paulo (USP), São Paulo, Brazil. Received 26 June 2008, accepted subject to revision 18 August 2008, accepted for publication November 2008. *Corresponding author: Email: [email protected] Introduction
used, the different sampling procedures and production conditions. Additional complicating factors are the multiplicity of experimental Given the exceptional biological properties attributed to it, royal jelly conditions, as well as the diversity of the analytical methods used (RJ) has considerable commercial appeal and is today utilised in and their continual evolution. many sectors, ranging from the pharmaceutical and food industries Knowledge of the composition of recently produced RJ is to the cosmetic and manufacturing sectors. This has resulted, among essential in order to define a standard composition, evaluate the other things, in large-scale importation in countries where production quality of commercial products and detect the presence of RJ in other is insufficient to meet domestic demand. Research capabilities thus need to be reinforced to permit both a reliable qualitative and At present some countries, like Switzerland (Bogdanov et al., quantitative evaluation of the different components and the 2004), Bulgaria, Brazil (Brasil Leis e decretos, 2001) and Uruguay implementation of analytical tests on commercially available products have defined national standards for this product. A group of the – RJ on its own or as an additive to new or traditional products – also International Honey Commission is dealing presently with royal jelly for the purpose of identifying possible adulteration. No official data exist about the RJ market (Grillenzoni, 2002), but China is unanimously acknowledged as being the leading Studies on royal jelly quality
world producer and exporter of RJ, which it sells at highly competitive prices. Chinese production of RJ is estimated as 2 000 In the 1980s a workgroup was formed in Italy which has devoted t/year (a quantity that represents over 60% of production much effort to the study of RJ (Lercker et al., 1981; Lercker et al., worldwide), almost all of which is exported to Japan, the United 1982; Lercker et al., 1984a; Lercker et al., 1984b; Lercker et al., States and Europe. Other countries like Korea, Taiwan and Japan are 1985; Lercker et al., 1986; Vecchi et al., 1988; Lercker et al., 1993; important producers and also exporters. Elsewhere in the world, RJ is Antinelli et al., 2003; Boselli et al., 2003; Lercker, 2003). The data produced mainly in Eastern Europe and to a lesser extent in Western presented in this article refer to the results obtained by the Italian Europe and in America: Mexico, in particular, is quite a big producer. group cited, completed by findings of researchers from other Numerous studies have been dedicated to RJ since as far back as the late 19th century (Planta, 1888; Lercker, 2003). However, Samples of recently produced, commercial grade RJ directly it is difficult to bring together the data col ected by different authors gathered from specialised beekeeping facilities located in different into an organic whole, as the data themselves are not always Italian regions were used both for the purpose of developing comparable due to the lack of homogeneity among the materials methods and conducting the analyses. The same samples were used Sabatini, Marcazzan, Caboni, Bogdanov, Almeida-Muradian to assess the changes occurring in RJ during storage. Determined by HPLC (Bloodworth et al., 1995; Apart from this project, other studies were also carried out. Genc and Aslan, 1999; Koshio and Almeida- Most of them were concerned with RJ authenticity. RJ adulteration is Muradian, 2003; Garcia-Amoedo and Almeida- the most important quality problem. Adulteration by the nursing Muradian, 2003, 2007; Pamplona et al., 2004) jellies for worker and drones is improbable because of the very little Determined by atomic absorption (Benfenati et al., amounts available for harvest. Adulteration with honey is more probable, causing an increase of the sugar values, the other values Acidity Titration method (Serra-Bonvehi, 1992) being lowered (Serra Bonvehi, 1991). The most important quality Sediment analysis Microscopical analysis (Ricciardelli d’Albore, 1986) criteria for RJ adulteration is 10-Hydroxy-2-Decenoic Acid (HDA). However, the composition limits, reported in the literature are very broad. 10-HDA content decreases with storage of RJ (Antinel i et al., Contamination
2003). This decrease is higher in honey containing RJ (Matsui, There are very few studies concerning the possible contamination of 1988). Thus, the determination of all fatty acids, as carried out in RJ. The content of RJ contaminants, compared to other bee products, the Italian studies (Lercker et al., 1981; Lercker et al., 1993), might is relatively low (Fleche et al., 1997). Recently, the problem of honey be the better approach that the determination of 10-HDA only. and RJ contamination by antibiotics has arisen. Although most studies It was recently reported that authenticity of RJ production concern residues in honey, antibiotic use in the colony can can be determined by measuring of the ratios stable isotopes of the contaminate also royal jelly (Matsuka and Nakamura, 1990). On the elements C and N (Stocker, 2003). The authenticity of production other hand, experience has shown that RJ residue analysis is difficult can be measured by determining the fatty acid composition of RJ and that old analysis methods are questionable. There are very few (Howe et al., 1985; Lercker et al., 1993). publications on antibiotic residues in RJ, mainly on chloramphenicol The geographical authenticity can be determined also by (Dharmananda, 2003; Reybroeck, 2003; Calvarese et al., 2006). The pollen analysis (Ricciardelli d'Albore et al., 1978; Ricciardelli first two papers do not report details, only in the last publication d'Albore, 1986). The 87Sr/86Sr ratios indicate also the geographic details on the methods and the contamination levels are given. The presence of chloramphenicol (CA) was detected in 29 out of 35 tested The amount of pollen, as well as visible wax and larvae samples imported in Italy, the concentrations ranging from 0.6 μg/kg particles should be minimal. RJ has relatively low concentration of to 28 μg /kg, with an average content of 6.1 μg/kg. As antibiotics are bacteria (Serra Bonvehi and Escola Jorda, 1991). not al owed for use in beekeeping, there is no MRL for honey or other bee products in the European Union. For CA in honey the EU has The parameters investigated concerned in the above established an MPRL of 0.3 μg/kg. By using method developed by mentioned studies concern the organoleptic characteristics and Calvarese and coworkers (Calvarese et al., 2006) this MPRL can also physicochemical properties as well as the following composition Determined by freeze-drying (Messia et al., Composition and quality criteria
2005), Karl Fischer (Ferioli et al., 2007), vacuum for royal jelly
oven, dessication (Garcia-Amoedo and Almeida- Organoleptic description and physical characteristics
Nitrogen determined with the Kjeldahl method RJ appears as a whitish substance with a gelatinous consistency, (Lercker et al., 1992-93; Garcia-Amoedo and often not homogenous due to the presence of undissolved granules of Almeida-Muradian, 2007). Free amino acids varying size. It has a distinctively sharp odour and taste. determined by ion chromatography (Boselli et It is partial y soluble in water and highly acidic (pH 3.4-4.5) with a density of 1.1 g/mL (Lercker, 2003). Determined by gas (Lercker et al., 1993) or Main components
Determined as free and total organic acids by The composition of the main constituents of RJ, proteins, gas chromatography (Lercker et al., 1992-93) or carbohydrates and lipids is reported in the literature (Takenaka and as total lipids, by solvent extraction (Karaali et Echigo, 1980; Bonomi et al., 1986; Pourtal ier et al., 1990; Lercker, 2003, Garcia-Amoedo and Almeida-Muradian, 2007). Quality and standardisation of Royal Jelly The values obtained by the various authors are fairly in agreement, present but often in highly variable concentrations. It is also possible notwithstanding the high variability displayed by some parameters to find oligosaccharides such as trehalose, maltose, gentiobiose, (sugars and lipids). It should be kept in mind that the reported isomaltose, raffinose, erlose, melezitose; though present in very small findings refer to different number of samples taken in different concentrations they are useful for identifying a characteristic pattern, places and at different times of production and that different which is comparable to that of honey and in some cases indicative of methods of sampling and analysis were used. Moreover, RJ is Our own analyses of RJ samples of different geographical Lipids and 10-Hydroxy-2-decenoic acid (10-HDA)
origins showed no differences in composition such as to distinguish This fraction is likewise present in fairly modest, variable concentrations (8-19% of dry matter), but no doubt represents the It may similarly be affirmed that environmental conditions do not significantly influence the main components. The lipid portion in fact consists primarily of organic acids (80 -90%), most of which free, with a rather unusual structure rarely encountered in nature: they are in fact mono- and dihydroxy acids Water content shows to be fairly uniform, greater than 60%, and and dicarboxylic acids with 8 and 10 carbon atoms, which show a with an activity (aw) above 0.92, in spite of which RJ displays characteristic arrangement (Lercker et al., 1992-93). considerable microbial stability. The constancy of the moisture Hydroxy acids with 10 carbon atoms (10-hydroxydecenoic content is basically assured, inside the hive, by the continuous and 10-hydroxy-2-decenoic acid) above all can be found in high provision of fresh supplies of this substance by nurse bees, by the concentrations. Not only may they be ascribed a role as a marker natural hygroscopicity of RJ and the entire colony’s efforts to component, but they have also been identified as responsible for maintain a level of ambient moisture; moreover the non solubility of important biological activities tied to the development strategies of some compounds can explain the variations in water content. The identification of this fraction – in particular as regards the Proteins
pattern and quantitative analysis of free organic acids – is believed to From a quantitative viewpoint, proteins (27-41%) represent the represent the criteria of choice for defining the genuineness of RJ and most important portion of the dry matter of RJ. the presence of RJ in other products, be they foods or cosmetics The amino acids present in the highest percentages were (Caboni et al., 1994). The analyses we performed showed that the proline, lysine, glutamic acid, β-alanine, phenylalanine, aspartate composition remained stable for as long as 2 years, regardless of and serine (Boselli et al., 2003). The concentration of series D amino whether the samples were stored at 4°C or at room temperature. acids was below the detection limit of the method (0.1mg/g of RJ) in A recent study (Antinelli et al., 2003) showed a 0.4 and 0.6% reduction in 10-hydroxy-2-decenoic acid in two RJ samples stored at The study aimed to assess how this parameter evolved room temperature for 12 months. It is difficult to evaluate such a during storage of the product. No significant changes were observed reduction in a sample in the control phase. Moreover it is difficult to in the overall concentration of free amino acids in RJ stored at 4°C use 10-hydroxy-2-decenoic-acid decrease as a freshness marker for 10 months. However, in the same samples stored at room because their variable amount on fresh RJ. Both HPLC and temperature, the proline and lysine content showed an increase in electrophoretic analysis of 10-HDA showed that samples of RJ from the first three months and after 6-10 months decreased to levels extra-european origin contain smaller amount of this compound; this slightly lower than those in the control samples. This suggests that, evidence was confirmed measuring total lipids after organic extraction in favourable temperature conditions, a proteolytic enzymatic Minerals
Carbohydrates
Ash content represents 0.8-3% of RJ (fresh matter) (Messia et al., On average this portion accounts for 30% of the dry matter of RJ. 2003; Garcia-Amoedo and Almeida-Muradian, 2007). The major However, while the components are highly constant in qualitative elements are, in descending order: K, Ca, Na, Mg, Zn, Fe, Cu and Mn terms, considerable variability may be observed from a quantitative (Nation and Robinson, 1971; Ivanov and Chervenakova, 1985; Benfenati et al., 1986), present in specific ratios such as K/Na and As in honey, the monosaccharides fructose and glucose are the main sugars. They often account for over 90% of the total The hypotheses regarding the quantitative presence of these metals sugars and, of the two, fructose is prevalent. Sucrose is always have focused on factors outside the colony (environment, Sabatini, Marcazzan, Caboni, Bogdanov, Almeida-Muradian procurement of food, production period) and to some extent internal Freshness definition
factors (biological factors tied to the bees). Another fundamental aspect lies in the possibility of defining a Authenticity
It has been noted that the macroscopic composition of RJ is The main quality factors of RJ have been described and studies have fairly stable on the whole but also variable, above all as far as certain revealed the importance of the lipid fraction as a marker and hence components are concerned. Thus it is not a suitable parameter for a criterion by which to determine the product’s genuineness. defining product freshness. Presently, 10-HDA is mostly used for routine testing of RJ For the latter purpose, experiments were conducted on RJ authenticity. However, the concentration of this acid varies in wide samples stored at 4 and 20°C over a period of 24 months to assess limits. Further studies are necessary to determine whether the changes in the content of the enzyme glucose oxidase. The results determination of the stable isotopes of the elements C and N obtained showed that the enzyme contained in RJ is influenced both (Stocker, 2003) is a promising approach for the determination of the by storage temperature and time. At 20°C it had decreased authenticity of production. Adulteration by honey results in a general significantly after one month and degraded completely after one diminution of proteins and lipids and a relative increase of sugars year. Even at 4°C there was an evident, albeit modest, reduction in Adulteration with more than 25% of yoghurt, egg white, The determination of glucose oxidase is analytically very water and corn starch slurry can be detected by the enhancement of simple and thus within the capabilities of all laboratories. This moisture, diminishing in lipid, protein and 10-HDA content as well as method could be used to evaluate the product’s freshness; however, the insolubility in alkaline medium. (Garcia-Amoedo and Almeida- further investigation must first be conducted into the natural variability of this component in the fresh product (Bosel i et al., Furthermore, microscopic analyses of RJ sediment, applied 2002). Marconi et al. (2002) quoted several experiments were according to the basic principles of melissopalynology (Louveaux et performed to evaluate the possibility of using furosine content as a al., 1978; Ricciardelli, 1986) and in particular the identification of the pol ens it contains, make it possible to define the geographical The value of furosine, a product of Maillard’s reaction, proved origins of the product and detect mixtures where they occur. Pollen very low (from 0 to 10 mg/100g of protein) in freshly produced RJ identification is made easier by the fact that only a few countries samples (Messia et al., 2003) but increased over time and in relation actually produce RJ and specialists are capable of formulating their to temperature. Specifically, the content rose to as high as 500 respective characteristic pollen associations. mg/100g of protein after 18 months’ storage at room temperature Another promising parameter for the evaluation of RJ and 50 mg/100g at 4°C. Samples taken from store shelves showed authenticity is the presence of apalbumin (Simuth et al., 2004). This values ranging from 40 to 100 mg/100g protein. By contrast, freeze- marker, if confirmed by further research, could gain high dried RJ showed strong tendency to form furosine during storage Table 1: Royal Jelly Composition
Fresh lyophilized
Quality and standardisation of Royal Jelly Table 1 shows some data from literature that could be used as a BOSELLI, E; CABONI, M F; LERCKER, G; MARCAZZAN, G L; preliminary proposal for fresh and lyophilized royal jelly standards. SABATINI, A G; BAGGIO, A; PRANDIN, L (2002) Valutazione di produzioni apistiche: gelatina reale e cera. Data come from many countries investigations and regulations but In Atti del convegno finale del Progetto Finalizzato AMA “Il for the establishment of a paper general standard, further ruolo della ricerca in apicoltura”. Istituto Nazionale di BOSELLI, E; CABONI, M F; SABATINI, A G; MARCAZZAN, G L; Given the product’s high water content, the composition values are LERCKER, G (2003) Determination and changes of free amino acids in royal jelly during storage. Apidologie also proposed for the freeze-dried sample. This enables a more direct comparison of data; plus RJ is also marketed in this form. BRASIL, LEIS E DECRETOS (2001) Instrução Normativa nº 3, de 19 Although the overall analytic data confirm that exposure to a de janeiro de 2001. Regulamentos técnicos de identidade e temperature of 4°C causes no alterations in RJ composition, recently qualidade de apitoxina, cera de abelha, geléia real, geléia it was also shown that only storage of RJ in frozen state prevents real liofilizada, pólen apícola, própolis e extrato de própolis. decomposition of biologically active RJ proteins and thus RJ should be frozen as soon as it is harvested (Li et al., 2007). CABONI, M F; SOLAZZO, P; LERCKER, G (1994) Proposta di un The next steps should be: 1. Validate the respective metodo per la valutazione quali-quantitativa della gelatina methods of analysis 2. Use the method and create a royal jelly reale nei prodotti alimentari. Bollettino dei Chimici Igienisti standard, based measurements on royal jelly samples produced in CALVARESE, S; FORTI, A F; SCORTICHINI, G; DILETTI, G (2006) different countries. To this end, the UNI (Italian certification body) is Chloramphenicol in royal jelly: analytical aspects and presently drawing up standards for these methods based on the occurrence in Italian imports. Apidologie 37: 673-678. DHARMANANDA, S (2003) Traces of Chloramphenicol in Chinese Bee Products: Origin, Development and Resolution, 1-5. Acknowledgements
FERIOLI, F; MARCAZZAN, G L; CABONI, M F (2007) Determination of The authors are grateful for the collaboration of the following (E)-10-hydroxy-2-decenoic acid content in pure royal jel y: a comparison between a new CZE method and HPLC. Journal of separation Science: 30, 1061 – 1069. Tseko Ivanov, Giovanni Lercker, Yanina Macebo, Emanuele Marconi, FLECHE, C; CLÉMENT, M C; ZEGGANE, S; FAUCON, J P (1997) Monique Morlot, Jun Nakamura, Giulio Sesta, Jozef Simuth and Contamination of bee products and risks for human health: the situation in France. Revue Scientifique et Technique de l’Office International des Epizooties 16: 609-619. GARCIA-AMOEDO, L H; ALMEIDA-MURADIAN, L B (2002) Comparação de metodologias para a determinação de References
umidade em geléia real. Química Nova 25: 676-679. GARCIA-AMOEDO, L H; ALMEIDA-MURADIAN, L B (2003) ANTINELLI, J F; ZEGGANE, S; DAVICO, R; ROGNONE, C; FAUCON, J Determination of trans 10-hydroxy-2-decenoic acid (10- P; LIZZANI, L (2003) Evaluation of (E)-10-hydroxydec-2- HDA) in Brazilian royal jelly. Ciencia y tecnología de enoic acid as a freshness parameter for royal jelly. GARCIA-AMOEDO, L H; ALMEIDA-MURADIAN, L B (2007) BENFENATI, L; SABATINI, A G; NANETTI, A (1986) Composizione in Physicochemical composition of pure and adulterated royal sali minerali della gelatina reale. Apicoltura 2: 129-143. BLOODWORTH, B C; HARN, C S; HOCK, C T; BOON, Y O (1995) GENC, M; ASLAN, A (1999) Determination of trans-10-hydroxy-2- Liquid chromatographic determination of trans-10-hydroxy decenoic acid content in pure royal jelly and royal jelly -2-decenoic acid content of commercial products products by column liquid chromatography. Journal of containing royal jelly. Journal of AOAC International GRILLENZONI, F G (2002) Prodotti dell’alveare: aspetti economici e BOGDANOV, S; BIERI, K; GREMAUD, G; IFF, D; KÄNZIG, A; SEILER, di mercato. In Sabatini, A G; Carpana, E (a cura di) I K; STÖCKLI, H; ZÜRCHER, K (2004) Swiss Food Manual: prodotti dell’apicoltura. LEADER II (Galizia, Portogal o, Gelée Royale. Bienenprodukte, BAG (Swiss Federal Office Italia); Portodemouros, Spagna; pp. 99-105. HOWE, S R; DIMICK, P S; BENTON, A W (1985) Composition of BONOMI, A; MARLETTO, F; LUCCELLI, L; ANGHINETTI, A; BONOMI, freshly harvested and commercial royal jelly. Journal of A; SABBIONI, A (1986) Composizione chimico- bromatologica della gelatina reale in rapporto alla flora IVANOV, T S; CHERVENAKOVA, I (1985) La quantité de macro et nettarifera e pollinifera. La Rivista della Società Italiana di micro éléments contenus par le miel, la gelée royale et le Scienza dell'Alimentazione 15(1-2): 53-62. pol en. In XXX Congres International d’Apiculture Nagoya, Apimondia, Bucarest Sabatini, Marcazzan, Caboni, Bogdanov, Almeida-Muradian KOSHIO, S; ALMEIDA-MURADIAN, L B (2003) Aplicação da CLAE para MESSIA, M C; CABONI, M F; MARCONI, E (2005) Storage stability determinação do ácido 10-hidróxi-2-decenóico (10-HDA) em assesment of freeze dried RJ by furosine determination. geléia real pura e adicionada a mel brasileiro. Química Nova Journal of Agriculture and Food Chemistry 53: 4440-4443. NATION, J L; ROBINSON, F A (1971) Concentration of some major KARAALI, A; MEYDANOGLU, F; EKE, D (1988) Studies on composition, and trace elements in honeybees, royal jelly and pollen, freeze-drying and storage of Turkish royal jel y. Journal of determined by atomic absorption spectrophotometry. Journal of Apicultural Research 10(1): 35-43. LERCKER, G (2003) La gelatina reale: composizione, autenticità ed PAMPLONA, L C; AZEDO, R A B; OLIVEIRA, K; GARCIA-AMOEDO, L adulterazione. In Atti del Convegno “Strategie per la H; ALMEIDA-MURADIAN, L B (2004) Physicochemical valorizzazione dei prodotti dell’alveare”. Università degli analyses indicated to the quality control of royal jelly with Studi del Molise; Campobasso; pp. 67-81. honey. Ciencia y tecnología de alimentos 24: 608-612. PLANTA, VON A (1888) Über den Futtersaft der Bienen. Hoppe- LERCKER, G; CAPELLA, P; CONTE, L S; RUINI, F; GIORDANI, G Seylers Zeitschrift fur Physiologische Chemie 12: 327-354. (1981) Components of royal jel y: I. Identification of the POURTALLIER, J; DAVICO, R; ROGNONE, M C (1990) Les analyses dans le contrôle de pureté de la gelée royale. Abeille de LERCKER, G; CAPELLA, P; CONTE, L S; RUINI, F; GIORDANI, G (1982) Components of royal jel y: II. The lipid fraction, REYBROECK, W (2003) Residues of antibiotics and sulphonamides in hydrocarbons and sterols. Journal of Apicultural Research honey on the Belgian market. Apiacta 38: 23-30. www.apimondia.org/apiacta/articles/2003/reybroek.pdf LERCKER, G; VECCHI, M A; PIANA, L; NANETTI, A; SABATINI, A G RICCIARDELLI D'ALBORE, G (1986) Analisi microscopica del miele e (1984a) Composition de la fraction lipidique de la gelée della gelatina reale: possibilità di applicazione e limiti. Atti royale de larves d’abeilles reines et ouvrières (Apis mellifera della Accademia gioenia di Scienze Naturali in Catania ligustica, Spinola) en fonction de l’âge des larves. Apidologie RICCIARDELLI D'ALBORE, G; BATTAGLINI, M; BERNARDINI, M LERCKER, G; VECCHI, M A; SABATINI, A G; NANETTI, A (1984b) (1978) Origine géographique de la Gelée royale. Controllo chimico analitico della gelatina reale. Rivista di SERRA BONVEHI, J (1990) Estudio de la proteina y aminoacidos LERCKER, G; CABONI, M F; VECCHI, M A; SABATINI, A G; NANETTI, libres de la jalea real. Anales de Bromatologia 42(2): 353- A; PIANA, L (1985) Composizione della frazione glucidica della gelatina reale e della gelatina delle api operaie in SERRA BONVEHI, J (1991) Study of adulteration of royal jelly with relazione all’età larvale. Apicoltura 1: 123-139. other honey bee products and water. Prod. Sanidad Anim. LERCKER, G; SAVIOLI, S; VECCHI, M A; SABATINI, A G; NANETTI, A; PIANA, L (1986) Carbohydrate determination of royal jelly SERRA BONVEHI, J (1992) Azucares, acidez y pH de la jalea real. by high resolution gas chromatography (HRGC). Food SERRA BONVEHI, J; ESCOLA JORDA, R (1991) Studie über die LERCKER, G; CABONI, M F; VECCHI, M A; SABATINI, A G; NANETTI, mikrobiologische Qualität und bakteriostatische Aktivität A (1993) Caratterizzazione dei principali costituenti della des Weiselfuttersaftes (Gelée Royale): Beeinflussung durch organische Säuren. Deutsche Lebensmittel- LI, J K; WANG, T; PENG, W J (2007) Comparative analysis of the effects of different storage conditions on major royal jelly SESTA, G (2006) Determination of sugars in royal jel y by HPLC. proteins. Journal of Apicultural Research 46 (2): 73-80. SIMUTH, J; BILIKOVA, K; KOVACOVA, E; KUZMOVA, Z; SCHRODER, LOUVEAUX, J; MAURIZIO, A; VORWOHL, G (1978) Methods of W (2004) Immunochemical approach to detection of melissopalynology. Bee World 59(4): 139-157. adulteration in honey: Physiologically active royal jelly MARCONI, E; CABONI, M F; MESSIA, M C; PANFILI, G (2002) protein stimulating TNF- alpha release is a regular Furosine: a suitable marker for assessing the freshness of component of honey. Journal of agricultural and food Royal Jelly. Journal of Agricultural and Food Chemistry STOCKER, A (2003) Isolation and characterisation of substances MATSUI, M (1988) Decreasing effect of honey on hydroxy acids in from Royal Jely. PhD Thesis; Université d'Orléans; Orléans (France). royal jelly products. Shokuhin Eiseigaku Zasshi TAKENAKA, T; ECHIGO, T (1980) General chemical composition of the royal jelly. Bulletin of the Faculty of Agriculture, MATSUKA, M (1980) Royal jelly. Honeybee Science 1(1): 31-41. MATSUKA, M; NAKAMURA, J (1990) Oxytetracycline residues in VECCHI, M A; SABATINI, A G; GRAZIA, L; TINI, V; ZAMBONELLI, C honey and royal jelly. Journal of Apicultural Research (1988) Il contenuto in vitamine come possibile elemento di caratterizzazione della gelatina reale. Apicoltura MESSIA, M C; CABONI, M F; MARCONI, E (2003) Valutazione della freschezza della gelatina reale. In Atti del Convegno WU, G; LI, Y; LIU, G (1991) The immunoregulative effect of royal “Strategie per la valorizzazione dei prodotti dell’alveare”. jel yacid, 778. Zhongguo Yaoke Daxue Xuebao Università degli Studi del Molise; Campobasso; pp. 83-92.

Source: http://ihc-platform.net/sabatinijaas2009.pdf