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.,
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
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
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
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
factors (biological factors tied to the bees).
Another fundamental aspect lies in the possibility of defining a
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;
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Given the product’s high water content, the composition values are
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also proposed for the freeze-dried sample. This enables a more
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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).
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methods of analysis 2. Use the method and create a royal jelly
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Natural Muscle Network Banned Substances & Drug Testing Guidelines OCB/NANBF/IFPA DRUG TESTING GUIDELINES Drug testing screening methods used at Organization of Competitive Bodybuilders (OCB), North American Natural Bodybuilding Federation (NANBF) and International Fitness & Physique Association (IFPA) events are a means to determine eligibility. If competitors can not successfully pass
COGER SAS79 rue des Morillons75015 ParisTel: 01 45 33 67 17: Fax: 01 45 32 71 04Frais de port30 € de frais de port pour les commandes < à 250 € HTFranco de port pour les commandes > à 250 € HT. Prod. No. Product Name Acarbose from Actinoplanes sp. ca. 100 FIP-AIU/mgAcarbose from Actinoplanes sp. ca. 100 FIP-AIU/mg contact dried, Acarbose from Actinoplanes sp. ca. 100 FIP-