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ORIGINAL RESEARCH ARTICLE
Quality and standardisation of Royal Jelly
Anna Gloria Sabatini
1*
, Gian Luigi Marcazzan
1
, Maria Fiorenza Caboni
2
,
Stefan Bogdanov
3
, 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: annagloria.sabatini@entecra.it
Journal of ApiProduct and ApiMedical Science
1(1): 1-6 (2009) © IBRA 2009
DOI: 10.3896/IBRA.4.1.01.04
Introduction
Given the exceptional biological properties attributed to it, royal jelly
(RJ) has considerable commercial appeal and is today utilised in
many sectors, ranging from the pharmaceutical and food industries
to the cosmetic and manufacturing sectors. This has resulted, among
other things, in large-scale importation in countries where production
is insufficient to meet domestic demand. Research capabilities thus
need to be reinforced to permit both a reliable qualitative and
quantitative evaluation of the different components and the
implementation of analytical tests on commercially available products
– RJ on its own or as an additive to new or traditional products – also
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
world producer and exporter of RJ, which it sells at highly
competitive prices. Chinese production of RJ is estimated as 2 000
t/year (a quantity that represents over 60% of production
worldwide), almost all of which is exported to Japan, the United
States and Europe. Other countries like Korea, Taiwan and Japan are
important producers and also exporters. Elsewhere in the world, RJ is
produced mainly in Eastern Europe and to a lesser extent in Western
Europe and in America: Mexico, in particular, is quite a big producer.
Numerous studies have been dedicated to RJ since as far
back as the late 19
th
century (Planta, 1888; Lercker, 2003). However,
it is difficult to bring together the data collected by different authors
into an organic whole, as the data themselves are not always
comparable due to the lack of homogeneity among the materials
used, the different sampling procedures and production conditions.
Additional complicating factors are the multiplicity of experimental
conditions, as well as the diversity of the analytical methods used
and their continual evolution.
Knowledge of the composition of recently produced RJ is
essential in order to define a standard composition, evaluate the
quality of commercial products and detect the presence of RJ in other
products which containing it.
At present some countries, like Switzerland (Bogdanov
et al
.,
2004), Bulgaria, Brazil (Brasil Leis e decretos, 2001) and Uruguay
have defined national standards for this product. A group of the
International Honey Commission is dealing presently with royal jelly
standardisation.
Studies on royal jelly quality
In the 1980s a workgroup was formed in Italy which has devoted
much effort to the study of RJ (Lercker
et al
., 1981; Lercker
et al
.,
1982; Lercker
et al
., 1984a; Lercker
et al
., 1984b; Lercker
et al.
,
1985; Lercker
et al
., 1986; Vecchi
et al
., 1988; Lercker
et al
., 1993;
Antinelli
et al
., 2003; Boselli
et al
., 2003; Lercker, 2003). The data
presented in this article refer to the results obtained by the Italian
group cited, completed by findings of researchers from other
countries.
Samples of recently produced, commercial grade RJ directly
gathered from specialised beekeeping facilities located in different
Italian regions were used both for the purpose of developing
methods and conducting the analyses. The same samples were used
to assess the changes occurring in RJ during storage.
Apart from this project, other studies were also carried out.
Most of them were concerned with RJ authenticity. RJ adulteration is
the most important quality problem. Adulteration by the nursing
jellies for worker and drones is improbable because of the very little
amounts available for harvest. Adulteration with honey is more
probable, causing an increase of the sugar values, the other values
being lowered (Serra Bonvehi, 1991). The most important quality
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 (Antinelli
et al
.,
2003). This decrease is higher in honey containing RJ (Matsui,
1988). Thus, the determination of all fatty acids, as carried out in
the Italian studies (Lercker
et al
., 1981; Lercker
et al
., 1993), might
be the better approach that the determination of 10-HDA only.
It was recently reported that authenticity of RJ production
can be determined by measuring of the ratios stable isotopes of the
elements C and N (Stocker, 2003). The authenticity of production
can be measured by determining the fatty acid composition of RJ
(Howe
et al
., 1985; Lercker
et al
., 1993).
The geographical authenticity can be determined also by
pollen analysis (Ricciardelli d'Albore
et al.
, 1978; Ricciardelli
d'Albore, 1986). The
87
Sr/
86
Sr ratios indicate also the geographic
regions of the samples (Stocker, 2003).
The amount of pollen, as well as visible wax and larvae
particles should be minimal. RJ has relatively low concentration of
bacteria (Serra Bonvehi and Escola Jorda, 1991).
The parameters investigated concerned in the above
mentioned studies concern the organoleptic characteristics and
physicochemical properties as well as the following composition
factors:
Water content Determined by freeze-drying (Messia
et al
.,
2005), Karl Fischer (Ferioli
et al
., 2007), vacuum
oven, dessication (Garcia-Amoedo and Almeida-
Muradian, 2002, 2007)
Total protein Nitrogen determined with the Kjeldahl method
(Lercker
et al
., 1992-93; Garcia-Amoedo and
Almeida-Muradian, 2007). Free amino acids
determined by ion chromatography (Boselli
et
al
., 2003)
Carbohydrates Determined by gas (Lercker
et al
., 1993)
or
liquid chromatographies (Sesta
,
2006)
Lipids Determined as free and total organic acids by
gas chromatography (Lercker
et al
., 1992-93) or
as total lipids, by solvent extraction (Karaali
et
al
., 1986; Garcia-Amoedo and Almeida-
Muradian, 2007)
10-HDA Determined by HPLC (Bloodworth
et al
., 1995;
Genc and Aslan, 1999; Koshio and Almeida-
Muradian, 2003; Garcia-Amoedo and Almeida-
Muradian, 2003, 2007; Pamplona
et al
., 2004)
Minerals Determined by atomic absorption (Benfenati
et al.
,
1986)
Acidity Titration method (Serra-Bonvehi, 1992)
Sediment analysis Microscopical analysis (Ricciardelli d’Albore, 1986)
Furosine, (Marconi
et al
., 2002)
Contamination
There are very few studies concerning the possible contamination of
RJ. The content of RJ contaminants, compared to other bee products,
is relatively low (Fleche
et al
., 1997). Recently, the problem of honey
and RJ contamination by antibiotics has arisen. Although most studies
concern residues in honey, antibiotic use in the colony can
contaminate also royal jelly (Matsuka and Nakamura, 1990). On the
other hand, experience has shown that RJ residue analysis is difficult
and that old analysis methods are questionable. There are very few
publications on antibiotic residues in RJ, mainly on chloramphenicol
(Dharmananda, 2003; Reybroeck, 2003; Calvarese
et al
., 2006). The
first two papers do not report details, only in the last publication
details on the methods and the contamination levels are given. The
presence of chloramphenicol (CA) was detected in 29 out of 35 tested
samples imported in Italy, the concentrations ranging from 0.6 μg/kg
to 28 μg /kg, with an average content of 6.1 μg/kg. As antibiotics are
not allowed 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
established an MPRL of 0.3 μg/kg. By using method developed by
Calvarese and coworkers (Calvarese
et al
., 2006) this MPRL can also
be used for royal jelly.
Composition and quality criteria
for royal jelly
Organoleptic description and physical characteristics
RJ appears as a whitish substance with a gelatinous consistency,
often not homogenous due to the presence of undissolved granules of
varying size. It has a distinctively sharp odour and taste.
It is partially soluble in water and highly acidic (pH 3.4-4.5)
with a density of 1.1 g/mL (Lercker, 2003).
Main components
The composition of the main constituents of RJ, proteins,
carbohydrates and lipids is reported in the literature (Takenaka and
Echigo, 1980; Bonomi
et al
., 1986; Pourtallier
et al
., 1990; Lercker,
2003, Garcia-Amoedo and Almeida
-Muradian, 2007).
2 Sabatini, Marcazzan, Caboni, Bogdanov, Almeida-Muradian
The values obtained by the various authors are fairly in agreement,
notwithstanding the high variability displayed by some parameters
(sugars and lipids). It should be kept in mind that the reported
findings refer to different number of samples taken in different
places and at different times of production and that different
methods of sampling and analysis were used. Moreover, RJ is
naturally inhomogeneous.
Our own analyses of RJ samples of different geographical
origins showed no differences in composition such as to distinguish
one product from another.
It may similarly be affirmed that environmental conditions
do not significantly influence the main components.
Water
Water content shows to be fairly uniform, greater than 60%, and
with an activity (a
w
) above 0.92, in spite of which RJ displays
considerable microbial stability. The constancy of the moisture
content is basically assured, inside the hive, by the continuous
provision of fresh supplies of this substance by nurse bees, by the
natural hygroscopicity of RJ and the entire colony’s efforts to
maintain a level of ambient moisture; moreover the non solubility of
some compounds can explain the variations in water content.
Proteins
From a quantitative viewpoint, proteins (27-41%) represent the
most important portion of the dry matter of RJ.
The amino acids present in the highest percentages were
proline, lysine, glutamic acid, β-alanine, phenylalanine, aspartate
and serine (Boselli
et al
., 2003). The concentration of series D amino
acids was below the detection limit of the method (0.1mg/g of RJ) in
all samples.
The study aimed to assess how this parameter evolved
during storage of the product. No significant changes were observed
in the overall concentration of free amino acids in RJ stored at 4°C
for 10 months. However, in the same samples stored at room
temperature, the proline and lysine content showed an increase in
the first three months and after 6-10 months decreased to levels
slightly lower than those in the control samples. This suggests that,
in favourable temperature conditions, a proteolytic enzymatic
activity continues to occur over time.
Carbohydrates
On average this portion accounts for 30% of the dry matter of RJ.
However, while the components are highly constant in qualitative
terms, considerable variability may be observed from a quantitative
standpoint.
As in honey, the monosaccharides fructose and glucose are
the main sugars. They often account for over 90% of the total
sugars and, of the two, fructose is prevalent. Sucrose is always
Quality and standardisation of Royal Jelly 3
present but often in highly variable concentrations. It is also possible
to find oligosaccharides such as trehalose, maltose, gentiobiose,
isomaltose, raffinose, erlose, melezitose; though present in very small
concentrations they are useful for identifying a characteristic pattern,
which is comparable to that of honey and in some cases indicative of
the genuineness of the product.
Lipids and 10-Hydroxy-2-decenoic acid (10-HDA)
This fraction is likewise present in fairly modest, variable
concentrations (8-19% of dry matter), but no doubt represents the
most important of RJ 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
and dicarboxylic acids with 8 and 10 carbon atoms, which show a
characteristic arrangement (Lercker
et al
., 1992-93).
Hydroxy acids with 10 carbon atoms (10-hydroxydecenoic
and 10-hydroxy-2-decenoic acid) above all can be found in high
concentrations. Not only may they be ascribed a role as a marker
component, but they have also been identified as responsible for
important biological activities tied to the development strategies of
the colony (Wu
et al. 1991)
The identification of this fraction – in particular as regards the
pattern and quantitative analysis of free organic acids – is believed to
represent the criteria of choice for defining the genuineness of RJ and
the presence of RJ in other products, be they foods or cosmetics
(Caboni
et al
., 1994). The analyses we performed showed that the
composition remained stable for as long as 2 years, regardless of
whether the samples were stored at 4°C or at room temperature.
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
room temperature for 12 months. It is difficult to evaluate such a
reduction in a sample in the control phase. Moreover it is difficult to
use 10-hydroxy-2-decenoic-acid decrease as a freshness marker
because their variable amount on fresh RJ. Both HPLC and
electrophoretic analysis of 10-HDA showed that samples of RJ from
extra-european origin contain smaller amount of this compound; this
evidence was confirmed measuring total lipids after organic extraction
(Ferioli
et al
., 2007).
Minerals
Ash content represents 0.8-3% of RJ (fresh matter) (Messia
et al
.,
2003; Garcia-Amoedo and Almeida-Muradian, 2007). The major
elements are, in descending order: K, Ca, Na, Mg, Zn, Fe, Cu and Mn
(Nation and Robinson, 1971; Ivanov and Chervenakova, 1985;
Benfenati
et al
., 1986), present in specific ratios such as K/Na and
Ca/Mg.
The hypotheses regarding the quantitative presence of these metals
have focused on factors outside the colony (environment,
procurement of food, production period) and to some extent internal
factors (biological factors tied to the bees).
Authenticity
The main quality factors of RJ have been described and studies have
revealed the importance of the lipid fraction as a marker and hence
a criterion by which to determine the product’s genuineness.
Presently, 10-HDA is mostly used for routine testing of RJ
authenticity. However, the concentration of this acid varies in wide
limits. Further studies are necessary to determine whether the
determination of the stable isotopes of the elements C and N
(Stocker, 2003) is a promising approach for the determination of the
authenticity of production. Adulteration by honey results in a general
diminution of proteins and lipids and a relative increase of sugars
(Serra-Bonvehi, 1991).
Adulteration with more than 25% of yoghurt, egg white,
water and corn starch slurry can be detected by the enhancement of
moisture, diminishing in lipid, protein and 10-HDA content as well as
the insolubility in alkaline medium. (Garcia-Amoedo and Almeida-
Muradian, 2007).
Furthermore, microscopic analyses of RJ sediment, applied
according to the basic principles of melissopalynology (Louveaux
et
al
.,
1978
; Ricciardelli, 1986) and in particular the identification of the
pollens it contains, make it possible to define the geographical
origins of the product and detect mixtures where they occur. Pollen
identification is made easier by the fact that only a few countries
actually produce RJ and specialists are capable of formulating their
respective characteristic pollen associations.
Another promising parameter for the evaluation of RJ
authenticity is the presence of apalbumin (Simuth
et al
., 2004). This
marker, if confirmed by further research, could gain high
importance.
4 Sabatini, Marcazzan, Caboni, Bogdanov, Almeida-Muradian
Freshness definition
Another fundamental aspect lies in the possibility of defining a
parameter of RJ freshness.
It has been noted that the macroscopic composition of RJ is
fairly stable on the whole but also variable, above all as far as certain
components are concerned. Thus it is not a suitable parameter for
defining product freshness.
For the latter purpose, experiments were conducted on RJ
samples stored at 4 and 20°C over a period of 24 months to assess
changes in the content of the enzyme glucose oxidase. The results
obtained showed that the enzyme contained in RJ is influenced both
by storage temperature and time. At 20°C it had decreased
significantly after one month and degraded completely after one
year. Even at 4°C there was an evident, albeit modest, reduction in
the enzyme.
The determination of glucose oxidase is analytically very
simple and thus within the capabilities of all laboratories. This
method could be used to evaluate the product’s freshness; however,
further investigation must first be conducted into the natural
variability of this component in the fresh product (Boselli
et al
.,
2002). Marconi
et al.
(2002) quoted several experiments were
performed to evaluate the possibility of using furosine content as a
marker for RJ freshness.
The value of furosine, a product of Maillard’s reaction, proved
very low (from 0 to 10 mg/100g of protein) in freshly produced RJ
samples (Messia
et al
., 2003) but increased over time and in relation
to temperature. Specifically, the content rose to as high as 500
mg/100g of protein after 18 months’ storage at room temperature
and 50 mg/100g at 4°C. Samples taken from store shelves showed
values ranging from 40 to 100 mg/100g protein. By contrast, freeze-
dried RJ showed strong tendency to form furosine during storage
(Messia
et al
., 2005).
Table 1: Royal Jelly Composition
Fresh lyophilized
Water % 60 – 70 < 5
Lipids % 3 – 8 8 – 19
10-Hydroxy-2-decenoic acid (10-HDA) % > 1,4 > 3,5
Protein % 9 – 18 27 – 41
Fructose + glucose+ sucrose % 7 – 18 -
Fructose % 3 – 13 -
Glucose % 4 – 8 -
Sucrose % 0,5 – 2,0 -
Ash % 0,8 - 3,0 2 – 5
pH
3,4 - 4,5 3,4 – 4,5
Acidity (ml 0.1N NaOH/g) 3,0 - 6,0 -
Furosine (mg/100g protein) < 50* -
Table 1 shows some data from literature that could be used as a
preliminary proposal for fresh and lyophilized royal jelly standards.
Data come from many countries investigations and regulations but
for the establishment of a paper general standard, further
investigation is needed.
Given the product’s high water content, the composition values are
also proposed for the freeze-dried sample. This enables a more
direct comparison of data; plus RJ is also marketed in this form.
Although the overall analytic data confirm that exposure to a
temperature of 4°C causes no alterations in RJ composition, recently
it was also shown that only storage of RJ in frozen state prevents
decomposition of biologically active RJ proteins and thus RJ should
be frozen as soon as it is harvested (Li
et al.,
2007).
The next steps should be: 1. Validate the respective
methods of analysis 2. Use the method and create a royal jelly
standard, based measurements on royal jelly samples produced in
different countries. To this end, the UNI (Italian certification body) is
presently drawing up standards for these methods based on the
available know-how.
Acknowledgements
The authors are grateful for the collaboration of the following
researchers:
Tseko Ivanov, Giovanni Lercker, Yanina Macebo, Emanuele Marconi,
Monique Morlot, Jun Nakamura, Giulio Sesta, Jozef Simuth and
Jürgen Wehlitz.
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