Part of this paper was presented during the Euro Food Chem

IX Conference, 2426 September 1997, Interlaken, Switzerland

J. Prodolliet 7 C. Hischenhuber (Y)
Nestec Ltd, Nestl Research Center, Vers-chez-les-Blanc,
CH-1000 Lausanne 26, Switzerland
Z Lebensm Unters Forsch A (1998) 207: 112 Q Springer-Verlag 1998
REVIEW
Jacques Prodolliet 7 Claudia Hischenhuber
Food authentication by carbohydrate chromatography
Received: 1 December 1997 / Revised version: 9 February 1998
Abstract This paper reviews the application of carbo-
hydrate chromatography for the assessment of the au-
thenticity of honey, maple syrup, fruit juice, mu-
shrooms, UHT milk, natural gums, and soluble coffee.
Examples are given for each commodity and the limita-
tions of the different techniques are discussed.
Key words Carbohydrate chromatography 7
Adulteration 7 Authenticity
Introduction
Economic adulteration has been practised for centuries
by unscrupulous farmers, manufacturers or traders who
seek substantial benefits by extending or substituting
expensive food products with low cost materials. This
has, of course, triggered the development of new analy-
tical methods for the assessment of food authenticity.
Various techniques such as GC, HPLC, stable isotope
analysis, electrophoresis, immunoassays, DNA analysis,
and NIR spectroscopy have been developed and many
different classes of compounds have been considered as
tracers of adulteration (e.g. proteins, carbohydrates,
fatty acids, pigments and organic acids). This paper re-
views the application of carbohydrate chromatography
as a specific tool for the assessment of authenticity.
However, one analytical technique is generally not suf-
ficient to detect all types of adulteration commonly
practised, and in most cases only a full battery of ana-
lyses can assess the authenticity of a product.
Carbohydrate chromatography has proven particu-
larly useful for the authentication of fruit juices, honeys
and maple syrups, as large amounts of carbohydrates,
mainly in the form of simple sugars such as fructose,
glucose and sucrose, are found in these commodities.
Not surprisingly, the most common technique of adul-
teration consists of adding inexpensive sweeteners, the
sugar compositions of which mimic those found in the
authentic products. Sweeteners can be detected by the
distortion of the natural sugar profile or by the pres-
ence of fingerprint oligosaccharides.
Different substitutes are used to adulterate com-
modities exhibiting a more complex carbohydrate com-
position. In certain cases, specific carbohydrates can
still be used as markers. Undeclared addition of coffee
husks in soluble coffee, detected by the presence of
high levels of xylose and mannitol, is a good example.
The use of carbohydrate chromatography has also
been reported for the detection of mislabelling of the
botanical and geographical origins of foods. For exam-
ple, distortion of the natural mannose to galactose ratio
in locust bean gum strongly suggests the addition of
guar gum.
Finally, the process by which a food product is man-
ufactured is sometimes precisely regulated by official
bodies. Examples where carbohydrate analysis allows
the manufacturing process to be identified have also
been reported, e.g. in discriminating between ultra
heat-treated (UHT) and sterilised milk according to
their lactulose contents.
The following sections give detailed examples of ad-
ulteration which can be detected using carbohydrate
markers and briefly discuss the chromatographic tech-
niques applied. The results presented were taken both
from the literature and from the authors own studies.
Authenticity of honey
Codex Alimentarius defines honey as the natural
sweet substance produced by honey bees from nectar of
blossoms or from secretions of living parts of plants or
excretions of plant sucking insects on the living part of
plants, which honey bees collect, transform and com-
2
bine with specific substances of their own, store and
leave in the honey comb to ripen and mature [1]. Hon-
ey has been appreciated since the stone age and, up to
the eighteenth century, was nearly the only concen-
trated sweetener available. The sale of adulterated or
artificial honey is widespread and has been reported for
many centuries.
Nowadays a number of inexpensive sweeteners and
syrups are commercially available for fraudulently re-
placing the natural carbohydrates of honey. The addi-
tion of sucrose can be detected by simply measuring its
level in the honey; even a small amount added will be
evaluated as being above the natural level. HPLC de-
termination of sucrose, glucose and fructose was colla-
boratively tested in 1979 [2]. As 8595% of the total
carbohydrate content of honey is composed of fructose
and glucose, the average ratio being 1.2: 1.0 [3], high
fructose syrups (HFS) and invert sugars (IS) are the
most favoured adulterants. Beet or cane total IS are
produced from refined beet or cane sucrose by either
acid or enzymatic hydrolysis resulting in a finished
product containing a 1: 1 ratio of glucose:fructose and
low levels of sucrose. HFS are mainly produced by the
enzymatic hydrolysis of either corn, potato, rice or
wheat starch. The resulting glucose syrup is partially
converted to fructose by enzymatic treatment. The fin-
ished products consists of a mixture of glucose and
fructose with ratios of about 0.81.4.
High fructose corn syrup (HFCS) can be detected by
carbon stable isotope ratio analysis (CSIRA). As an al-
ternative, various chromatographic methods based on
the consideration that the di- and oligosaccharide pro-
files of honey and syrups are different have been pro-
posed. Some of these methods are also applicable to
the detection of beet IS that cannot be distinguished
easily from honey by stable isotope analysis tech-
niques.
Doner et al. [4] measured the maltose/isomaltose ra-
tios of honeys and HFCS by GC and showed that ratios
above 0.51 indicated adulteration. Kushnir [5] de-
scribed a TLC separation of oligosaccharides after
clean-up on a charcoal-Celite column, while Lipp et al.
[6] separated these substances by reversed-phase
HPLC. Both methods detected low percentages of
HFCS in honey. More recently, methods using high-
performance anion-exchange chromatography with
pulsed amperometric detection (HPAE-PAD) [7] and
capillary GC [8] have been developed by Low and co-
workers. The HPAE-PAD method involved charcoal-
Celite column clean-up and separated specific marker
peaks for cane IS and HFCS. Capillary GC was per-
formed on diluted, freeze-dried and silylated samples.
Specific marker peaks for IS that were either absent or
present in low amounts in authentic honey were re-
ported. As a matter of fact, all of these procedures yield
quite complex chromatograms due to the presence of a
large number of naturally occurring oligosaccharides in
authentic honeys and, therefore, careful interpretation
of the results is necessary.
Fig. 1A, B HPLC chromatograms of honeys. A Honeydew. B
Acacia. F Fructose; G glucose; S sucrose; TU turanose; MpMA
maltose; TR trehalose; I isomaltose; E erlose; ME melezitose; R
raffinose; x maltulose, nigerose. Reprinted from Mitt Geb
Lebensmittelunters Hyg [12] with the kind permission of the
Bundesamt fr Gesundheit Facheinheit Lebensmittel und
Gebrauchsgegenstnde, Bern
The principal source of honey is nectar, and blossom
honey is preferred in most parts of the world [911].
However, in central Europe honeydew honey prevails
and is more highly prized than blossom honey [911].
Therefore, mislabelling of the origin of a honey is an-
other authenticity issue.
Honeydew honey can be distinguished from blossom
honey by its higher pollen count, electrical conductivity
and salt content, a deeper colour, and by the presence
of spores of sooty moulds, pieces of fungal hyphae and
algal cells [1, 4]. Moreover, it is dextrorotatory, while
blossom honey is invariably laevorotatory [911]. This
is due to major differences in the carbohydrate profile.
Indeed, it was found by many authors that honeydew
honey contains lower levels of fructose and glucose, but
higher levels of oligosaccharides, in particular the tris-
accharides melezitose and raffinose [914].
Bogdanov and Baumann [12] determined the mono-,
di- and trisaccharide profiles of 118 blossom honeys
and 38 honeydew honeys of different origins, all sold
on the Swiss market. Carbohydrates were separated by
HPLC on an amino column using a mixture of acetoni-
trile and water as eluent, and were detected by a refrac-
tive index (RI) detector. Melezitose and raffinose were
found to be very characteristic of honeydew honey.
Melezitose ranged from 1.0% to 22.0% (average 5.3%)
in honeydew honey, and from 0.1% to 1.0% (average
0.5%) in blossom honey. Raffinose ranged from 0.1%
to 1.5% (average 0.5%) in honeydew honey, and was
not detected in any of the blossom honeys analysed.
Examples of the chromatograms are given in Fig. 1.
These results were confirmed by Fldhzi [13] on 34
blossom honeys from different floral sources and one
honeydew honey, using a similar HPLC technique. An-
other trisaccharide, namely erlose, has also been sug-
gested as a tracer of honeydew honey. However, large
amounts of this carbohydrate were found in blossom
honey from alfalfa, alsike and trefoil [12, 15]. In the lat-
ter study, the carbohydrates were analysed by HPAE-
PAD. The technique allows a good separation of 20
honey oligosaccharides and is more sensitive than
3
Fig. 2 Separation of unifloral honeys using canonical
discriminant analysis. Numbered O Group centroids; c bramble,
69 samples; } ling, five samples; x oil seed rape, eight samples; o
white clover, five samples; g hawthorn, two samples; l
willow-herb, one sample. Samples A and B contained 21 and 25%
bramble, respectively. Reprinted from J Chromatogr [18] with
kind permission of Elsevier, Amsterdam
HPLC-RI. The number of species of flowers around
the world that are significant sources of blossom honey
is very large, each giving very distinct organoleptic
characteristics to the final product. In most cases, the
floral source is stated because consumers prefer the fla-
vour, aroma, colour or texture of the honey from a par-
ticular flower. These so-called unifloral honeys, such as
orange blossom or acacia honey, command a premium
price and are an ideal target for stretching with cheaper
honey. For these reasons, reliable analytical tools are
needed to judge the source of each honey.
The most widely used method of authenticating the
particular floral source is based on identification of the
pollen present in the honey [9]. However, this tech-
nique requires a high level of expertise, is very time
consuming and suffers from several limitations [9].
Moreover, pollen is commercially available and a
fraudster could filter out all the pollen and add back a
pollen of his choice [9, 16]. Organoleptic evaluation
also serves to indicate the floral origin, but is too sub-
jective. Analysis of flavours, flavonoids, amino acids,
methyl anthranilate and other trace organics has also
proved fairly useful [1, 4]. In an early study, carbohy-
drate analysis by capillary GC failed to discriminate be-
tween honeys from different floral sources (alfalfa, al-
sike, canola, red clover, sweet clover, trefoil). All sam-
ples exhibited a similar oligosaccharide profile [17].
However, the number of unifloral honeys was very lim-
ited (one per source), and no multivariate statistical
analysis of the data was performed. More recently,
Goodall et al. [18] determined the carbohydrate profile
of 91 unifloral honeys from six different origins by
HPAE-PAD. Canonical discriminant analysis, perform-
ed on the 40 separated, but unidentified oligosacchar-
ides, proved successful in discriminating between hon-
eys from ling, oil seed rape and bramble (Fig. 2). How-
ever, the technique does not apply to all floral types. It
is worth mentioning that acacia honey presented by far
the highest fructose/glucose ratios among all the uniflo-
ral honeys analysed in Fldhzis study [13].
Authenticity of maple syrup
Maple syrup is produced from the sap of the sugar ma-
ple tree (Acer saccharum). Collected during the end of
the winter season, this sap is concentrated to about 677
Brix. As much as 35 l sap is required to produce about
1 l pure syrup. The major components of maple syrup
are the carbohydrates, which account for about 98% of
the total soluble solids. Sucrose is the major carbohy-
drate present, and its concentration ranges from 88%
to 100% of the total carbohydrates depending on the
syrups age and other natural, storage and processing
factors [19]. Other constituents of maple syrup are glu-
cose, fructose, oligosaccharides, organic acids, minerals,
amino acids and vitamins [19, 20]. Profitable adultera-
tion can be readily accomplished by simply adding
inexpensive sweeteners such as cane or beet sugar,
HFCS or beet medium invert sugar (BMIS).
Several methods of assessing authenticity have been
developed, including conductivity measurements, CSI-
RA [21, 22], site-specific natural isotope fractionation
nuclear magnetic resonance spectroscopy (SNIF-NMR)
[22], and mono-, di- and oligosaccharide profiling [20].
Using carbohydrate profiling it is not possible to de-
tect pure cane or beet sugar the methods of choice for
this type of adulteration are CSIRA and SNIF-NMR
but it is an interesting tool to detect HFCS and BMIS.
As non-aged pure maple syrup has a combined glucose
and fructose concentration of less than 1112% [19],
samples with monosaccharide levels greater than 12%
may be indicative of adulteration. Another approach
was reported by Stuckel and Low [19]. They showed
that oligosaccharide fingerprinting by HPAE-PAD can
be used to detect HFCS and BMIS in maple syrup. The
method was tested on 80 authentic maple syrup sam-
ples representing the major geographical regions of
production and 30 samples spiked with HFCS or BMIS.
A detection limit of about 5% for each of the sweeten-
ers was obtained.
Authenticity of fruit juice
Addition of sugar
About 98% of the total soluble solids in fruit juices are
carbohydrates. It is therefore obvious that the most
profitable adulteration of fruit juices involves partial
replacement of the natural sugars with less expensive
ones. Besides sucrose, HFS and IS, BMIS and cane me-
dium invert sugar (CMIS) are popular adulterants.
These materials are produced by either acid or enzy-
matic hydrolysis of refined sucrose resulting in a fin-
4
Fig. 3 Chromatograms of orange juice spiked with various levels
(020%) of beet medium invert sugar (BMIS). Reprinted from J
AOAC Int [31] with kind permission of AOAC International,
Arlington, USA
ished product containing a 1: 1: 2 ratio of glucose:fruc-
tose:sucrose, thus approximating to the sugar distribu-
tion of orange juice.
When assessing the authenticity of fruit juices, the
glucose: fructose: sucrose distribution is routinely
checked and compared to generally accepted guide val-
ues [23, 24]. This gives information about whether
sweeteners that unbalance the natural sugar proportion
have been added. For this analysis several HPLC pro-
cedures involving ion-exchange chromatography with
RI detection [25] or HPAE-PAD [26] are available.
Other methods published in the course of the last 6
years rely on the determination of unusual di-, and/or
tri- and tetrasaccharides formed during the manufac-
ture of syrups. Oligosaccharides may be formed chemi-
cally by the action of acid on sucrose, fructose and glu-
cose (reversion reactions), others by the use of hy-
drolitic enzymes during syrup production. The first
method introduced that used oligosaccharide finger-
printing was a HPAE-PAD method to detect BMIS in
orange juice [27]. The procedure involved a quite labo-
rious clean-up step to remove organic acids and mono-
saccharides, HPAE with gradient elution using two
analytical columns in series and PAD. Later on, the
clean-up step to eliminate monosaccharides was omit-
ted, chromatography was shortened from 3 h to 96 min,
and the method was extended to the detection of other
inexpensive sweeteners such as HFCS and cane and
beet IS [28]. The technique was also applied to other
citrus fruits such as grapefruit [29]. The original proce-
dure was further adapted by other research groups, e.g.
by White and Cancalon [30], who introduced a column
switching technique: the monosaccharides were eluted
from the first column to waste and only the di- and ol-
igosaccharides were transferred to the second analytical
column. Iuliano [31] decreased overall analysis time to
45 min by isocratic elution of the oligosaccharides after
diverting sucrose, glucose and fructose to waste after
the first analytical column. Figure 3 shows chromato-
grams of orange juice spiked with various levels of
BMIS.
However, it must be emphasised that oligosacchar-
ide profiling has several limitations. Besides the general
limitation that it will not detect the addition of pure su-
crose, there are two other important drawbacks. The
concentration of marker oligosaccharides can vary con-
siderably depending on the method of preparation of
the adulterants and consequently, the detection limit
will vary accordingly. The most worrying limitation of
this method can be summarised by the slogan heat or
beet?. Juice concentrates that have undergone impor-
tant heat treatments yield complex chromatograms
5
Fig. 4 Capillary GC chromatogram of an apple juice adulterated
with high fructose syrup from inulin (HFIS). HFIS1
(RTp17.4 min), HFIS2 (RTp23.8 min); HFIS3 (RTp28.5 min);
marker peaks for the presence of HFIS
making interpretation quite difficult. While stable iso-
tope analysis techniques (CSIRA and SNIF-NMR) suf-
fer from other limitations, they do allow the detection
of added beet or cane sucrose as well as the detection
of IS in heat-treated concentrates.
To improve oligosaccharide profiling, Low [32, 33]
developed an alternative method using capillary GC
with flame ionization detection. Specific marker peaks
were used to detect adulterants in orange and apple
juice, i.e. two unidentified disaccharides for tracing IS
and two peaks of isomaltose to trace HFS. The method
involved a column clean-up that was omitted later on
[34], freeze-drying of the sample and trimethylsilylation
of the carbohydrates. Capillary GC was performed on a
0.25 mm!30 m DB-5 column. In pure orange and ap-
ple juices the marker peaks were virtually absent and a
detection limit for HFS and IS of 5% was claimed. At
the beginning of 1996, Lows GC procedure gained
considerable popularity, when strange oligosaccharide
peaks could be observed in a number of apple juice
concentrates. These peaks were finally identified as
fructulin, a high fructose syrup from inulin (HFIS) that
had been used by unscrupulous suppliers to adulterate
apple concentrates [34]. HFIS has a high fructose/glu-
cose ratio which makes this material a good adulterant
for apple juice, which has a fructose/glucose ratio ex-
ceeding 2. Figure 4 shows a chromatogram of a com-
mercial apple juice adulterated with HFIS. The operat-
ing conditions were according to Low and Hammond
[34] without column clean-up and with hydrogen as the
carrier gas for GC. When compared to an HFIS stand-
ard, the adulteration corresponded to about 30% of the
total sugar present. There is no doubt that this oligosac-
charide profiling technique is a valuable tool for fruit
juice authenticity assessment.
Fig. 5AC Capillary GC chromatograms of apple juice
concentrates. A Authentic, untreated. B Authentic, heat treated.
C Sample B spiked with 10% invert sugar (IS). IS1
(RTp38.6 min) and IS2 (RTp39.8 min); marker peaks for the
presence of IS. Peak height ratios IS2/IS1: sample B 1.31, sample
C 1.70
However, the method suffers from limitations simi-
lar to the HPAE-PAD technique discussed earlier.
Heat-treated authentic apple juice concentrates may
show the same marker peaks as IS; only the peak ratios
differ to some extent. The marker peaks generated by
heat have a peak ratio close to 1, while those generated
by IS have a ratio greater than 3. Considerable efforts
have been undertaken to establish analytical conditions
that allow distinguishing between heat and beet [35].
Taking into consideration the reproducibility of the
evaluation of these small marker peaks and fluctuations
in the concentration of the marker oligosaccharides
present in various IS preparations and in heated apple
juices, the detection limit may vary between 5% and
more than 30% of added IS. Figure 5 shows the chro-
matograms of a heat-treated authentic apple concen-
trate and of the same concentrate spiked with 10% IS
containing quite high levels of marker oligosaccharides.
The added concentration corresponds in this case to
the detection limit.
In other juice concentrates, such as berry juice con-
centrates that are produced using enzymes, the detec-
tion of IS is almost impossible. Peaks showing the same
ratio as those originating from IS are observed in auth-
entic juices. However, in some types of berry juice the
detection of HFS is possible. Fig. 6 shows a chromato-
gram of an authentic blackcurrant concentrate and of
the same concentrate spiked with 10% of HFCS. The
second marker peak (isomaltose 2) was virtually absent
in all authentic blackcurrant juices analysed, thus allow-
ing the detection of HFCS. The detection of the differ-
ent adulterants must be carefully evaluated for each
type of juice. For example, the detection of low
amounts of HFS in pineapple juices is difficult, as low
levels of isomaltose may occur naturally in pineapples.
Summarising, it can be said that no single method
available allows the detection of all forms of sugar ad-
dition to fruit juices, but oligosaccharide profiling is a
6
Fig. 6 Capillary GC chromatograms of blackcurrant juice
concentrates. A Authentic, B sample A spiked with 10% high
fructose corn syrup (HFCS). Isomaltose 1 (RTp40.8 min) and
isomaltose 2 (RTp44.2 min); marker peaks for the presence of
HFCS. Arrows show retention times of marker peaks of invert
sugar
method that should be included in the authenticity as-
sessment of juices. Taking as an example apple juice,
Lees et al. [36] demonstrated clearly, that a whole set of
analyses is necessary to cover most types of adultera-
tion. More information will be obtained by carefully as-
sessing the authenticity of a limited number of well-de-
fined batches of fruit concentrates than by testing many
batches with a single method.
Addition of another type of fruit
Another fraud is the partial replacement of one type of
fruit juice/pulp with one which is less expensive, e.g. ad-
dition of apple or pear to expensive berry juices/pulps
or fruit preparations. In these cases, sorbitol present in
apples, pears and other fruits, but virtually absent in
berry fruits such as raspberry, strawberry, blackcurrant,
red currant or blueberry, is an excellent tracer for adul-
teration. An interesting alternative to the enzymatic
sorbitol assay is the simultaneous separation of sorbi-
tol, glucose, fructose and saccharose by HPLC with RI
detection [25] or HPAE-PAD [26].
Addition of pulpwash
In many countries, such as the United States and coun-
tries of the European Union (EU), the addition of
pulpwash to pure juice concentrates is not allowed.
Pulpwash is defined as the water-extracted soluble fruit
solids recovered in the presence of water from unfer-
mented excess fruit pulp removed during the produc-
tion of citrus juice products. US federal regulations al-
low the in-line addition of pulpwash to freshly ex-
tracted juice, while in the EU both in-line and off-line
addition is prohibited.
Various methods for the detection of orange pulp-
wash based on the quantification of components that
occur at higher levels in the pulp and peel than in the
juice have been proposed. However, due to large natu-
ral fluctuations and differences in juice extraction tech-
nology, the detection of pulpwash remains an analytical
challenge. The most well-known methods involve the
determination of water-soluble pectin, calcium, spectro-
photometric determination of the flavonoid glycosides,
HPLC determination of hesperidin and narirutin, or
evaluation of the UV/VIS absorbance and the fluores-
cence at distinct wavelengths.
An alternative to the labour-intensive carbazole
method for the determination of water-soluble pectin
[37] using HPAE-PAD [38] has been proposed. In this
method, the sample is filtered and the water-soluble
pectin is completely hydrolysed with pectolytic en-
zymes to produce free galacturonic acid. The galactu-
ronic acid is then determined by HPAE-PAD. Smaller
pectin fragments formed during enzymatic treatment in
the production of pulpwash concentrates (off-line pulp-
wash) and peel extracts are taken into account with this
procedure, while they are lost when applying the carba-
zole method. In a recent paper this method was applied
to a large number of samples and the results compared
with those obtained with the carbazole method [39]. In
addition, total galacturonic acid is measured in the ex-
tracts before the carbazole reaction. Large differences
between results obtained on the juice and on the ex-
tract would indicate the presence of enzymatically
treated pulpwash. A capillary GC method for the de-
termination of galacturonic acid in orange juice had
been described 10 years earlier by Kauschus and Thier
[40]: the polysaccharides were cleaved by methanolysis
and the resulting galacturonic acid was silylated and
analysed by capillary GC. The chromatograms were
rather complex, silylated galacturonic acid yielding four
peaks.
Another HPAE-PAD method that allows the detec-
tion of enzymatically treated pulpwash or peel extracts
in citrus juices [41, 42] by analysing the oligogalactu-
ronide pattern has been proposed.
Adulteration of mushrooms
Some culinary products, notably soups and sauces, de-
rive their excellent taste and flavour from the use of
wild mushrooms as essential ingredients. Among the
commercially available wild mushrooms, two boletus
species, namely Boletus edulis (cep, king boletus) and
Suillus luteus (brown-yellow boletus, slippery jack), are
undoubtedly the most important. However, B. edulis is
tastier and about four times more expensive than S. lu-
teus. Substitution of the latter fungi for B. edulis is,
therefore, very appealing [43].
7
B. edulis and S. luteus are sufficiently different to be
visually or microscopically identifiable. This is certainly
true for whole or large chunks of fungi. However, ex-
amination of granulates or powders used by the food
industry, in which most of the morphological character-
istics are lost, is much more difficult. More objective
analytical methods are required.
Mushrooms contain, in general, fairly high amounts
of sugar alcohols, mainly mannitol and arabitol [44].
However, their relative proportions differ from one
type of mushroom to another. Stijve [43] measured the
mannitol and arabitol contents of 20 samples of both
boletus species by high-performance thin-layer chroma-
tography (HPTLC). Mannitol ranged from 0 to 4.0%
on dry matter (o.d.m.) (average 2.4%) in B. edulis, and
from 2.0% to 7.5% o.d.m. (average 5.1%) in S. luteus.
Arabitol ranged from 0.3% to 2.0% o.d.m. (average
0.9%) in B. edulis, and from 4.7% to 20.0% o.d.m. (av-
erage 9.7%) in S. luteus. Therefore, high levels of both
sugar alcohols could be indicative of the presence of S.
luteus in B. edulis. However, the technique would only
detect high amounts ( 120%) of the cheaper fungi.
Using a very similar HPTLC technique, Andary et
al. analysed samples of Suillus granulatus, Suillus bel-
linii and S. luteus [45]. All three species presented high
levels of mannitol (4.47.0% o.d.m.) and arabitol
(12.820.0% o.d.m.), and the results obtained for S. lu-
teus fell within the upper range of Stijves data.
Substitution of chanterelle mushrooms (Cantharellus
cibarius) with the cheaper false chanterelle (Hygro-
phoropsis aurantiaca) is another potential authenticity
issue. Laub [46] suggested using the arabitol/total sol-
uble carbohydrate ratio as a tracer for the presence of
false chanterelle. He found that the ratio ranged from
0.25% to 3.00% o.d.m. (average 1.34%, np14) in chan-
terelle mushrooms, and from 27.9% to 55.8% o.d.m.
(average 40.7%, np5) in the false chanterelle. He con-
cluded that the technique could detect as little as 10%
of false chanterelle in the authentic product. Laub and
Woller [47] also suggested that a mannitol content be-
low 300 mg/100 ml infusion could indicate poor manu-
facturing practices in the canning of cultivated white
mushrooms (Agaricus bisporus).
Differentiation between UHT and sterilised milks
The EU gives specific requirements for the manufac-
ture of heat-treated milk and, in particular, defines
what a UHT and a sterilised milk must be [48]. The
Codex Alimentarius has also proposed other defini-
tions [49]. One of the main characteristics is that UHT
milk is sterilised by a continuous flow process and asep-
tically filled into sterile containers, whereas sterilised
milk is produced by in-container sterilisation. The aim
of the two treatments is to destroy all residual spoilage
micro-organisms and their spores [48]. However, it is
known that the in-container process causes a greater
chemical change in the milk than the UHT process for
Table 1 Typical levels of lactulose in UHT and sterilised milks
Study UHT milk
mg/100 ml
Sterilised
milk
mg/100 ml
Martinez-Castro and Olano [57] 1030 80200
Geier and Klostermeyer [50] 1072 87137
Andrews [51] 572 69120
Olano et al. [52] 1075 75175
Andrews and Morant [53] 1072 57173
the same sterilising effect [5052], leading to differ-
ences in organoleptic characteristics [49]. Indeed, steril-
ised milk is generally associated with a lower organo-
leptic acceptability, a stronger cooked defect and a
darker colour [53]. Therefore, indicators by which the
two types of milk may be identified are required.
Lactulose is a disaccharide formed by the isomerisa-
tion of lactose during heat treatment of milk. It has
been shown to occur in different concentrations in
UHT and sterilised milk (Table 1). For this reason, the
EU and the International Dairy Federation (IDF) are
considering the use of lactulose as an indicator for dis-
tinguishing between the two products. However, to our
knowledge, no official standards have been issued so
far, and limits below or above which milk should be
considered as UHT or sterilised are still under debate
[54]. Nevertheless, limits of 60 and 70 mg/100 ml have
been proposed by the IDF [55] and Andrews [56], re-
spectively.
Enzymatic, GC and HPLC methods have been re-
ported for the determination of lactulose in heat-
treated milk [50, 52, 58]. The provisional standards is-
sued by the International Organization for Standardiza-
tion (ISO) and the IDF both describe an HPLC meth-
od, in which lactulose is analysed on an ion-exchange
material in the lead form and quantified by RI detec-
tion [59, 60]. The procedure has been subjected to sev-
eral collaborative studies [61]. The quantification of
lactulose is complicated by the presence of huge
amounts of lactose. Recently, HPAE-PAD has been
proposed as an alternative to the official methods [62].
Using this technique, better separation of lactulose
from lactose is possible (Fig. 7), and it is much more
sensitive. Sample preparation is also more straightfor-
ward. It has been applied to a wide variety of milk
products [62] and certainly deserves further attention.
Authenticity of natural gums
Hydrocolloids are heteropolysaccharide food additives,
incorporated into a very diverse range of food formula-
tions to impart a wide variety of characteristics to the
finished product. They are mainly employed as thicken-
ers, gelling agents and stabilisers, but also as emulsif-
iers, flavour encapsulators, sugar crystallisation inhibi-
tors, or simply as food ingredients. Most of the hydro-
8
Fig. 7 HPAE-PAD chromatogram of UHT milk. Column:
CarboPac PA1, eluent: gradient of 10 mM NaOH and 0.5 mM
Zn(AcO)
2
; lactulose concentation: 27 mg/100 ml
Table 2 Carbohydrate composition for different gum exudates (%)
Species Galactose Arabinose Rhamnose Glucuronic
acid
4-O-methyl
glucuronic acid
References
Acacia senegal 3056 1646 724 1228 0.52 [63, 70, 72, 73,
7578]
Acacia seyal 3238 4151 14 6.517 15.5 [70, 72, 73, 76]
colloids used by the food industry are of natural origin,
such as seaweed extracts (e.g. carrageenans), plant exu-
dates (e.g. gum arabic), plant cell wall extracts (e.g.
pectin) or seed mucilages (e.g. locust bean gum). Hy-
drocolloids can be very expensive and as natural prod-
ucts may be subject to serious shortages. Therefore, the
incentive to adulterate one hydrocolloid by a cheaper
and/or more readily available substitute is always pres-
ent, and fraud has been a common practice in the gum
industry [6365] for many years. Gum arabic and locust
bean gum are still favoured targets.
Gum arabic
The gum exudate from Acacia senegal trees, commonly
known as gum arabic, originates in Africa. The main
producing countries are Sudan, Nigeria, Chad, Ethiopia
and Senegal. It is an approved food additive (E 414),
principally used in confectionery and beverages. The
Joint FAO/WHO Expert Committee on Food Addi-
tives (JECFA) defines gum arabic as a dried exuda-
tion obtained from the stems and branches of Acacia
senegal (L.) Willdenow or closely related species of
Acacia (fam. Leguminosae) [66]. The Committee has
recently revised the specification for gum arabic [66],
taking into account concerns expressed about the po-
tential adulteration of commercial gum arabic with
gums from non-Acacia species [67]. For this reason,
identification tests are described to ensure the absence
of mannose, xylose and galacturonic acid, three sugars
characteristic of common adulterants in gum arabic [66,
67]. However, they did not include limits for specific ro-
tation and nitrogen, two important parameters present
in the previous specification [68].
There has been an extensive polemic in the litera-
ture concerning the meaning of closely related species
of Acacia and different opinions were expressed about
what gum arabic should be [63, 6973]. In particular,
some authors think that gum A. seyal (gum talha),
which is the most commercially abundant Acacia gum
after A. senegal, should not be considered as gum arab-
ic [69, 71, 73, 74] and, therefore, its presence in com-
mercial samples is a fraud. They argue that A. seyal has
never been subjected to any form of toxicological eval-
uation [69, 71]. In fact, A. seyal did not fall within the
limits set for specific rotation and nitrogen in the 1990
JECFA specification [68] and, consequently, could not
be regarded as gum arabic. However, it now completely
meets the new JECFA specification [66] and, accord-
ingly, should be considered as safe [67] and accepted as
food-grade gum arabic. Food manufacturers who want
to stick to more stringent specifications and to purchase
pure A. senegal gum free from A. seyal can nevertheless
rely on different analytical techniques for the control of
their raw material. Some are based on the difference in
the carbohydrate composition of the two gums (Table
2).
All the data show that A. seyal has much lower
rhamnose and higher arabinose contents than A. sene-
gal. However, due to large natural variations in the
concentration of the two monosaccharides, the method
would only detect high amounts of gum talha. Other
parameters should be included in order to improve the
capability of distinguishing between the two gums. In
this connection, principal component analysis perform-
ed on nine physical and chemical parameters (Fig. 8),
including all constitutive carbohydrates analysed by
both HPLC and TLC, proved successful in separating
gums from A. senegal and A. seyal [76]. Recently, an
immunological technique, using an anti-arabinogalac-
tan protein/arabinogalactan monoclonal antibody, has
been proposed to screen for the presence of gum talha
in commercial gum arabic [73].
Locust bean gum
Locust or carob bean gum is primarily the refined en-
dosperm of the seed of the carob tree, Ceratonia sili-
9
Fig. 8 Separation of Acacia senegal and Acacia seyal gums by
PCA. Parameters: specific rotation, nitrogen, viscosity, equivalent
weight, galactose, arabinose, rhamnose, glucuronic acid,
4-O-methyl glucuronic acid. Axes: first and second principal
components, 45% and 19% of total variance, respectively. L
Vulgares; l A. Senegal; c A. Seyal; g Gummiferae. Reprinted
from Food Hydrocoll [76] with kind permission of Oxford
University Press, Oxford
qua, a leguminous tree grown in Mediterranean coun-
tries. It is an approved food additive (E 410) used in
dairy, dessert, bakery and meat products. The price of
locust bean gum (LBG) is relatively high, and varies
greatly depending on the world demand and on some-
what unreliable crops. Therefore, the market is wide
open for fraud. Adulteration of LBG with a much
cheaper gum, namely guar gum, has been reported [65].
Guar gum is obtained from the seed of another legumi-
nous tree, Cyamopsis tetragonolobus, that grows mainly
in the Indian subcontinent. It is also an approved food
additive (E 412), but is available at a much cheaper
price than LBG.
Microscopy [79], double-diffusion lectin assay [80]
and capillary electrophoresis [65] have been suggested
for the detection of guar gum in LBG. Distortion in the
natural carbohydrate profile of LBG can also be indica-
tive of the presence of guar gum. Indeed, both gums
consist mainly of high molecular weight galactoman-
nans. However, the relative proportions of mannose
and galactose, as measured by any chromatographic
technique after acid hydrolysis or methanolysis of the
product [8184], are very different. The carbohydrate
composition of commercial samples of refined LBG
and guar gums from different suppliers was deter-
mined. The gums were dispersed in 12 M H
2
SO
4
and
incubated at 30 7C for 1 h. The samples were then di-
luted with water to reach 0.414 M H
2
SO
4
, and auto-
Table 3 Carbohydrate compositionof commercial refined locust
bean and guar gums (%)
Carbohydrate Locust bean
a
Guar
b
Mannose Range 59.369.9 45.656.5
Average
c
63.9 (3.0) 53.0 (2.6)
Galactose Range 16.018.2 28.637.2
Average 16.8 (0.6) 30.9 (1.9)
M/G
d
Range 3.584.20 1.491.84
Average 3.80 (0.21) 1.72 (0.09)
Arabinose Range 0.801.37 1.292.19
Average 1.01 (0.15) 1.76 (0.22)
Glucose Range 1.492.65 1.474.54
Average 2.05 (0.38) 2.41 (0.79)
a
np12
b
np19
c
Standard deviation in parentheses
d
Mannose: galactose ratio
Fig. 9A, B HPAE-PAD chromatograms of locust bean gums. A
authentic, B suspect. Column: CarboPac PA1, eluent: gradient of
100 mM NaOH and water
claved for 1 h at 125 7C [81]. The constituent monosac-
charides were analysed by HPAE-PAD [82]. The re-
sults are given in Table 3 and a typical chromatogram
of LBG is shown in Fig. 9A.
The total galactomannan content of both gums is
very similar. However, LBG exhibits higher levels of
mannose and lower concentrations of galactose than
guar gum. Consequently, the mannose/galactose ratio
in LBG is about twice that measured in guar gum. In
the course of our survey, one sample sold as pure LBG
presented a mannose/galactose ratio of 1.68 (Fig. 9B).
The latter is typical for guar gum (Table 3) and it is
obvious that the product had been mislabelled. Howev-
er, it should be pointed out that the technique is limited
to the detection of high levels of guar gum.
10
Adulteration of soluble coffee
Coffee trees thrive between latitude 307 north and lati-
tude 307 south, at altitudes ranging from sea level to
more than 2000 m, in a globe-encircling geographical
region known as the coffee belt. The main coffee
producing countries are Brazil, Colombia, Indonesia,
Mexico and the Ivory Coast.
What is green coffee? The International Coffee Or-
ganisation, an inter-governmental body which used to
regulate the world supply and price of coffee beans, de-
fines green coffee as all coffee in the naked bean form
before roasting, roasted coffee as green coffee
roasted to any degree and including ground coffee,
and soluble coffee as the dried water-soluble solids
derived from roasted coffee [85]. ISO [86], the EU
[87] and the United States [88] also define green coffee
in similar terms. Therefore, internationally accepted
definitions clearly establish that a commercial product
sold as pure soluble coffee must not contain any mate-
rial derived from sources other than green coffee
beans.
Whatever its final use, coffee remains an expensive
raw material and over the years many fraudsters have
been tempted to replace it with cheaper substitutes and
to falsify the product declaration. In particular, it has
been reported that commercial products were in fact
adulterated with coffee husks/parchments, cereals,
malt, maltodextrins and caramelised sugar [8995]. The
free and total carbohydrate profiles (after acid hydroly-
sis) of soluble coffee were used for detecting and even-
tually characterising the coffee substitutes. Marker car-
bohydrates include free fructose, free glucose, sucrose,
mannitol, total glucose and total xylose.
Different analytical methods were applied for the
determination of carbohydrates in soluble coffee, in-
cluding HPLC with post-column derivatisation [89, 95],
GC [90] and HPAE-PAD [9194, 96, 97]. The latter
technique is simple, precise and sensitive, and allows a
complete separation of all major carbohydrates in one
single run (Fig. 10). It has been collaboratively tested
within the Association of Soluble Coffee Manufactur-
ers of the European Community (AFCASOLE) [96,
97]. The method has now been published as Interna-
tional Standard ISO 11292: 1995 [98], and has been ad-
opted as a First Action by AOAC International [97].
Carbohydrate profiles of a wide variety of commer-
cial products, all sold as pure soluble coffees, were de-
termined [8995]. Pure soluble coffee is characterised
by low free carbohydrate contents and high amounts of
total galactose and total mannose (Fig. 10A). Products
adulterated with coffee husks or parchments present
high levels of free mannitol, free fructose, total glucose
and total xylose (Fig. 10B); occasionally, sucrose may
be present in relatively high concentrations. Products
adulterated with starch-containing substitutes (e.g. cer-
eals or malt), maltodextrins or caramelised sugar exhi-
bit high amounts of free fructose, free glucose and su-
crose as well as huge levels of total glucose (Fig. 10C).
Fig. 10AC Total carbohydrate profiles of commercial soluble
coffees. A Pure. B Adulterated with coffee husks/parchments.
C Adulterated with cereals. 1 mannitol; 2 arabinose/rhamnose; 3
galactose; 4 glucose; 5 xylose; 6 mannose. Technique:
HPAE-PAD, column: CarboPac PA1, eluent; water
Analysis of the total xylose content in a wide selec-
tion of green coffee beans and the assessment of its fate
during processing allowed the determination of a maxi-
mum total xylose limit of 0.40%, above which a soluble
coffee should be considered as adulterated [93]. Maxi-
mum limits of 0.30% and 2.10% were also proposed for
free mannitol and total glucose contents, respectively
[93]. These limits have been recently taken into account
for the establishment of the Code of Practice for the
Soluble Coffee Industry in the United Kingdom [99],
which, in addition, set up a maximum acceptable con-
tent for free fructose of 0.60%.
Acknowledgements The authors wish to thank C. Martinez, J.-
M. Durgnat, T. Stijve, E. Prior and R. Acheson for their help.
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