HANSEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO.
6, 2001 1851
FOOD COMPOSITION AND ADDITIVES
Enzyme Assay for Identification of Pectin and Pectin Derivatives,
Based on Recombinant Pectate Lyase
KARIN M. HANSEN, ANNETTE B. THUESEN, and JØRGEN R. SØDERBERG1
CP Kelco ApS, DK-4623, Lille Skensved, Denmark
A simple method was developed for fast identifica-
tion of pectin, based on a recombinant endo-
pectate lyase cloned from Aspergillus niger. When
pectin was demethylated and treated with pectate
lyase, $-elimination occurred, resulting in a double
bond between C-4 and C-5 in the galacturonic acid
residue of the released nonreducing end. The for-
mation of double bonds produced an increase in
light absorption, which was detected at 235 nm.
The assay was tested on pectin of different origins
(apple, orange, sugar beet, sunflower, celery,
lemon), pectin derivatives (amidated pectin), and
speciality types such as low molecular weight and
low %DE (degree of esterification, percentage of
galacturonic acid groups esterified with methanol)
pectin. The highest response was given by pectate
(pectin with %DE< 5) and the lowest by pectin ex-
tracted from sugar beet. No other gums (carboxy-
methylcellulose, carrageenan, locust bean gum,
tragacanth, gellan, tamarind, xanthan, amylogum,
sodium alginate, or agar) gave any response. Mem-
bers of IPPA (International Pectin Producers Asso-
ciation) have evaluated the validity of the assay in
a ring test. All members of the Association were
able to identify pectin from other gums in a blind
test. The method can replace more laborious and
ambiguous identification tests which exist today.
ectins are polysaccharides obtained from primary cell
P walls and intercellular regions of higher plants (1).
Commercial pectins are produced by aqueous extraction
of edible plant material, usually citrus fruits or apples; other
sources such as sugar beet (2), sunflower heads (3, 4), and
mango (5) may also be used. The most important derivatives
of pectin in food technology are amidated pectins (6), pro-
duced by deesterification of pectins with ammonia (7, 8).
Pectins are used widely, preferably as gelling agents, but also
as texturizers, emulsifiers, thickeners, and stabilizers (9).
Pectin consists mainly of linear chains, of
α(1→4)-D-galacturonic acid residues, methylated to different
Received March 14, 2000. Accepted by AH January 14, 2001.
1
Author to whom correspondence should be addressed; e-mail:
jsoe.derberg@herc.com.
degrees (9, 10), and interrupted at certain intervals by α(1→2)
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linked L-rhamnose residues (11). Pectins isolated from some
plants, such as beet, also carry acetyl groups on secondary hy-
droxyls of the galacturonic acids (12). In specific areas of the
pectin polymer, the backbone is branched. In these areas, the
polymer consists of the alternating disaccharide →4)
α-D-galpA-(1→2)α-L-Rhap-(1→, where some of the
rhamnose residues are substituted with side-chains consisting
mainly of arabinose and galactose (13–15).
The regulatory status of pectin is described in the following
publications: JECFA (Joint FAO/WHO Expert Committee on
Food Additives) 1992; FCC (Food Chemicals Codex), 4th Ed.,
Washington, DC, 1996, including supplements; EU Directive of
November 11, 1998; USP XXIV United States Pharmacopeia
(USP). These publications contain identity tests with which pec-
tin must comply when used in food applications as additives; for
example, in jams and jellies, and in stabilizing acidified milk
drinks, or in pharmaceuticals with reference to the USP. The
identity tests are also a confirmation of purity and are often de-
manded for allowing importation in many countries.
Identification of pectin has been difficult, and only methods
measuring functionality are used. As the range of pectins cov-
ered by the specifications in JECFA is enlarged, it becomes
more and more difficult to use identification methods based on
functionality. Specifications now include specialties such as
amidated pectins, sugar beet pectins, and a much wider range of
degrees of methyl esterification. Furthermore, functionality of
pectin products overlaps with that of other gums and stabilizers
as such functionality tests become less specific.
Several commercial pectins, which fall within the description,
are insoluble because they are low in degree of esterification or
are prepared as calcium salts. The identification tests of pectin
(A–F) described by JECFA (16) presume a material soluble in
water that will gel in 10% solution upon cooling (B). Many com-
mercial citrus and apple pectins are now purified so that they do
not gel at this concentration. Unless specially modified, sugar
beet pectin is not capable of gelling under any of the prescribed
conditions. To overcome difficulties due to the low level of cal-
cium in some pectins, which caused failure of test D (gelling of
sodium pectate formed by the alkaline hydrolysis in water), cal-
cium ions should be added to the solution.
A simple and unambigous method for identification of pec-
tin would therefore be very valuable. Such a method has been
developed based on endo-pectate lyase (E.C. 4.2.2.2). Pectate
lyase splits the glycosidic bonds of a galacturonide chain by
1852 HANSEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001

Table 1. Response of pectin and pectin derivatives in tinction coefficient g235 = 4600M/cm (20). Two blanks were
identification assay based on $-eliminative reaction with prepared, one containing only pectin, Tris–HCl buffer, and
pectate lyasea,b ion-exchanged water (2 + 1 + 2), and the other containing only
)[unsaturated Tris–HCl, enzyme, and ion exchanged water (1 + 1 + 3). One
%DE/ product]c unit was defined as the enzyme activity which produced 1
Sample %DAc %AGA MW %DA × 105 µmol unsaturated product/min.

Apple 63/2 68 93000 — 2.47

Pectin Samples Used for Determination of Method
Sensitivity
Orange 65/2 77 83000 — 2.50
Sugar beet 56/28 65 72000 — 1.67 High methyl ester (HM)-pectins of different origins: lemon,
Sunflower 52/12 76 92000 — 3.23
apple, celery, sunflower, sugar beet, and orange (Table 1);
amidated pectin: 25% degree of amidation (DA); speciality
Celery 65/4 73 118000 — 2.98

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pectins: calcium-sensitive pectin (CSP), noncalcium-sensitive
Lemon 69/0 82 125000 — 2.01 pectin (NCSP); pectin Z (%DE 58, MW 25 000); pectin Q
Amidated pectin 23/0 73 — 25 2.93 (%DE 2, MW 20 000); pectin P (%DE 3, MW 5000), pectin R
NCSP 75/0 83 91000 — 2.59 (%DE 12, MW 103 000). (All of these pectins were provided
CSP 66/0 83 138000 — 2.79 by CP Kelco ApS, Lille Skensved, Denmark.)
Pectin R 12/0 84 103000 — 5.52 Other Gums Used to Determine Method Specificity
Pectate P 3/0 89 5000 — 4.64
Agar, locust bean gum (LBG), carrageenan: cottonii (CP
Pectin Z 58/0 82 25000 — 2.61
Kelco ApS, Lille Skensved, Denmark), tragacanth (Sigma
Pectate Q 2/0 85 20000 — 6.08 Chemical Co., St. Louis, MO), gellan (CP Kelco ApS, San
a
Diego, CA), tamarind, carboxymethylcellulose (CMC; Hercules
All samples were analyzed as double determinations (n = 2).
b GmbH, Aqualon Division, Düsseldorf, Germany), xanthan
%DE (degree of esterification), %DAc (degree of acetylation), AGA
(anhydro galacturonic acid), MW (molecular weight), %DA (degree (Rode & Rode, Hedensted, Denmark), sodium alginate (Satia,
of amidation). Degussa, Baupte, France), amylogum (Avebe, Malmö, Sweden).
c
Concentration (mol/liter) of unsaturated product produced.
Samples Used for Validation by Ring Test (IPPA
Members)
Apple pectin type HM (Herbstreith & Fox KG), lemon
trans-elimination of hydrogen from the 4 and 5 carbon posi- pectin type HM (Obipektin), lime pectin type HM (Danisco),
tions of the galacturonosyl moiety. This results in a double orange pectin type HM (Danisco), sugar beet pectin type HM
bond, which causes an increase in absorbance at 235 nm (17). (CP Kelco ApS), apple type (LM; Obipektin), apple type
A similar reaction is catalyzed by pectin lyase, but pectate amidated (LMA; SKW), lemon/lime type LM (Citrus
lyase is distinguished by its preference for a glycosidic linkage Colloids), sodium pectate (Citrus Colloids), carrageenan (CP
next to a free carboxyl group, rather than to an esterified Kelco ApS), gum tamarind (CP Kelco ApS).
group (18). Therefore, pectin is deesterified before the pectate Note: HM = high methyl ester; LM = low methyl ester;
lyase treatment to make it more susceptible to degradation. LMA = low methyl ester amidated.

Experimental Pectin Identification Assay

Pectate Lyase A 0.05 g amount of sample to be identified was moistened

The recombinant pectate lyase cloned from Aspergillus
niger (19) was kindly provided by the Department of Molecu-
lar Genetics of Industrial Microorganisms, Wageningen Uni-
versity (Wageningen, The Netherlands).
Assay for Determination of Pectate Lyase Activity
A 0.05% aqueous solution was prepared, consisting of pec-
tin [MW 103 000, degree of esterification (%DE, percentage
of galacturonic acid groups substituted with methyl) 12],
pH 7.00. The pectate lyase to be tested was diluted in
Tris–HCl buffer [50mM Tris (hydroxymethyl)
aminomethane, containing 1mM CaCl2, pH 7.0]. Pectin was
mixed with Tris–HCl buffer, diluted pectate lyase, and
ion-exchanged water (2 + 1 + 1 + 1) at room temperature. The
absorbance was then measured continuously at 235 nm, ex-
by addition of 250 µL 100% 2-propanol. A 50 mL volume of
ion-exchanged water was added on a magnetic stirrer and pH
was adjusted to 12 with 0.5N NaOH. The sample was left at
room temperature, for 15 min without stirring; the pH was
lowered to 7.0 by addition of 0.5N HCl. Sample concentration
was adjusted to 0.05% by addition of ion-exchanged water.
The pectin lyase was diluted in Tris–HCl buffer [50mM
Tris (hydroxylmethyl) aminomethane, containing 1mM
CaCl2, pH 7.0], to a concentration of 0.035 units/mL.The sam-
ple to be identified was mixed with Tris–HCl buffer, enzyme,
and ion-exchanged water (2 + 1 + 1 + 1), in a quartz cuvette,
the absorbance at 235 nm was immediately determined; the
preparation was left at room temperature for 10 min, and the
absorbance was measured again. Two blanks, one containing
the sample to be identified, Tris–HCl buffer, and
ion-exchanged water (1 + 2 + 2), and one containing Tris–HCl
 HANSEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 1853

Table 2. Response of gums other than pectin, tested in In contrast to other types of interlaboratory tests, IPPA ring
identification assay based on $-eliminative reaction with tests often deal with test methods that will not yield a single nu-
pectate lyasea
merical figure as a test result. For example, the test result could be
Sample )[unsaturated product]b × 105 a visual description of some kind such as a precipitation or gel
formation. Many of these more visual characterization methods
Agar –(0.02) for pectin are very difficult to interpret unless the laboratory staff
Locust bean gum (LBG) –(0.30) has much experience working with pectin. Therefore, the recruit-
ment policy for IPPA’s ring test is that all IPPA members, and
Carrageenan (Cottonii) 0.09
only those laboratories, will participate in the test.
Tragacanth 0.00
Organization of the experiment is entrusted to a single lab-
Gellan 0.11
oratory, in this case CP Kelco, but in close cooperation with
Tamarind –(0.04)
the technical chairman of IPPA, who is the chief excutive offi-

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Carboxymethylcellulose (CMC) –(0.02)
Xanthan 0.33
Sodium alginate 0.00
Amylogum 0.15
a
All samples were analyzed as double determinations (n = 2).
b
Concentration (mol/liter) of unsaturated product produced.
buffer, enzyme, and ion-exchanged water (1 + 1 + 3) were also
prepared; absorbance was measured for both samples at 0 and
10 min. From the increase in absorbance (absorbance at
10 min – absorbance at 0 min = ) abs.), the concentration of
unsaturated product produced can be calculated as ) abs./I–
g235 = )[unsaturated product], where I is thickness of the sam-
ple and g is the extinction coefficient (molar absorption coeffi-
cient), which is 4600M/cm (20).
Results and Discussion
Method Sensitivity and Specificity
All of the pectins and pectin derivatives showed an increase
in absorbance at 235 nm, resulting from the $-eliminative at-
tack with pectate lyase (Table 1). The highest response was
given by a pectate (%DE < 5) and the lowest by sugar beet pec-
tin. The difference in response between the different types of
pectins and pectin derivatives is related to mode of action for
the pectate lyase. The lowest response, given by sugar beet pec-
tin, was significant (1.67 × 10–5) compared to that of the other
gums which were tested: agar, carrageenan, locust bean gum,
tragacanth, gellan, tamarind, xanthan, amylogum, sodium
alginate, and carboxymethylcellulose, which showed almost no
response (<0.5 × 10–5; Table 2).
Method Evaluation by Ring Test Among IPPA
Members
Many different factors such as operator, equipment, cali-
bration, and environment can have an impact on test results.
Even though test material as well as procedures are identical,
some unavoidable random errors will be identified in all test
methods. Realizing this, IPPA members have agreed that be-
fore implementation of a new test regarding pectin or pectin
functionality, an interlaboratory test (ring test) among the
members of IPPA must be performed.
cer (CEO) of the test. The CEO collects results of the test from
participating laboratories, decodes samples, and sends results
to the organizers. To guarantee that this test was capable of
identifying samples of different origin, produced by different
methods, all members of IPPA delivered a set of 2 different
pectin samples, prepared at their facility, to the organizer.
Identically coded sets of samples, including 2 other gums, i.e.,
carrageenan and tamarind, were then dispatched to the labora-
tories listed in Table 3. Tests were performed according to the
procedure given by the organizer.
Although a numerical value is measured in this test, it is not
a quantitative test, but rather a qualitative test in which a posi-
tive numerical value, >0.5 × 10–5, indicates the presence of
pectin. For all pectin samples analyzed, results were
>1.5 × 10–5 (Tables 1 and 3), and for other gums, values
<0.5 × 10–5 (Tables 2 and 3). Pectin concentration of 0.02% in
final solution (used in this test) should result in values
>1.5 × 10–5. Decreasing the pectin concentration will of
course decrease the response value, but samples resulting in
values <0.5 × 10–5 cannot be identified as pectin. All laborato-
ries (Table 3) found all pectins positive, detecting more than
2.0 × 10–5M unsaturated product produced. Although the nu-
merical values differred, all were significant as an identity
test, in contrast to results for carrageenan and tamarind gum
(also containing galacturonic acid groups) which were <0.5 ×
10–5M unsaturated product, stated as 0.
Conclusions
Results showed that this method is capable of replacing the
existing identity test based on functional properties, many of
which pectin shares with other gums. This test is fast and reli-
able for all pectin material irrespective of origin and type.
Acknowledgments
We thank the members of IPPA (Citrus Colloids, UK;
Danisco, Denmark; Herbstreith and Fox, Germany;
Obipektin, Switzerland; SKW Biosystems, France) who
kindly provided samples and participated in testing the assay.
European Union financially supported this work as a part of
the project AIR2-CT94-1345.
1854 HANSEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001

Table 3. Validation of pectin identification test by members of IPPAa

IPPA members

Citrus Colloids Danisco Herbstreith & Fox CP Kelco ApS Obipektin SKW

Sample )[unsaturated product]b x 105

HM pectin
Apple 4.6 4.1 2.7 2.2 2.4 3.5
Lemon 4.3 4.2 3.7 2.8 2.8 4.7
Lime 4.7 3.9 3.7 2.7 3.4 4.0
Orange 4.1 5.6 2.7 2.9 3.0 4.9

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Sugar beet 7.6 3.4 4.0 2.6 3.1 6.0
LM pectin

Apple 6.9 7.1 3.1 4.4 3.1 5.0

Apple (amide) 4.3 3.6 2.2 2.5 2.1 3.7
Lemon/lime 6.4 7.1 3.5 4.5 3.7 5.7
Sodium pectate 8.7 7.8 4.1 10.0 4.4 7.1
Other gums

Carrageenan 0 0 0 0 0 0
Gum tamarind 0 0 0 0 0 0

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