International Journal of Food Science and Technology 2012 1

Original article
Pectic oligosaccharides production from orange peel waste by
enzymatic hydrolysis

Martina Martı́nez Sabajanes, Remedios Yáñez, José L. Alonso* & Juan C. Parajó
Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain
(Received 22 July 2011; Accepted in revised form 3 November 2011)

Summary Orange peel waste (OPW) was evaluated as a raw material for the enzymatic production of pectic
oligosaccharides. In a preliminary step, free sugars and other water-soluble compounds were extracted from
OPW to obtain a substrate for enzymatic hydrolysis (EHS), to achieve two objectives: (i) removal of sugars
and impurities, and (ii) production of a sugar-rich stream suitable for further utilisation, according to the
‘biorefinery’ philosophy. EHS was treated with hydrolytic enzymes (pectinases and cellulases), and the effects
of selected operational variables (enzyme charges and reaction time) on selected dependent variables
(measuring the conversions of polysaccharides into mono- and oligosaccharides, and liquor recovery) were
assessed. Mathematical models were developed for this purpose. Under selected conditions, the models
predicted that 100 kg of EHS yielded 7.5 kg of gluco-oligosaccharides, 4.5 kg of galacto-oligosaccharides,
6.3 kg of arabino-oligosaccharides and 13 kg of oligogalacturonides (contained in 991.6 kg of liquor).
Keywords Cellulases, enzymatic hydrolysis, orange peel waste, pectic oligosaccharides, pectinases, prebiotics.

2005; Gullón et al., 2009). The prebiotic potential of

Introduction
Pectic oligosaccharides (POS) can be obtained from
pectin-rich by-products such as sugar beet pulp, apple
pomace or citrus peel by chemical and ⁄ or enzymatic
processing.
Pectin is a heteropolysaccharide mainly made up of
three structural elements: homogalacturonan (HG),
rhamnogalacturonan I (RG-I) and rhamnogalacturo-
nan II (RG-II). HG consists of a backbone of a-
(1 fi 4)-linked galacturonic acid (GalA) residues,
which can be partially methyl-esterified at C-6 and
acetyl-esterified at O-2 and ⁄ or O-3. RG-I is a ramified
polymer made up of chains containing alternate units
of GalA and rhamnose (Rha), where arabinan, galac-
tan and arabinogalactan (type I and II) branches can be
attached. RG-II is a complex polymer composed of
GalA, rhamnose, galactose (Gal) and some unusual
sugars (Ralet et al., 2005).
Recent studies reported healthy effects for pectins and
POS, including regulation of lipid and glucose metab-
olism with decreased glycemic response and blood
cholesterol levels, anticancer and immunological prop-
erties, anti-obesity effects, antibacterial and antioxidant
properties (Olano-Martı́n et al., 2001; Rastall et al.,
*Correspondent: Fax: +34 988 38 70 01;
e-mail: xluis@uvigo.es
POS is also under evaluation by means of in vitro
fermentation assays using both individual cells and
faecal inocula, and, even, some studies were also carried
out in humans (Fanaro et al., 2005; Magne et al., 2008).
Experiments carried out using POS derived from high-
methoxy citrus pectin, low-methoxy apple pectin and
orange peel enhanced the growth of bifidobacteria and
lactobacilli, while limiting the growth of pathogenic
genera (Olano-Martı́n et al., 2002; Manderson et al.,
2005; Mandalari et al., 2007). The prebiotic effect of
pectins and POS depend on the physicochemical char-
acteristics of substrates, and particularly, on the molec-
ular weight distribution and degree of esterification
(Gullón et al., 2009). For instance, it was demonstrated
that POS with low degree of methylation show higher
prebiotic index than high degree of methylation POS
(Olano-Martı́n et al., 2002). Al-Tamimi et al. (2006)
reported that low molecular weight arabino-oligosac-
charides (AOS) provoked higher increases in bifidobac-
teria than other AOS. Some authors have insinuated
that POS could be a new class of prebiotics, but further
work is necessary before using POS as prebiotics in
human foods.
Several cheap pectin sources (including the aforemen-
tioned agroindustrial by-products) and processing tech-
nologies have been employed for POS production.
Martı́nez et al. (2009a) studied the potential of sugar

doi:10.1111/j.1365-2621.2011.02903.x
 2012 The Authors. International Journal of Food Science and Technology  2012 Institute of Food Science and Technology
2 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.
beep pulp as a raw material for POS production by
hydrothermal processing, whereas Manderson et al.
(2005) employed HNO3 solutions for producing POS
from orange albedo.
The use of enzymes for the same purpose has been
reported; for example, pectinolytic enzymes were used to
obtain POS from bergamot peel (Mandalari et al., 2007)
and sugar beet pulp (Martı́nez et al., 2009b), whereas
Olano-Martı́n et al. (2001) reported on the continuous
production of POS in an enzyme membrane reactor
using pectin from citrus and apple.
Spain is the main orange producer in Europe,
exceeding 3.3 million metric tons per year. About 50%
of this amount is squeezed for juice, generating huge
amounts of easily fermentable wastes that may cause
environmental problems. The solid residue from juice
extraction [here denoted as OPW (orange peel wastes)]
may account for up to 40–60% of the fruit weight and is
made up of peel, segment membranes and other
components (Grohmann & Baldwin, 1992). Currently,
these solids are dried and used as low-value cattle feed
or as organic fertilizers; but drying is costly because of
their high moisture content. In some cases, other
compounds (for example pectin) are commercially
obtained from orange peel. However, the great world-
wide supply of orange-processing residues and the
increasing demand for new functional ingredients foster
the interest in new processes for POS production from
OPW.
This work deals with the production POS mixtures
from OPW following a two-step process: sugar extrac-
tion and enzymatic hydrolysis of the extracted solid. The
effects of selected operational variables [polygalacturon-
ase loading, cellulase to polygalacturonase ratio and
reaction time (t)] on selected dependent variables (con-
versions of polysaccharides into mono- and oligosac-
charides, and liquor recovery) were assessed to maximise
the production of POS. Empirical models suitable to
reproduce and to predict the composition of enzymatic
hydrolysates for defined operational conditions (within
the experimental domain considered) were developed,
validated and employed to assess the selection of
optimal operational conditions.
Materials and methods
Raw material
Orange peel wastes samples were kindly supplied by
Indulleida S.A. (Lleida, Spain). To avoid variations in
the composition of the raw material, all the samples
were mixed to obtain a single lot, which was stored at
)18 C until use. When necessary, aliquots from this lot
were defrosted, milled and subjected to analytical
determinations or processed according to the methods
explained later.
International Journal of Food Science and Technology 2012
International Journal of Food Science and Technology  2012 Institute of Food Science and Technology
Analysis of the raw material
Orange peel wastes samples with particle size below
0.5 mm were used for analysis. Moisture and ashes were
determined according to the standard methods ISO
638:1978 and ISO 776, respectively. Elemental nitrogen
was determined with a Thermo Finnegan Flash EATM
1112 analyzer (operational conditions: He flow,
130 mL min)1; O2 flow, 100 mL min)1; oven tempera-
ture, 50 C). All determinations were made by triplicate.
Protein content was obtained by multiplying the ele-
mental N content by 6.25.
An OPW sample was extracted until exhaustion with
water at room temperature in a stirred tank (10-stage
cross-flow operation using 10 g water per gram initial
solid per stage) giving two streams: ‘extractives’
(obtained by mixing the aqueous phases coming from
the individual extraction stages) and ‘water-insoluble
material’ (the exhausted solid phase). Both phases were
then characterised according to the methods reported by
Martı́nez et al. (2010). Extractives were analysed for
their sugar content (glucose, fructose and arabinose),
whereas the water-insoluble material for their content of
glucan, galacturonan, arabinan and galactan. Moreover,
the water-insoluble material was assayed for their pectin
content by acid extraction and alcohol precipitation
(Hwang et al., 1998). The GalA content of pectin was
determined using the method reported by Blumenkrantz
& Asboe-Hansen (1973). The acetylation and methyla-
tion degrees were assayed according to Levigne et al.
(2002).
Manufacture of enzymatic hydrolysis substrates
Although the water-insoluble fraction obtained accord-
ing to the method described in the previous section
would be suitable for enzymatic hydrolysis, the exhaus-
tive extraction procedure is only reasonable for analyt-
ical duties. Preliminary experiments (data not shown)
proved that a simpler and cheaper extraction method
(involving three stages) enabled the manufacture of
suitable hydrolysis substrates (as much as 85% of free
sugars were removed from OPW using this approach).
On this basis, substrates for enzymatic hydrolysis were
manufactured by extracting OPW with water for
15 min at room temperature in a 2-L tank, using a
liquor to solid ratio = 10 g water per gram oven-dry
OPW. Solids were recovered by centrifugation and
extracted with water two additional times (using 2 g
water per gram oven-dry OPW) at same temperature
and for same time as before. The resulting solids were
employed as enzymatic hydrolysis substrates and
denoted as EHS.
The three liquid phases coming from the individual
extraction stages were mixed and assayed for composi-
tion using the methods cited earlier.
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 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.
Enzymatic hydrolysis
Enzymatic hydrolyses of EHS were carried out at
37 C and pH 5 (50 mm sodium acetate buffer)
(Martı́nez et al., 2009b) in Erlenmeyer flasks of 100-
mL working volume placed in orbital shakers
(150 r.p.m.) using two commercial enzyme prepara-
tions at selected loadings (see operational conditions
later). Previous experiments (data not shown) con-
firmed synergistic effects between both enzymatic
preparations for POS production. The commercial
enzyme concentrates (‘Celluclast 1.5L’ cellulases from
Trichoderma reesei and ‘Viscozyme L’ pectinases from
Aspergillus aculeatus) were kindly provided by Novo
Nordisk Bioindustrial (Madrid, Spain). The cellulase
and polygalacturonase activities of the respective con-
centrates were determined according to the methodol-
ogy used by Martı́nez et al. (2009b).
Enzymatic assays were performed for up to 20 h at
a fixed liquor to solid ratio of 12 g g)1, using
Viscozyme L loadings leading to polygalacturonase
to solid ratios (PSR) in the range 5–15 U g)1 and
Celluclast 1.5-L loadings resulting in cellulase to
polygalacturonase ratios (CPR) in the range 0.25–
1.75 FPU U)1. For the sake of simplicity, the possible
contributions of Celluclast 1.5L to the polygalaturon-
ase activity and of Viscozyme L to the cellulase
activity were neglected. Before enzyme supplementa-
tion, EHS samples were suspended in sodium acetate
buffer (50 mm) and sterilised for 15 min at 121 C. At
the end of enzymatic processing, liquors were sepa-
rated by centrifugation, heated at 100 C for 5 min to
inactivate the enzymes, weighed and analysed as
described later.
Experimental design and mathematical modelling
To assess the POS production by enzymatic hydroly-
sis, a set of preliminary experiments (data not shown)
was carried out to identify the most influential
operational variables and their range of practical
interest. Because of the complex composition of the
raw material, gluco-oligosaccharides from glucan (here
denoted, GOS) were also generated in the reaction
media.
The effects of selected operational variables (PSR;
cellulase to polygalacturonase ratio, denoted CPR and
reaction time, denoted t) on dependent variables
selected to measure the progress of hydrolytic reactions
[glucan conversion into glucose or y1, glucan conver-
sion into GOS or y2, galactan conversion into galactose
or y3,, galactan conversion into galacto-oligosaccha-
rides (GaOS) or y4, arabinan conversion into arabinose
or y5, arabinan conversion into AOS or y6, galacturo-
nan conversion into GalA or y7, galacturonan conver-
sion into oligogalacturonides (OGalA) or y8 and the
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International Journal of Food Science and Technology  2012 Institute of Food Science and Technology
3
mass of recovered liquors denoted y9] were measured.
To assess the interrelationship between dependent and
independent variables by means of the response surface
methodology (RSM), an experimental plan correspond-
ing to a factorial, incomplete, centred, second-order
experimental design was selected. Mathematical mod-
elling, of which RSM is one, provides a precise map
leading to successful optimisation (Nwabueze, 2010).
RSM has been previously used, for instance, in
enzymatic processing of sugar beet pulp (Martı́nez
et al., 2009b) and in chemical pectin extraction (Klie-
mann et al., 2009). The empirical equation giving the
interdependence between variables followed the gener-
alised expression:
X XX
yj ¼ b0j þ bij xj þ bikj xj xk ð1Þ
i i k
where yj represents the dependent variable defined
earlier (j: 1–9), b0j....bikj are the regression coefficients
calculated from the experimental data by multiple
regression using the least-squares method and xi or xk
(i or k: 1–3, k ‡ i) are the dimensionless, normalised,
independent variables with variation ranges ()1,1)
related linearly to the PSR, CPR an t.
Table S1 lists the operational conditions correspond-
ing to experiments 1–15 (expressed as terms of dimen-
sional and dimensionless independent variables).
Analysis of enzymatic hydrolysates
Aliquots of liquors from enzymatic media were centri-
fuged, filtered through 0.45-lm membranes and analy-
sed for sugars by HPLC. Glucose, fructose, galactose
and arabinose were determined using an Aminex HPX-
87P column, whereas GalA was analysed using an
Aminex HPX-87H column. Based on the enzymatic
method for determination of uronic acids and methanol
in pectin described by Anthon & Barrett (2008), a
second sample of liquors was subjected to enzymatic
posthydrolysis with polygalacturonases and cellulases
under the following operational conditions: T = 37 C,
t = 40 h, agitation speed = 150 r.p.m., endopolygalac-
turonase loading = 45 U g)1 liquor, cellulase load-
ing = 5 FPU g)1 liquor. Sodium acetate buffer
(50 mm) was employed to keep the pH at 5. The
increase in the concentrations of monosaccharides
caused by enzymatic posthydrolysis provided a measure
of the oligomer concentration. It can be noted that this
operational mode enabled the determination of mono-
mers making part of oligomeric compounds derived
from the enzymatic processing, but did not provide
information on the type of linkages among monomers,
oligomer structure or distribution of molecular weights.
The total oligomers contained in selected samples
(denoted OS) were assayed for methylation degree using
the same method cited earlier.
International Journal of Food Science and Technology 2012
4 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.
High-performance size exclusion chromatography
The molecular weight distribution of oligomers obtained
under selected conditions was determined by high-
performance size exclusion chromatography (HPSEC)
using a Agilent instrument fitted with a refractive index
detector and a MCIGEL CK02S column (20 mmi.d.
· 250 mm; Mitsubishi Chemical Co., Minato-Ku, Tokyo,
Japan) kept at 80 C. Deionised water (1.0 mL min)1)
was used as a mobile phase.
Fitting of data
The experimental data were fitted to the proposed
models by minimising the sum of the deviation squares
between experimental and predicted values using
commercial software (Microsoft Excel; Microsoft,
Redmond, WA, USA).
Results and discussion
Composition of raw material
The composition of the raw material is discussed in
terms of the extractable fraction and the water-insoluble
material.
Table S2 shows that the extractable fraction ac-
counted for 60.4 wt% of oven-dry OPW. The major
components were glucose (33.8 wt% oven-dry OPW),
fructose (29.1 wt%) and sucrose (7.7 wt%). The
water-insoluble material, accounting for 39.6 wt% of
oven-dry OPW, was mainly composed of glucan
(21.7 wt% of oven-dry insoluble material) and galac-
turonan (31.0 wt% of oven-dry insoluble material).
Arabinan and galactan were present in similar
amounts (7.8 and 7.5 wt% of oven-dry insoluble
material, respectively), whereas pectin accounted for
31.4 wt% of oven-dry insoluble material. Pectin con-
tained 70.9 g GalA per 100 g pectin, with a methyl-
ation degree of 44%.
Other fractions such as protein, lignin and ash,
appeared in low proportions and were not relevant for
the purposes of this study. The acetyl group content of
pectin was 0.91% (corresponding to an acetylation
degree of 3%), a value lower than the ones reported for
other pectin-rich substrates such as sugar beet (Martı́nez
et al., 2009a,b), but close to the results reported for
orange peel pectin (Ralet et al., 2002; Zykwinska et al.,
2008).
In general, the compositional data fall within the
range reported in literature (Grohmann et al., 1995;
Wilkins et al., 2005) although significant variations were
observed in the contents of sucrose, glucose and
fructose, possibly because of changes during ripening
(Grohmann et al., 1995).
International Journal of Food Science and Technology 2012
International Journal of Food Science and Technology  2012 Institute of Food Science and Technology
Substrate preparation by aqueous extraction of orange peel
wastes
EHS, partially free from sugars and other extractable
impurities, were obtained after three-stage water extrac-
tion at a yield of 48.8 kg per 100 kg oven-dry OPW and
contained the following polysaccharides (in oven-dry
basis): galacturonan, 21.1%; glucan, 19.9%; galactan,
8.01%; arabinan; 6.5%; fructosyl moieties, 4.5% and
xylan, 4.5%. Moreover, the water processing stages
resulted in the extraction of 37.5 kg sugars per 100 kg
dry OPW. More details about this treatment can be
found in Martı́nez et al. (2010).
Enzymatic hydrolysis
Although this work is focused on the manufacture of
oligosaccharides from EHS (measured by variables y2,
y4, y6 and y8) by enzymatic hydrolysis, the action of
enzymes also resulted in the generation of monomers
(measured by variables y1, y3, y5 and y7). Table S3
shows the experimental results determined for these
variables and for y8 (measuring the recovery of liquor).
Table 4 shows the regression coefficients, the signifi-
cance of each term (based on a Student’s t-test), as well
as the statistical parameters measuring the correlation
(R2) and significance of models (based on the Fisher’s F-
test). As can be seen, all the models were statistically
significant, with R2 in the range 0.95–0.99, except in the
case of variable y5. The main aspects concerning
experimental data and model predictions are discussed
in the next sections.
Glucan conversion into glucose and gluco-oligosaccharides
Table S3 shows the experimental values for both
glucan conversion into glucose (y1) and glucan con-
version into GOS (y2). As no glucose decomposition
reactions are expected to occur under the operational
conditions used in experiments, it can be assumed that
glucan was hydrolysed to yield just glucose and GOS.
From the data shown in Table S3, it can be inferred
that the percentage of glucan hydrolysed (defined as
the sum of y1 and y2) varied between 29.1% and
76.7% of the stoichiometric amount. In many exper-
iments, y1 and y2 showed closely related results.
According to the values of the regression coefficients
(see Table S4), increases in enzyme charges and ⁄ or in
reaction time resulted in increased glucose generation
because of the b-glucosidase activity provided by both
enzyme preparations (Ruiz-Teran et al., 2001), achiev-
ing an experimental maximum (41.9% conversion) in
experiment 14.
Figure S1 shows the calculated dependence of y2
(glucan conversion into GOS) on PSR and CPR at the
three levels considered for the reaction time. Variable y2
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 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.
varied in the range 14.7–36.9%, and it was significantly
affected by the three independent variables (see values of
regression coefficients in Table S4). The effects of CPR
depended on the PSR considered: at low PSR, increased
values of CPR resulted in increased GOS production;
whereas when operation is carried out using PSR, higher
CPR did not affect the production GOS, as the enzyme
charge favoured the generation of glucose from GOS,
particularly at prolonged reaction times. The variation
pattern was in agreement with results reported for the
enzymatic hydrolysis of sugar beet pulp (Martı́nez et al.,
2009b). The model predicted a maximum y2 value of
39.60% for conditions defined by t = 20 h,
PSR = 9.50 U g)1 and CPR = 1.08 FPU U)1.
Galactan conversion into galactose and
galacto-oligosaccharides
Viscozyme is a multienzyme complex that includes
hemicellulases and arabinanases among other activities
(Lee et al., 2008). According to the data in Table S3,
GaOS were the major products derived from galactan
hydrolysis. The overall percentage of galactan solubi-
lised (calculated as the sum of y3 and y4) varied in the
range 46.5–73.8%; whereas the galactan conversion into
galactose (y3) varied in the range 5.4–30.7% and was
significantly affected just by PSR and reaction time (see
Table S4). Its maximum value was obtained in experi-
ment 14 (operating at the highest values considered for
PSR and t).
According to the data in Table S3, y4 (measuring the
galactan conversion into oligosaccharides) was mainly
affected by CPR and t, with a maximum experimental
value (54.9%) reached in experiment 3. Figure S2 shows
the calculated dependence of y4 on CPR and t for
experiments carried out at PSR = 5, 10 and 15 U g)1.
CPR showed a positive effect on the conversion of
galactan into GaOS, with more pronounced effects
along the first half of its variation, confirming the
presence of galactanase activity in the Celluclast com-
plex. In a related study, Mandalari et al. (2006) found
galactanase activity in a cellulase preparation. The effect
of t depended on PSR: in experiments carried out at low
PSR, longer t resulted in improved production of GaOS;
but operating at high PSR, the opposite behaviour was
observed, because of the generation of Gal from GaOS.
The model predicted a maximum in y4 (close to 60%)
for conditions defined by PSR = 5 U g)1, CPR =
1.75 FPU U)1 and t = 20 h.
Arabinan conversion into arabinose and arabino-
oligosaccharides
The AOS generation from the EHS confirmed the
presence of arabinanase activities of the enzymatic
complexes employed in this work. The experimental
results determined for variables y5 (arabinan conversion
into arabinose) and y6 (arabinan conversion into AOS)
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5
are listed in Table S3. The arabinan solubilisation
(calculated as the sum of y5 and y6) varied in the range
70.5–100% (experiments 2 and 14, respectively), con-
firming the high susceptibility of this fraction to the
enzymatic hydrolysis. AOS were the major reaction
products, whereas the presence of arabinose was almost
negligible (see data in Table S3). This variation pattern
is similar to the one reported for sugar beet pulp
processing (Martı́nez et al., 2009b).
Figure S3 shows the calculated dependence of y6 on t
and PSR for experiments carried out at CPR of 0.25, 1
and 1.75 FPU U)1. The model predicted quantitative
arabinan conversion into AOS under the severest
operational conditions assayed.
Galacturonan conversion into galacturonic acid and
oligogalacturonides
The experimental results obtained for variables y7
(galacturonan conversion into GalA) and y8 (galactu-
ronan conversion into OGaU) are listed in Table S3.
Galacturonan solubilisation (defined as the sum of y7
and y8) varied in the range 58.7–92.6% (see Table S3).
Variable y7 increased from 7.7% (experiment 2, operat-
ing at low enzyme loadings and short reaction times) up
to 48.0% (experiment 14, in which the substrate was
treated for 20 h at a high enzyme charge). PSR and t
were the most influential variables, whereas CPR caused
negligible effects, a fact ascribed to the same reason
already discussed for arabinose.
Except in experiment 14, OGalA was the main
product of galacturonan hydrolysis. Variable y8 reached
its maximum experimental value (62.0%) in experiment
3 (performed at PSR = 5 U g)1 and CPR =
1 FPU U)1 during 20 h). Figure S4 shows the predicted
dependence of y8 on CPR and PSR for experiments
carried out at t = 6, 13 and 20 h. Increased CPR
values resulted in slightly increased values of y8, no
matter of the reaction time. However, the effects caused
by PSR depended on t: as discussed for galactan
hydrolysis, PSR showed a positive effect on OGalA
generation at short reaction times; but longer treat-
ments resulted in an opposite behaviour owing to the
increased participation of oligosaccharide hydrolysis
reactions. At intermediate values reaction times, the
galacturonan conversion into OGalA varied in a
narrow range (55–60%).
Liquor recovery
Variable y9 (mass of recovered liquors) provided a
comparative assessment on the processability of the
enzymatic reaction media. According to Table S3, y9
varied in the range 69.7–88.6 g (values achieved in
experiments 1 and 14, respectively), with a strong influ-
ence of the treatment severity (defined by the enzyme
charges and the reaction time). The percentage of mass
recovery in liquid phase at the end of treatments varied in
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6 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.
the range 65–82%. The coefficients in Table S4 showed
that y9 was significantly affected by all the independent
variables: when the severity of the treatments increased,
pectin solubilisation was promoted and, as a consequence,
the water retention capacity of the solids decreased. The
maximum predicted value y9 corresponded to the severest
operational conditions (PSR = 15 U g)1, CPR =
1.75 FPU U)1 and t = 20 h).
Experimental assays for model validation
Additional assays, using randomly selected operational
conditions (experiment 16: x1 = )0.6, x2 = )0.3,
x3 = 0.7; experiment 17: x1 = )0.3, x2 = 0.5, x3 =
0.6 and experiment 18: x1 = 0.7, x2 = 0.2, x3 = )0.67),
were carried out for model validation. As can be observed
(see Fig. S5), the close agreement between experimental
and predicted values confirmed the suitability of the
models for data prediction. Except for variable y2, models
predicted the experimental results with absolute devia-
tions <10% (dashed lines in Fig. S5).
Selection of operational conditions
The set of mathematical models enabled the calculation
of a given objective function (for instance, maximum
production of AOS or maximum production of OGalA)
along the experimental domain. In our case, the
objective was assumed to be the maximum yield of
total oligomers (OS, defined as g oligomers per 100 g
EHS). This parameter was calculated using the equa-
tion:
CGlucan CGalactan CArabinan
OSð%Þ ¼  y2 þ  y4 þ  y6
100 100 100
CGalacturonan
þ  y8
100
ð2Þ
where CGlucan, CGalactan, CArabinan and CGalacturonan are
the glucan, galactan, arabinan and galacturonan con-
tents of EHS, expressed as g per 100 of oven-dry EHS
(see Table S2).
According to the models, the operational conditions
leading to the maximum OS were x1 = )1, x2 = 0.67
and x3 = 1, which corresponded to PSR = 5 U g)1,
CPR = 1.5 FPU U)1 and t = 20 h. Under these con-
ditions, models predicted that 991.6 kg of liquors
containing 31.3 kg of oligosaccharides can be obtained
from 100 kg of EHS, with the following distribution:
7.5 kg of GOS, 4.5 kg GaOS, 6.3 kg of AOS and 13 kg
of OGalA. Because of the action of pectinmethylester-
ases present in Viscozyme, the methylation degree of
OGalA (23%) was lower than the one determined for
pectin (44%).
International Journal of Food Science and Technology 2012
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Table S5 shows the detailed composition of both
hydrolysates and spent solids obtained in experiments
carried out under the selected operational conditions
(PSR = 5 U g)1, CPR = 1.5 FPU U)1 and t = 20 h).
To provide an assessment on the molecular weight
distribution of oligomers resulting from enzymatic
hydrolysis, samples obtained under the optimal condi-
tions were analysed by HPSEC and compared with
standards of AOS [degrees of polymerisation (DP) in the
range 2–8], OGalA (DP 2-3) and GalA. The experimen-
tal results (shown in Fig. S6) showed that the sample
contained compounds eluting at the same time than the
standards, as well as additional peaks (which are
ascribed to AOS and OGalA with different DP and ⁄ or
substitution pattern).
Conclusion
Orange peel wastes was characterised and evaluated as a
raw material for the enzymatic production of POS.
Aqueous extraction of OPW resulted in liquors con-
taining glucose, fructose and sucrose (at yields of 16.9,
13.7 and 6.6 kg per 100 kg of oven-dry OPW, respec-
tively). In the further enzymatic processing of solid
substrates obtained by water extraction of OPW
(denoted as EHS), the effects of the enzyme loadings
(polygalacturonases and cellulases) and reaction time on
the conversion of polysaccharides into monosaccharides
and oligomers were assessed using an empirical model.
Under selected conditions, the models predicted that
991.6 kg of liquors containing 31.3 kg of oligosaccha-
rides can be obtained from 100 kg of EHS, with the
following distribution: 7.5 kg of GOS, 4.5 kg GaOS,
6.3 kg of AOS and 13 kg of OGalA. Monosaccharides
(at an oligosaccharide ⁄ monosaccharide ratio of 2 kg per
kg) and nonsaccharide, nonvolatile compounds (up to
24.7 kg per 100 kg of nonvolatile solutes) were also
present in liquors.
Acknowledgments
Authors are grateful to the ‘Ministry of Science and
Innovation’ of Spain (Project ref: CTQ2008-
05322 ⁄ PPQ, partially funded by the FEDER funds of
the European Union) for the financial support of this
work. Dr. R. Yáñez is grateful to the ‘Xunta de Galicia’
for her ‘Isidro Parga Pondal’ contract and M. Martı́nez
to the ‘University of Vigo’ by her grant.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Calculated dependence of the cellulose
conversion into gluco-oligosaccharides (variable y2) on
the polygalacturonase to solid ratio and on the cellulase
to polygalacturonase ratio for experiments lasting 6, 13
or 20 h.
Figure S2. Calculated dependence of the galactan
conversion into galacto-oligosaccharides (variable y4)
on the reaction time (t) and on the cellulase to
polygalacturonase ratio for experiments carried out at
PSR = 5, 10 or 15 U g)1.
Figure S3. Calculated dependence of the arabinan
conversion into arabino-oligosaccharides (variable y6)
on the reaction time and on the polygalacturonase
to solid ratio for experiments carried out at cellulase
to polygalacturonase ratios = 0.25, 1 or 1.75
FPU U)1.
Figure S4. Calculated dependence of the galacturonan
conversion into oligogalacturonides (variable y8) on the
polygalacturonase to solid ratio and on the cellulase to
polygalacturonase ratio for experiments carried out at 6,
13 or 20 h.
Figure S5. Interrelationship between experimental and
predicted results in experiments carried out for model
validation (experiment 16: x1 = )0.6, x2 = )0.3,
x3 = 0.7; experiment 17: x1 = )0.3, x2 = 0.5,
International Journal of Food Science and Technology 2012
8 Pectic oligosaccharides production from orange peel waste M. Martı́nez Sabajanes et al.

x3 = 0.6 and experiment 18: x1 = 0.7, x2 = 0.2,
x3 = )0.67).
Figure S6. High-performance size exclusion chroma-
tography elution profile of (a) standard mixtures and (b)
hydrolysates obtained under selected conditions
(PSR = 5 U g)1, cellulase to polygalacturonase
ratios = 1.5 FPU g)1, t = 20 h).
Table S1. Operational conditions expressed in terms
of the dimensional and dimensionless, normalised vari-
ables.
Table S2. Composition of the raw material.
Table S3. Experimental results obtained in experi-
ments 1–15.
Table S4. Regression coefficients and significance
(based on a t-test) and statistical parameters measuring
the correlation and significance of models obtained for
variables y1 to y9, in the set of experiments 1–15 (see
experimental conditions in Table 1).
Table S5. Composition of hydrolysates and spent
solids obtained under selected conditions.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.

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