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Pectic oligosaccharides from agricultural byproducts: production, characterization and

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Crit Rev Biotechnol, Early Online: 113
! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.996732

REVIEW ARTICLE

Pectic oligosaccharides from agricultural by-products: production,

characterization and health benefits
Neha Babbar1,2, Winnie Dejonghe1, Monica Gatti2, Stefano Sforza2, and Elst Kathy1
Separation & Conversion Technology, VITO-Flemish Institute for Technological Research, Boeretang, Mol, Belgium and 2Department of Food
Science, University of Parma, Parco Area delle Scienze, Parma, Italy

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Abstract

Keywords

Pectin containing agricultural by-products are potential sources of a new class of prebiotics
known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG,
a-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic
acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin
containing agricultural by-products like sugar beet, apple, olive and citrus by chemical,
enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galactooligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive
research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent.
This review comprehensively covers different facets of POS, including the nature and chemistry of
pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for
facilitating selective extraction of pectin, identification and characterization of POS, health
benefits and important applications of POS in food and feed. This review has been compiled to
establish a platform for future research in the purification and characterization of POS and for
in vivo and in vitro studies of important POS, so that they could be commercially exploited.

Agricultural residues, health benefits,

pectic oligosaccharides, pectic substances,
prebiotic

Introduction
Pectin is a complex and heterogeneous polysaccharide present
within the primary cell wall and intercellular regions of higher
plants (Chen et al., 2013). Pectin comprises a family of acidic
polymers, known as homogalacturonan (HG) and rhamnogalacturonan (RG) with several neutral sugars/polymers such as
arabinans, galactans and arabinogalactans (attached as side
chains) (Obro et al., 2004; Strasser & Amado, 2001). The
extraction of these neutral and acidic polymers in the form of
pectic oligosaccharide (POS) is a promising step towards the
manufacture of prebiotics from agricultural by-products
(Munoz et al., 2012; Westphal et al., 2010). Pectic oligosaccharides (POS) are non-digestible oligosaccharides which
beneficially affect the host by selectively stimulating the
growth and/or activity of one or a limited number of bacteria in
the colon (Bifidobacteria and Lactobacilli) (Baldan et al.,
2003; Garthoff et al., 2010; Gibson & Roberfroid, 1995;
Manderson et al., 2005; Mussatto & Mancilha, 2007;
Roberfroid, 1996). Pectic oligosaccharides have been reported
to suppress the activity of entero-putrefactive and pathogenic
organisms (Baldan et al., 2003; Garthoff et al., 2010; Gibson &
Roberfroid, 1995; Manderson et al., 2005; Mussatto &
Mancilha, 2007; Roberfroid, 1996). The colonic fermentation
of prebiotic POS results in the generation of short-chain fatty

Address for correspondence: Dr Elst Kathy, Tel: +32-14335617. Fax:

+32-14321186. E-mail: kathy.elst@vito.be

History
Received 9 January 2014
Revised 12 September 2014
Accepted 12 September 2014
Published online 2 February 2015

acids (SCFA), which exerts a number of health effects like

inhibition of pathogenic bacteria, relief of constipation,
reduction in blood glucose levels, improvement in mineral
absorption, decreased incidence of colonic cancer and modulation of the immune system (Gullon et al., 2013). The
literature also suggests that POS can act as phytoalexin elicitor,
flowering inducer and antibacterial agent in plants (Iwasaki
et al., 1998).
Agricultural by-products have been studied extensively
for bioethanol production (Brienzo et al., 2009; Oberoi
et al., 2011a), enzyme synthesis (Dhillon et al., 2011; Oberoi
et al., 2012) and protein enriched cattle feed (Laufenberg et al.,
2003). Some agricultural by-products like apple pomace, sugar
beet pulp, berry pomace also contain significant amounts of
pectin (Martinez et al., 2010; Munoz et al., 2012). The
production of POS from these agricultural residues is an
interesting way to reuse waste streams for both environmental
and economic benefits. The most common and well
known POS are arabinogalacto-oligosaccharides, arabinoxylo-oligosaccharides, arabino-oligosaccharides, galacto-oligosaccharides, oligo-galactouronides and rhamnogalacturonanoligosaccharides (Concha-Olmos & Zuniga-Hansen, 2012;
Martinez et al., 2009). This review has been compiled to
provide information on the nature and chemistry of pectin;
potential sources of pectin; various pre-treatment methods
for the production of POS from pectin containing agricultural
by-products; purification and characterization of POS; health
benefits of POS and potential application of these compounds
in food and feed industry.

N. Babbar et al.

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Pectin
Pectin is a complex macromolecule made up of several
monosaccharides containing diverse linkages. The structure of
pectin is hypothesized to be made up of smooth
homogalacturonic (polygalacturonic acid) and branched
hairy rhamnogalacturonic regions (in which most of the
neutral sugars are located) (Yapo et al., 2007). A schematic
representation of the structure of pectin and the constituent
sugars in each region is presented in Figure 1. Four main pectic
components have been identified, namely, homogalacturonan
(HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II
(RG-II) and xylogalacturonan (XG) (Caffall & Mohnen, 2009;
Gullon et al., 1989; Ralet et al., 2001; Voragen et al., 2009;
Yapo et al., 2007) All these pectic components are connected
by either covalent or ionic cross links (Schols & Voragen,
2002). The most abundant pectic polysaccharide HG is made
of galacturonic acid (Galp) residues with a-1,4-linkages and
comprises more than 65% pectin (Yapo et al., 2007). It can be
partly methyl-esterified at C-6 and possibly partly acetyl
esterified at O-2 and O-3 (Ralet et al., 2001).
The RGI backbone is composed of [!2)-a-L-Rhap(1 ! 4)- a-D-GalpA-(!1] repeats (Westphal et al., 2010).
Rhamnose is a minor component of the pectin backbone and
introduces a kink into the straight chain. The length and

quantity of HG and RG-I components can vary in different

plants (Gullon et al., 2013). For instance, in sugar beet pectin,
the length of HG polymer is shorter than that of pectin of
citrus and apple. On the other hand, RG-I is more abundant in
sugar beet pulp than in citrus and apple (Gullon et al., 2013).
Rhamnogalacturonan I has a number of side chains in the
form of sugars and branched oligosaccharides attached to its
backbone (Willats et al., 2001). The length of these side
chains can vary from single neutral glycosyl to polymeric side
chains of different types viz. (1 ! 5)-a-L-arabinans, (1 ! 4)b-D-galactans, arabinogalactans-I, arabinogalactans-II (Obro
et al., 2004). Rhamnogalacturonan II is a structurally complex
pectin and accounts for more than 10% pectin (ONeill et al.,
1990). The building blocks of RG-II are galacturonic acid,
rhamnose, galactose and unusual neutral sugars. The structure
of RG-II is characterized as a distinct region within HG that
contains a cluster of side chains of rare sugar residues, such as
apiose, aceric acid, 3-deoxy-lyxo-2-heptuloasaric (DHA) and
3-deoxy-manno-2-octulosonic acid (Zandleven et al., 2007).
Xylo-galacturonan (XGA) is a substituted HG with a single
unit of b-D-Xylp-(1 ! 3) side chain (Voragen et al., 2009).
The presence of XGA has been mainly identified in reproductive organs or storage tissues such as in the cell walls of
peas, soybeans, watermelons, apples, pears, onions, potatoes,
pine pollen and cotton seed (Wong, 2008; Zandleven et al.,
2007).

Figure 1. Structure of pectic polysaccharide.

Pectic oligosaccharides from agricultural by-products

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Pectin complexes

olive oil generates huge quantities of olive pomace. Cell wall

material from olive pomace comprises a number of molecular
components, with considerable quantities of pectic polysaccharides (39%), cellulose (30%), hemicellulosic polymers rich
in xylans and glucuronoxylans (14%), xyloglucans (15%) and
mannans (2%) (Jimenez et al., 1994, 2001).
The pectic polysaccharides of olive pomace are unique due
to the presence of arabinan. The degree of methyl esterification and acetylation in olive pomace was determined to be 48
and 11%, respectively, by Cardoso et al (Cardoso et al., 2003)
which suggests high gelling properties of pectin. Due to
high arabinan and galacturonic acid, olive pomace can be
considered as a potential source of POS (Coimbra et al., 2010;
Munoz et al., 2012; Rodrguez et al., 2007). Hydrothermal
processing of olive pomace has been reported to produce
tetra-, tri- and di-galacturonic acid and different structures of
neutral and acidic xylo-oligosaccharides (Munoz et al., 2012).

There are three type of pectin complexes, namely HG calcium,

RG-II borate and uronyl ester complex. The HG calcium
complex is formed by two unesterified HG chains, whereby the
carboxyl groups of two GalpA residues form a negatively
charged pocket that binds with a Ca2+ cation. A minimum of 10
continuous unesterified GalpA residues are needed to build a
stable cross-link between the chains (Vincken et al., 2003).
Calcium cross linking of HG contributes to the cell wall
strength by bringing blocks of unmethylesterified HG chains
into a tightly packed conformation (Caffall & Mohnen, 2009).
The second pectin crosslink is known as the borate diol ester
(RG-II borate). This is formed by two RG-II molecules with
boron. Only the apiofuranosyl residues of the 2-O-methyl-Dxylose containing side chains in each subunits of the dimer can
participate in the cross-linking. Borate-diol esters can also
crosslink two HG chains as RG-II is an integral part of HG
chain. Cations such as Ca2+ Pb2+, Sr2+ and La3+ promote
dimer formation in vitro (Caffall & Mohnen, 2009).
Homogoalacturonan can also cross-link to other components
by uronyl ester. Approximately 2% of GalpA residues can be
cross-linked this way. Homogalacturonan is mainly found in
plant cell walls in a methyl-esterified form and it is clear that
these molecules hold enormous potential for cross-linking
(Caffall & Mohnen, 2009).

Sugar beet (Beta vulgaris) pulp

Sugar beet pulp is a by-product of the sugar refining industry
and is used mostly as animal feed. It is combined with
molasses and dried to give a high energy feed for ruminants.
Sugar beet pulp polysaccharides consist approximately of
2224% cellulose, 30% hemicellulose, 1525% pectin, 3% ash
and 5.9% lignin (Sun & Hughes, 1999). Beet pulp contains
low amounts of protein, lignin and fat. The combination of
shorter HG chain length, high degree of acetylation and the
higher concentration of side chains (containing neutral sugars)
contributes to the poor gelling properties of sugar beet pectin.
Production of POS from sugar beet pulp (SBP) has been
successfully carried out by various researchers (ConchaOlmos & Zuniga-Hansen, 2012; Leijdekkers et al., 2013).
Al-Tamimi et al. (2006) isolated sugar beet arabinan (MW
570010 000 Da) and arabino-oligosaccharides from sugar
beet pulp. Kuhnel et al. (2010) characterized branched
arabino-oligosaccharides [having an a-(1,5)-linked backbone
of L-arabinosyl residues] from sugar beet pulp produced by a
mixture of arabinohydrolases.

Potential sources of pectic oligosaccharides

Pectin containing by-products, in addition to their conventional uses, can also be exploited for POS production. Table 1
illustrates the quantity of pectin rich agricultural by-products
produced in Europe along with the content of HG and RG
sugars. Further, Table 2 gives a detail insight of the pectin
content as well as acidic and neutral sugar composition of
important agricultural by-products.
Olive (Olea europaea) pomace
Olive pomace is the by-product of olive oil processing. Spain is
the leading producer of olive oil in the world. The production of

Table 1. Pectin content and side chain composition of agricultural by-products.

% Total pectin
Source

Total production
(tones)a
4b

Pectin
content (%)

HG

RG I

NSC

RG II

References

0.3

(Martinez et al., 2010; Eurostat, 2012;

El-Nawawi & Shehata, 1987)
(Voragen et al., 2009; Koodziejczyk
et al., 2007; Schemin et al., 2005)
(Yapo et al., 2007; Voragen et al., 2009;
Faostat, 2012; Guillon et al., 1989)
(Coimbra et al., 2010; Faostat, 2012)
(Eurostat, 2012; Turquis et al., 1999)
(Monsoor & Proctor, 2001)
(Eurostat, 2012; Alexander & Sulebele,
1973)

Citrus waste

8.0  10

30

77

Apple pulp

3.8  105c

20.9

36

47

10

Sugar beet pulp

9.1  107

16.2

29

48

34.4
15
16.31
2734

20

75

38.8

Olive pomace
Potato pulp
Soy hull
Onion skin

6d

1.6  10
1.3  105e

8.5  104f

HG, Homogalacturonan; RG, Rhamnogalacturonan; NSC, Neutral side chains

Waste statistics Europe, on wet basis.
b
Lemon peels (sum of waste produced during lemon juice and lemon jam processing).
c
Pulp produced during apple juice processing.
d
Pomace produced during olive oil production.
e
Potato peel (Sum of waste produced during steaming, drying, cutting and slicing.
f
Sum of waste produced during processing of dried onions, whole, cut, sliced, broken.
a

N. Babbar et al.

Crit Rev Biotechnol, Early Online: 113

Table 2. Pectic polysaccharide composition of agricultural by-products.

Source
a

Orange peel
Chicory roota
Citrus peela
Cauliflowera,b
Endive pulpa
Beet pulpa
Apple pulpc
Limec
Soy hullc
Grape skinc

GalpA

Ara

Rha

Fuc

Man

Xyl

Gal

References

31
23.2
25.8
16
20
25
61.1
82.3
68.72
15.4

7.78
7.2
8.4
7.5
8.4
22.5
3.2
5.1

7.0

1.4
0.9
1
1.2
1.6
4.6
5.1

1.1

0.3
0.6
0.4
0.2
0.2

0.1

1.7
3
1.2
1.6
1.4

0.4

4.29
3.3
3.7
2.4
2.5
1.9
2.7
0.2

1.1

7.47
3.9
6.4
4.3
5.0
5.4
16.0
7.5

7.5

(Martinez et al., 2010)

(Zykwinska et al., 2008)
(Zykwinska et al., 2008)
(Zykwinska et al., 2008)
(Zykwinska et al., 2008)
(Zykwinska et al., 2008)
(Bonin et al., 2002)
(Bonin et al., 2002)
(Monsoor & Proctor, 2001)
(Lecas & Brillquet, 1994)

% dry matter.
Cauliflower florets and buds.
% pectin.
GalpA, Galacturonic acid; Ara, Arabinose; Rha, Rhamnose; Fuc, Fucose; Man, Mannose; Xyl, Xylose; Gal, Galactose.

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Potato (Solanum tuberosum) pulp

Potato pulp is a by-product of the potato industry. Potato pulp
consists of pectic polysaccharides (56%), starch (12%),
proteins (5%), ash (4%) fat (0.3 %) and water (9%). The
pectin of potato pulp contains a high proportion of RG-I with
long galactan side chains (75%) (Khodaei & Karboune, 2013;
Thomassen & Meyer, 2010). Previous studies have reported
successful extraction of HG and RG-I oligosaccharides from
potato pulp (Byg et al., 2012; Thomassen et al., 2011). The
RG-I in potato pulp contains a complex arabinogalactan
structure which can be converted to POS (Obro et al., 2004).
Citrus waste
Citrus fruits are the most important fruits grown and
consumed all over the world (Aggarwal & Sandhu, 2004).
The waste from the orange juice processing industry ranges
between 4060% of the fruit weight and is made up of peel
and segment membranes (Grohmann & Baldwin, 1992).
Citrus reticulata, an important tropical crop, contains 10%
cellulose, 4.28% hemicellulose, 0.56% lignin, 5.78% protein,
22.6% pectin and 3.23% ash (Oberoi et al., 2011b). The
presence of low lignin makes citrus by-products ideal for use
in the area of bioprocessing. Some information on POS
production has been reported in orange peel wastes (Martinez
et al., 2010). Cancalon (1992) found significant amounts of
oligosaccharides of DP42 in naturally fermented citrus
juices. The presence of transfructosidase activity (present in
invertases) catalyses the synthesis of various oligosaccharides
during such fermentations. Kang et al. (2009) evaluated the
positive effects of POS produced by irradiation (10 kGy/h)
from citrus pectin on levels of serum triglyceride, total
cholesterol and LDL-cholesterol in the blood of mice fed
high-cholesterol diets.
Apple (Malus domestica) pomace
Apple pomace a by-product of the apple processing industry
(Watt et al., 1999) accounts for 2535% of the dry mass of an
apple (Gullon et al., 2007b). It contains 7% protein, 1.4% ash,
8.3% pectin, 58.3% neutral polysaccharides (cellulose and
hemicellulose) (Voragen et al., 2009). The residue of apple
contains highly branched RG and XG polysaccharide (Schols
et al., 1995). These polysaccharides can be further degraded

to produce oligomers of desired chain length. Watt et al.

(1999) and Renard et al. (1995b) obtained fucogalactoxyloglucan oligosaccharides from apple pomace by alkaline pretreatment. Gullon et al. (2007a) found 3245% of alcohol
soluble compounds in apple pomace (inclusive of monosaccharides, oligosaccharides and malic acid). Oligosaccharides
were mainly present in the form of gluco-oligosaccharides,
xylo-oligosaccharides and arabino-oligosaccharides.
Others
Oligo-galacturonic acid (DP 612) from tomato processing
waste isolated by acid hydrolysis was found to be potent plant
growth promoter (Suzuki et al., 2002). Hydrolysis of
Lucerene (Medicago sativa) led to the production of acidic
oligosaccharides (Aspinall et al., 1968). Montella et al. (2013)
isolated galacto-oligosaccharides and xyloglucans from hazel
nut skin by alkaline and water extraction. Bilberries and black
currants, important crops in Scandinavian countries contain
pectin. Due to the formation of pectin gel after mashing, some
pectinolytic enzymes are added to the mash to release the
juice. After degradation, some polysaccharides remain in the
mash in the form of RG-II which can be used for POS
production (Hilz et al., 2006). Zykwinska et al. (2008)
obtained POS of different molecular weight from chicory
roots, citrus peel, cauliflower floret/leaves and sugar beet
pulp. Pectic oligomers obtained by hydrolyzing the soybean
polysaccharides were of RG origin (Nakamura et al., 2002).
Cello-oligosaccharides (cellopentaose, cellotetraose, cellotriose and cellobiose) and galactooligosaccharides (galactotetraose, galactotriose) from carrot pomace were obtained after
alkaline pre-treatment (Yoon et al., 2005).

Processes for the production of pectic

oligosaccharides
Pectic polysaccharides are covalently cross linked and
therefore certain pre-treatment is required to separate HG,
RG-I and RG-II from each other. Pectic oligosaccharides can
be obtained by depolymerization of suitable raw materials by
different pre-treatment methods viz. enzymatic, chemical and
physical (Byun et al., 2006; Chen et al., 2013; Combo et al.,
2012; Martinez et al., 2009). Table 3 comprehensively covers
different pre-treatment approaches for the extraction of POS
from different agricultural by-products.

Hydrothermal

Enz (Viscozyme L,
Pectinase and
combinations)
Enz (Araf, Abn and
mixtures)

Sugar beet pulp

Sugar beet pulp

Enz (XG: XG hydrolase,

PGA: Endo-PG, apple
MHR: RG hydrolase)
Enz (EPG-M2, Pectinase,
Viscozyme L, Pectinex
ultra SP-L, Pectinex
62 L, Macer 8 FJ)

Hydrothermal

XG, PGA, Apple MHR

Polygalacturonic
acid (model)

PH (Enz)b

287 min at 160  C;

357 min at 163  C
45  C up to 48 h

Directc

3540  C, pH 3.85.0

Directc

16 h at 37  C

453533 K at 10 MPa

Directc
PH (Acid)b

PH (Acid)b
Directc

10 kGy/h at 14  C
72 h at 80  C

PH (Enz)b

288 min at 160  C

PH (Acid)b

PH (Acid)
PH (Enz)b

Type

15 min at 170 C

Conditions

Identification with Oligomer

standards (di- and tri-PGA)

2 N TFA at 121 C for 2 h

Olivex at 40  C, pH 5.0 for
24 h
Viscozyme L 45 U/g,
Cellulase - 5 FPU/g 45 h,
37  C, pH 5
Viscozyme L 45 U/g 40 h,
37  C, pH 5
1 h, 30  C in 72% (w/w)
H2SO4, followed by 3 h,
100  C in 1 M H2SO4.
1 h, 30  C in 72% (w/w) H2SO4
followed by 3 h, 100  C in
1 M H2SO4;
Quantification by arabinose
oligomers

MW assessment by oligogalacturonides and maltodextrins

of 9801630 D
3 h, 120  C in 2 M TFA
Structural information with MS

Method

Quantification of oligosaccharides

EPG-M2, 2 h:
58% (w/w) DP3
18% (w/w) DP2
13% (w/w) DP1 of total
polysaccharides
Mixture DP 210

DP 620

Mixtures of DP 16 depending
on conditions

31.2% (w/w) POS 29.9% (w/w)

POS of oven dried SBP

25.1% (w/w) POS of oven dried

raw material.

23% (w/w) POS of total polysaccharides (30001000 Da)

Yield

(Miyazawa &
Funazukuri, 2004)

(Combo et al., 2012)

(Coenen et al., 2008)

(Kang et al., 2009)

(Renard et al., 1995a)

(Kuhnel et al., 2010)

(Leijdekkers et al., 2013)

(Martinez et al., 2009)

(Martinez et al., 2010)

(Munoz et al., 2012)

References

Semi-solid by-product of virgin olive oil processing.

PH, Post-hydrolysis (Quantification of the additional monomers formed by post-hydrolyzing the mixture).
c
Direct, Direct quantification/identification of the oligomers present in the mixture.
Araf, arabinofuranosidase; Abn, arabinohydrolase; AOS, arabino-oligosaccharides; DHPM, dynamic high pressure microfluidization; DP, degree of polymerization; EA, endo-arabinase; Enz, enzymatic; EndoPG,
endopolygalacturonase; ExA, exo-arabinase; GO, gluco-oligosaccharides; GalO, galacto-oligosaccharides; GalacidO, galacturonic acid oligosaccharides; HNO3, nitric acid; MHR, modified hairy regions; MS,
mass spectrometer; MW, molecular weight; POS, pectic oligosaccharide; PGA, polygalacturonic acid; RG, rhamnogalacturonase; SBP, sugarbeet pulp; XG, xylogalacturonan

Poly-galacturonic
acid (model)

Irradiation
Acid (HCl 0.1 M)

Citrus pectin
Pectin (apple, sugar
beet and citrus)

Sugar beet Arabinan

Hydrothermal

Steam

Type

Orange peel

Alperujo

Substrate

Pre-treatment

Table 3. Different pre-treatment methods, quantification, identification and yield of POS from various agricultural by-products.

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DOI: 10.3109/07388551.2014.996732

Pectic oligosaccharides from agricultural by-products

5

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Figure 2. Mode of action of different

enzymes on pectin moiety. PME, Pectin
methylesterase; PAE, Pectinacetyl esterase;
PG, Polygalacturanase; RGL,
Rhamnogalacturonanlyasc; RGAE,
Rhamnogalacturonan acetyl esterase; RG,
Rhamnogalacturonan hydrolase; AF,
Arabinofuraosidase; EA, Endo-arabinase;
Endo-Gal, Endo-galactanase; bGal,
b-Galactosidase.

Enzymatic processes
Various enzymes have been widely used for the production of
POS because of their specificity and selectivity. In addition, the
use of enzymes over other pre-treatment methods is regarded
as safe due to minimum adverse chemical modifications of
products (Kim & Rajapakse, 2005). Some specific pectin
degrading enzymes have been used, which acts synergistically
to produce POS (Combo et al., 2012; Concha-Olmos &
Zuniga-Hansen, 2012; Mandalari et al., 2007; Martinez et al.,
2009; Pedrolli et al., 2012; Voragen et al., 2009). Pedrolli et al.
(2012) reviewed the type of enzymes needed for the production
of POS. Figure 2 illustrates various pectic enzymes active on
smooth and hairy regions of pectin. The methyl esters and
acetyl groups from galacturonic acid residues are removed by
pectin methyl esterase (PME) and pectin acetyl esterase (PAE),
respectively (Shevchik & Hugouvieux, 1997). Both enzymes
act before endo-polygalacturonase (Endo-PG). Endo-polygalacturonase (endo-PG) is able to cleave the glycosidic bond of
the a-(1 ! 4)-polygalacturonan in a random fashion (Cameron
et al., 2009). Endo-polygalacturonase generally prefers a nonesterified substrate and shows decreasing activity with
an increasing degree of methyl esterification (Parenicova
et al., 2000). Exo-polygalacturonase (Exo-PG) attacks the
substrate from the non-reducing end and is able to remove
terminally (1-) linked Gal A residues from HG chains (Kester
et al., 1999).
The RG subunit of the ramified hairy regions can be
degraded sequentially by rhamnogalacturonan hydrolase (RG),
and rhamnogalacturonanlyase (RGL) both acting on a-D-1,4GalpA-a-L-1,2-Rhap and a-L-1,2-Rhap-a-D-1,4-GalpA linkage of the RG backbone, respectively. Rhamnogalacturonan
acetyl esterase (RGAE) is an exo-acting pectinase active on the
acetyl groups and also removes terminal rhamnosyl residues
(Mutter et al., 1994). The removal of side chains from RGI can
be achieved by a cocktail of various enzymes such as: (i)
arabinofuranosidase B (Araf), which removes terminal arabinose residues from the arabinan side-chains of pectins
(Westphal et al., 2010), (ii) endoarabinase (EA) hydrolyze

the linear regions of the arabinan backbone and release a

mixture of arabinose oligomers (Beldman et al., 1997) (iii)
exoarabinases (ExA) releases arabinose, arabinobiose
(Carapito et al., 2009), arabinotriose (Kaji & Shimokawa,
1984) to from linear a-linked arabinan. These enzymes act in a
synergistic fashion, leading to a rapid degradation of the
arabinans. On the other hand, the relatively long (1 ! 4)-linked
galactan side-chains can be degraded by endogalactanase
while, b-galactosidase is able to remove terminal galactose
residues from galactans or arabinogalactans (Pedrolli et al.,
2012). Eight neutral branched arabino-oligosaccharides
(a-1,5-linked backbone of L-arabinofuranosyl residues) from
sugar beet arabinan was obtained by a mixture of arabinohydrolases, abn 1 (endo-arabinase), abn2 (exo-arabinase) and abn
4 (arabinofuranosidase) (Westphal et al., 2010). Holck et al.
(2011) separated sugar beet pectin into HG and RG-I by
sequentially applying enzymes viz. pectin lyase, b-galactosidase-1, b-galactosidase-2, galactanase, arabinofuranosidase
and arabinanase.
The process parameters, such as time, temperature,
enzyme concentration, absence and presence of particular
enzyme influence oligosaccharides production (Martinez
et al., 2009). Leijdekkers et al. (2013) and Kuhnel et al.
(2010) concluded that branched arabino-oligosaccharides can
be produced if the enzyme mixture lacks arabinofuranosidase.
Same authors observed that the higher enzyme loadings
results in increased arabinan conversion to arabinose. The
presence of galacturonic acid and low DP oligomers indicated
the presence of Exo-PG which cleaves the polygalacturonic
acid oligomers (Combo et al., 2012; Leijdekkers et al., 2013).
The absence of RG rhamnohydrolase and RG galacturonase
led to the production of recalcitrant oligosaccharides
(Leijdekkers et al., 2013) while the presence of rhamnogalacturonase resulted in the production of rhamnogalacturonan
oligomers (Renard et al., 1995b). Feruloylated arabinose di,
tri, hexa, hepta and octa saccharides, as well as feruloylated
galactose disaccharides, were obtained after the hydrolysis of
sugar beet pulp with driselase (Colquhoun et al., 1994). Potato
pulp was hydrolyzed with pectin lyase, polygalacturonase and

DOI: 10.3109/07388551.2014.996732

pectin methyl esterase for the production of HG and RG-I

oligosaccharides (Thomassen et al., 2011).

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Physical and chemical processes

For POS production, physical pre-treatments like hydrothermal, dynamic high pressure microfluidization (DHPM)
and irradiation have been tried. During hydrothermal pretreatment, pectin is partially hydrolyzed and oligosaccharides
can be effectively released from the biomass. Arabino and
galacto-oligosaccharides were successfully produced from
various agro-residues (Gomez et al., 2013; Martinez et al.,
2009; Munoz et al., 2012; Takano & Sato, 2010) by
hydrothermal hydrolysis. Another physical pre-treatment
DHPM, which is based on the principal of powerful shear,
turbulence, impaction and cavitation, has been used for POS
production from apple pectin (Chen et al., 2013). Various DP
of POS were obtained and identified as oligo-galacturonides,
arabino-oligosaccharides and galacto-oligosaccharides.
Chemical hydrolysis of pectin for the production of POS
has not been studied extensively except for the alkaline pretreatment which is generally used for the production of RG-I
pectin (Sila et al., 2009). Zykwinska et al. (2006) used
alkaline extraction for the production of RG-I oligosaccharides from potato pulp. There are some disadvantages of
chemical hydrolysis processes, as these are generally not safe
for the environment and there is also a limitation to achieve
the desired degree of polymerization (Kim & Rajapakse,
2005).

Characterization and purification of POS

As mentioned previously, pectin is often pre-treated to
produce POS of varying DPs (Table 3). The effect of this
degradation results in fragments which are in the range of a
broad set of analytical techniques (Schols & Voragen, 2002),
ranging from liquid chromatography, to capillary electrophoresis (CE), gas chromatography (GC) and mass spectrometry
(MS). Liquid chromatographic analyses are the most commonly used and are often conducted using high performance
anion exchange chromatography with pulsed amperometric
detection (HPAEC-PAD). Sugar oligosaccharides are separated based on their charge differences with HPAEC, with the
separation being performed at pH 12. The negatively charged
sugars bind to the column and elute through competitive
binding with an increasing salt gradient (Kabel et al., 2001;
Lee, 1996). A series of galacturonic acid oligomers (DP1
DP10), formed by depolymerization of polygalacturonic acid,
were identified on HPAEC-PAD (Combo et al., 2012).
Another possibility recently receiving more and more attention is the use of hydrophilic interaction liquid chromatography (HILIC). POS oligomers have recently been efficiently
separated, identified and quantified using HILIC with online
electrospray ionization ion trap mass spectrometry (ESI-ITMSn) and evaporative light scattering detection (ELSD)
(Remoroza et al., 2014). The molecular weight of POS can
also be estimated with size exclusion chromatography (SEC)
(Combo et al., 2013). The presence of tetra-, tri- and digalacturonic acid, prepared from olive by-products, were
confirmed by adsorption/SEC and identified by HPLC, GC,
ESI-MS and ESI-MS/MS (Munoz et al., 2012). Beside

Pectic oligosaccharides from agricultural by-products

chromatography, matrix-assisted laser desorption ionization

mass spectrometry, due to its tolerance to residual salts, ease
of simple sample preparation and the high speed of analysis,
is often used for offline MS analysis in order to identify the
DP and the composition of the separated oligomers (Daas
et al., 1998). Arabino-oligosaccharides prepared from sugar
beet pulp were identified by MALDI-TOF MS and HPAECPAD (Westphal et al., 2010).
Purification of POS can be accomplished by membrane
based separation or other chromatography based purification
techniques described elsewhere in this article (Garna et al.,
2006; Leijdekkers et al., 2013). Holck et al. (2011) employed
a regenerated cellulose membrane of 3 kDa molecular weight
cut-off for POS purification. Munoz et al. (2012) obtained
POS by ultrafiltration through 1000, 3000, 5000 and 10 000
Da cellulose regenerated molecular weight cut-off membranes. Different techniques based either on the membrane
based technology or those based on chromatography are
employed for separation of POS of different DPs. It is
important to select the membrane and its cut-off on the basis
of molecular weight of a specific compound. Similarly, the
chemistry of the resin/matrix used during chromatography is
important for separation of a specific POS of a desired DP. In
brief, the separation and purification techniques are generally
chosen according to the compound/mixture of compounds to
be separated from a mixture.

Health benefits of POS

The health effects imparted by oligosaccharides make them
active ingredients of functional foods which are similar in
appearance to conventional foods that are consumed as part of
a normal diet and have physiological benefits and/or reduce the
risk of chronic disease beyond basic nutritional functions
(Clydesdale, 1997). As food ingredients, prebiotics have an
acceptable odor and are low-calorie, this allows their utilization in anti-obesity diets. It has been stated that the prebiotic
effect of POS depends upon the molecular weight of the
fractions (Chen et al., 2013; Garna et al., 2006). Olano-Martin
et al. (2002) were the first to compare the effect of pectin and
POS on the growth of pure cultures of various species
indigenous to the gastrointestinal tract. Several authors have
reported that low molecular weight POS have a prebiotic
potential better than high molecular weight POS (Al-Tamimi
et al., 2006). In vitro studies have given a clear indication that
POS can be successfully used to promote bifidogenic flora.
Pectic oligosaccharides of DP 37 were produced enzymatically from bergamot peel and successfully evaluated for their
prebiotic properties in fecal batch cultures (Mandalari et al.,
2007). Chen et al. (2013) used apple pectin POS and found a
decrease in the number of Bacteroides and Clostridia. The
fermentative capability of some intestinal strains viz.
Bacteroides, Bifidobacterium, Clostridium, Klebsiella and
E. coli was tested on POS prepared from soy arabinogalacturonan, sugar beet arabinan, wheat flour arabinoxylan, polygalacturonan and rhamnogalacturonan fraction from apple.
Except for Bacteroides, all other species were able to ferment
in vitro (Van Laere et al., 2000). Small oligomers of
galacturonic acid (with DP 27) were responsible for inhibiting
the adherence of bacteria to epithelial cells, the initial and

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N. Babbar et al.

crucial stage of infection (Guggenbichler et al., 1997). The

in vitro fermentability of apple pomace oligosaccharides
showed an increase in short-chain fatty acids and increased
bacterial growth (Gullon et al., 2011). Holck et al. (2011)
reported that the long-chain arabino-oligosaccharides from
sugar beet pulp have a large bifidiogenic effect in comparison
to short-chain arabino-oligosaccharides. An increase in
Eubacterium rectale population and butyrate levels was
observed with the use of orange peel POS (Manderson et al.,
2005). A stimulation of Bifidobacteria and Lactobacilli
population was seen with the use of low molecular weight
arabino-oligosaccharides (Al-Tamimi et al., 2006).
Pectic oligosaccharides have also been shown to possess
antioxidant activity (Kang et al., 2006) and have a significant
effect lowering the serum levels of total cholesterol (p50.01)
and triglycerides (p50.05) and the inhibition in the accumulation of body fat (Li et al., 2010). Pectic oligosaccharides have
been reported to protect against cardiovascular diseases in vivo
(Li et al., 2010). Treatment with haw POS at higher doses
(150300 mg/kg) significantly suppressed weight gain in mice
(Li et al., 2010). Little literature is available on the evaluation
of POS isolated from agro-residues for their health benefits.
Table 4 summarizes the biological and prebiotic effects of POS
from agro-processing residues. There have been reports that
POS regulates lipid and glucose metabolism with decreased
glycemic response and blood cholesterol levels (Garna et al.,
2006). Pectic derived acidic oligosaccharides (pAOS) have
been evaluated for their genotoxic potential and the safety of
pAOS for human consumption was tested by Garthoff et al.
(2010). An increase in Bifidobacteria populations and a
considerable decrease in the Clostridium lituseburense/
Clostridium histolyticum group was observed in HIV patients
after ingesting a mixture of POS (Gori et al., 2011).
Other health promoting effects of POS are the protection of
colonic cells against Shigella toxins (Hotchkiss et al., 2003;
Olano-Martin et al., 2003a), prevention of adhesion of
uropathogenic microorganisms (Guggenbichler et al., 1997)
and the stimulation of apoptosis of human colonic adenocarcinoma cells (Olano-Martin et al., 2003b). In vivo, the
synergistic empowerment of immunomodulation caused by
galacto-oligosaccharides (GalOS) and fructo-oligosaccharides
(FOS) was studied by Vos et al. (2007). Makker et al. (2002)
have reported inhibition of tumor growth and metastasis by
galactan oligomers. Anti-tumor activity of the galacturonide
(1 kDa) oligosaccharide, obtained from citrus pectin, was
successfully tested on mouse and human tumor cells (Makker
et al., 2002). However, a precise study on the effect of individual
arabino-oligomer, galacto-oligomer, arabino-oligomer is lacking, because POS are generally produced in a mixture and the
complex nature of POSs makes them difficult to separate.
The disadvantages of in vitro methods are the absence of
synergistic, antagonistic, and/or competitive effects as well as
the absence of an immune system. In the field of prebiotics,
POS are an exciting new development as they can be
manufactured from low cost agricultural by-products.
POS in the food industry
Information on the prebiotic activity of POS stated above is
mainly from in vitro models representing the human colon.

Crit Rev Biotechnol, Early Online: 113

However, the mechanisms operating in vivo need to be

elucidated to interpret if these studies can be extended to
human needs as well. Worldwide awareness of consumers
towards diet and health has opened new opportunities for food
industries in research and development of functional foods.
Foods that contain pre- and pro-biotics are drawing the
special attention of consumers and are a potentially exciting
component of the food market. Different prebiotics can be
used for the fortification of different food products to design
functional foods for special target groups. Moreover, prebiotics from other sources have been successfully tested for
their stability at high temperature and low pH and can
therefore be added to bakery product, pasteurized juices and
acidic foods like yogurts (Charalampopoulous & Rastall,
2012). The importance of prebiotic foods lies in their active
stimulation of growth of beneficial bacteria, thereby adding
to potential health and nutritional benefits (Panesar et al.,
2014). However, to further substantiate the claim of the
prebiotic efficacy and other health benefits of POS, more
rigorous in vitro investigations are required and in vivo studies
will validate the claim. Potential applications of prebiotics (both the food and the non-food) in general are
listed in Figure 3.
POS in the feed industry
As antibiotics are prohibited in many countries due to transfer
of the genes which resists anti-microbial/antibiotic action
from animal to human microbiota (Mathur & Singh, 2005).
Consequently, an alternative is needed that could enhance the
natural defense mechanisms of animals. Poultry flocks are the
main infection sources of Camphylobacter jejuni (Corry &
Atabay, 2001), Clostridia and Salmonella infections. Pigs
have been found to be more prone to E. coli infections.
Oligosaccharides have been found to prevent this kind of
invasion by binding to the microbes carbohydrate-binding
proteins and pathogens are cleared by the physiological
mechanism characteristic of the specific tissue (Choct, 2009;
Crittenden, 2006; Zopf & Roth, 1996). In addition, in diets
containing reasonable quantities of carbohydrates, saccharolytic fermentation prevails, thus the pH of the GIT remains
stable and subsequently reduces the onset of Clostridia
infections, as a more alkaline pH is required by species of
this genus. Inulin, added to rabbit feed, was fermented in the
caecum produced SCFA, and reduced the risk of clostridiosis
(Maertens et al., 2004). Pectic oligosaccharides are reported
to possess this activity and have been tested against some
pathogens or toxins (Olano-Martin et al., 2003a). Ganan et al.
(2010) found that POS significantly inhibits cell invasion.
Gaggia et al. (2010) have reviewed, in detail, the application
of prebiotics in animal feeding. As mentioned previously,
there are a number of reports available on the application of
POS in food and pharmaceutical industry. However, their
potential in the feed industry is yet to be exploited. There is
only limited information available on the use of POS in
animal feeds to promote the health of the animal or acting as
therapeutic agents. In vitro studies show that POS have a
potential to be used as feed additives. However, extensive
in vivo studies may be required in different animal models due
to the complex structure of the GIT and diverse microflora.

210 and 714

10100 KDag; 4100 KDah

14001700 KDa

210 (Branched and Linear)

DP 5 and 9i; DP 5j; DP 10k; DP 5l

Neutral sugar fractions:
1 KDa (Oligogalacturonides)

Sugar beetc

Potato pulp

Bergamot peel

AraGalOS from Soy

POS from haw pectin

Mixture of POS

Acidic (galacturonic acid) POS

Acidic (galacturonic acid) POS

Sugar beet Arabinan

Pectin

Bifidobacterium and Eubacterium

rectalea
(Bifidobacterium, Bacteroids,
Lactobacilli)a, Clostridiad
(Bifidobacterium and
Lactobacilli)a
(Bifidobacterium, Lactobacilli,
Eubacteria)a and Clostridiab
Assimilation of Bacteroides
Decreased serum cholesterol,
inhibition of accumulation of
body fat
Bifidobacterium growth in HIV
patients, reduction in fecal
pathogens
Improved immune response
Reduced allergic asthma,
(Bifidobacterium and
Lactobacilli)a, Clostridiab
(Lactobacilli and
Bifidobacterium)a
Increase in growth of
Bifidobacterium;
Growth inhibition of human cancer
cells

In vivo/in vitro effects

DP, Degree of polymerization; POS, Pectic Oligosaccharides; SCFA, Short-chain fatty acids.
Increase in numbers.
Highest production.
c
Feruloylated and non-ferulolylated arabino-oligosaccharides.
d
Decrease in numbers.
e
Increased with decreasing molecular weight.
f
Decreased with decreasing molecular weight.
g
Homogalacturonan.
h
b-1,4 galactan chains of Rhamnogalacturonan.
i
Oligogalacturonides.
j
Methylated Oligorhamnogalacturonides.
k
Oligorhamnogalacturonides.
l
Oligogalactosides.
m
KDa, Kilo Dalton.

Citrus pectinm

Mixture of POS

Effective DP/Molecular weight

Orange peel

Source/Type of POS

Table 4. Health benefits of POS extracted from different sources.

Quantitative real-time PCR

Acetate, propionate and

butyrate

SCFA produced/others
Lactic, propionic, butyricb,
Acetic
Acetatese and propionatesf

Fluorescence in situ
hybridization

Fluorescence in situ
hybridization
Fluorescence in situ
hybridization

Enumeration of bacterial
growth/others

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References

(Huang et al., 2012)

(Onumpai et al., 2011)

(Vigsnaes et al., 2011)

(Olano-Martin et al., 2003b)

(Magne et al., 2008)

(Li et al., 2010)

(Olano-Martin et al., 2002)

(Kang et al., 2006)

(Mandalari et al., 2007)

(Thomassen et al., 2011)

(Al-Tamimi et al., 2006)

(Manderson et al., 2005)

DOI: 10.3109/07388551.2014.996732

Pectic oligosaccharides from agricultural by-products

9

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10

N. Babbar et al.

Crit Rev Biotechnol, Early Online: 113

Figure 3. Potential applications of prebiotics.

Market demand of prebiotics

According to the Global Industry Analysts (GIA) report, the
European and the US market for prebiotics is projected to
reach $1.17 billion and $225.31 million, respectively, by the
year 2015. While the European market is driven by the
expansion of prebiotic ingredient manufacturers into new
application areas such as meat and snack products, the US
market is driven by continued demand for fructans, which
includes both inulin as well as fructo-oligosaccharides. The
global market for prebiotics is projected to reach US$4.8
billion by 2018, driven by the rising awareness of health and
nutrition, growing consumer acceptance of the benefits of
prebiotics, expanding applications and rapid innovations in
prebiotics based food products. The potential in the world
market for emerging prebiotics in terms of their production
and purification is yet to be completely realized and can be
optimized from cellulosic and pectic biomass pre-treatments.
Fructans represent the largest product market worldwide.
Prominence of GOS (Galacto oligosaccharides) is growing
led by the inherent benefits offered by this class of
oligosaccharides in their versatility for use in a wide range
of products including clear beverages, juices and bakery
products. Since pectic oligosaccharides are not yet commercial, it is difficult to predict their contribution to the prebiotic
industry in economic terms. However, it is felt that the POS
are likely to contribute significantly to the prebiotic market in
the years to come. Development of POS from relatively
cheaper by-products such as agro-residues for application in
food, feed and pharmaceutical industry will set new directions
for future research.

Concluding remarks
Hopefully, this review has thrown light on some of the
important aspects of POS and their beneficial effects on
human health. Pectic oligosaccharides belong to an important
category of prebiotics which are also known for prevention
and treatment of various chronic diseases, such as constipation, hepatic encephalopathy, cancer etc. However, to improve

the economics of prebiotic production, technologies based on

bio-utilization of agro-residues need to be further strengthened. In addition, the characterization and purification of
individual oligosaccharide from POS needs further studies to
confirm which POS is responsible for the prebiotic effect and
other health benefits in humans as well as animals. Thus, POS
could be valuable in the development of nutritional and drug
therapies to combat different health ailments. At this stage, it
is also important to conduct extensive research on the
application of POS as biopreservatives, natural therapeutics
and immune building molecules. Their contribution in feeds
which have a direct impact on the quantity and quality of meat
needs extensive research.

Declaration of interest
Authors have no conflict of interest. The authors acknowledge
the work supported by European commission (NOSHAN,
contract no. 312140 FP7 and RESFOOD, contract no. 308316
FP7).

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