Pectic oligosaccharides: Manufacture and functional properties

Trends in Food Science & Technology 30 (2013) 153e161

Review

Pectic oligosaccharides: Manufacture and functional properties
na, B. Go
mezb,c, B. Gullo M. Mart
ınez-Sabajanesb,c,
b,c and ~ezb,c, J.C. Parajo R. Y
an J.L. Alonsob,c,* a

CBQF, Escola Superior de Biotecnologia,
lica Portuguesa, Rua Dr. Anto
nio Universidade Cato Bernardino de Almeida, 4200-072 Porto, Portugal b Department of Chemical Engineering, Faculty of Science, University of Vigo, Campus Ourense, As Lagoas, 32004 Ourense, Spain c ~as, 32901 CITI-Tecnopole, San Cipri
an de Vin Ourense, Spain (Tel.: D34 988387233; fax: D34 988387001; e-mail: [email protected]) There is an increased interest on the identification, production, purification, evaluation and commercialization of new prebiotics with improved properties. Partial hydrolysis of pectin by chemical and/or enzymatic methods leads to the production of pectin-derived oligosaccharides (POS), which have been proposed as a new class of prebiotics. This work reviews the scientific information available on the production, chemical characterization, purification and properties of POS, with special focus on their in vitro and in vivo effects.

Introduction The colonic microbiota plays an important role on the host health. According to the International Scientific Association for Probiotics and Prebiotics, “a dietary prebiotic is a selectively fermented ingredient that results in specific

* Corresponding author. 0924-2244/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tifs.2013.01.006

changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health”. The most usual prebiotics are Non-Digestible Oligosaccharides (NDOs), which resist digestion and reach the colon intact. The colonic fermentation of prebiotic oligosaccharides results in the generation of Short Chain Fatty Acids (SCFA), which exert a number of healthy effects, including relief of constipation, reduction of the blood glucose level, improvement of mineral absorption, regulation of lipid metabolism, decreased incidence of colonic cancer and modulation of the immune system. Other beneficial effects caused by prebiotics include the inhibition of pathogenic bacteria and the “barrier effect” that limits the number of available adhesion sites. There is an increased interest in the identification, evaluation and commercialization of new products with improved functional properties, for example higher ability for modulating microbiota and for causing effects on the distal part of the colon (where there are increased risks of colon cancer and ulcerative colitis). Resistant starch, xylooligosaccharides, soya oligosaccharides, mannooligosaccharides and pectin-derived oligosaccharides (POS) are representative examples of these new products. Pectic oligosaccharides (POS) have been proposed as a new class of prebiotics capable of exerting a number of health-promoting effects (Hotchkiss et al., 2004), including:  Protection of colonic cells against Shiga toxins (OlanoMartin, Williams, Gibson, & Rastall, 2003).  Prevention of the adhesion of uropathogenic microorganisms (Guggenbichler, Bettignies-Dutz, Meissner, Schellmoser, & Jurenitsch, 1997).  Stimulation of apoptosis of human colonic adenocarcinoma cells (Olano-Martin, Rimbach, Gibson, & Rastall, 2003).  In vivo synergistic empowerment of immunomodulation caused by galactooligosaccharides (GalOS) and fructooligosaccharides (FOS) (Vos et al., 2007).  Potential for cardiovascular protection in vivo (Li et al., 2010), reduction of damage by heavy metals, antiobesity effects, dermatological applications and antitoxic, antiinfection, antibacterial and antioxidant properties.

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Additionally, in vivo and in vitro studies have confirmed that acidic POS are not cytotoxic or mutagenic, being suitable for use in foods for children and babies (Garthoff et al., 2010). This work provides an updated overview on the scientific information concerning the production, chemical characterization and properties of POS. Their in vitro and in vivo effects are also discussed. Pectins as sources of POS POS are obtained by pectin depolymerization. Pectins are ramified heteropolymers made up of a linear backbone of a(1e4)-linked D-galacturonic acid (GalA) units (which can be randomly acetylated and/or methylated). These linear structures make part of the “smooth region” of pectins, which is occasionally interrupted by “hairy regions” characterized by ramifications made up of neutral sugars. Fig. 1 shows a simplified structure of pectins, which are made up of the following major components:  Homogalacturonan (HG), a polymer formed by GalA units, containing free or esterified carboxyl groups, which can be partly substituted by sugars.  Rhamnogalacturonan type I (RG-I), a polymer made up of alternate units of rhamnose (Rha) and GalA ramified principally with arabinan, galactan and arabinogalactan.  Rhamnogalacturonan type II (RG-II), a polymer of GalA with a complex ramification pattern. Table 1 lists the pectin contents of typical feedstocks, among which the most important are citrus peels and apple pulp. Other substrates (such as sugar beet pulp, peels of mango or peach and pumpkin pulp) also have high pectin contents. HG from different raw materials may present significant structural differences, which are influential on their properties and applications. For example, the HG chains in sugar beet pectin are shorter than the ones of citrus and apple, whereas RG-I is more abundant in sugar beet pulp than in citrus peel or apple pulp. Pectins from spinach and sugar beet contain significant amounts of esterified ferulic acid, a favourable feature for multifunctional effects based on antioxidant activity. Industrial manufacture of pectins Pectin is commercially produced by extraction of the native feedstock with acidic solutions and further alcohol precipitation. Commercial pectins are usually classified according to their methylation degree, as it is considered as the most influential factor on the physicochemical properties (Sila et al., 2009). Applications of pectins Pectins are food ingredients widely used as thickeners or as gelling and stabilizing agents. Pectin from industrial sugar beet processing shows limited gelling ability and is

principally employed as an emulsifier (Funami et al., 2011). Other applications of pectins include the formulation of pharmaceutical products for treating gastrointestinal disorders, diabetes, high blood pressure or high blood cholesterol (Matsumoto et al., 2008). Pectin derivatives have also been employed in vaccine manufacture (Szu, Bystricky, Hinojosa-Ahumada, Egan, & Robbins, 1994). Production, purification and fractionation of POS In this work, the acronym POS is employed in a generic way to denotate the various types of oligomers (substituted or non-substituted) obtained by pectin depolymerization. According to this idea, POS includes oligogalacturonides (OGalA), galactooligosaccharides (GalOS), arabinooligosaccharides (AraOS), rhamnogalacturonoligosaccharides (RhaGalAOS), xylooligogalacturonides (XylOGalA) and arabinogalactooligosaccharides (AraGalOS). POS production POS can be obtained by depolymerization of suitable raw materials or purified pectins by partial enzymatic hydrolysis (Combo, Aguedo, Goffin, Wathelet, & Paquot, 2012; Concha & Z
u~niga, 2012; Mandalari et al., 2006; Mart
ınez, Gull
on, Y
a~nez, Alonso, & Paraj
o, 2009), acid hydrolysis (Grohmann, Cameron, & Buslig, 1995), hydrothermal processing (Mart
ınez, Gull
on, Y
a~nez, Alonso, & Paraj
o, 2010), dynamic high-pressure microfluidization (Chen et al., 2013) or photochemical reaction in media containing TiO2 (Burana-Osot, Soonthornchareonnon, Hosoyama, Linhardt, & Toida, 2010). Chemical methods The partial breakdown of the various pectin fractions (HG, RGI, RGII and side chains) can be achieved by processing with externally added acids or by aqueous processing (hydrothermal treatments). In both cases, hydronium ions (from externally added acids, in situ generated acids or water autoionization) are the catalytic species. a) Processing with externally added acids The information reported on this topic is scarce. Manderson et al. (2005) hydrolyzed orange albedo with HNO3 solutions, whereas Coenen, Kabel, Schols, and Voragen (2008) treated apple pulp with solutions containing HCl and TFA to obtain a complex mixture of saccharides. Renard, Lahaye, Mutter, Voragen, and Thibault (1997) obtained a product rich in RhaGalAOS from deesterified beet pulp using HCl solutions. The same approach has been employed to obtain products with Mw in the range 15400e19800 (Hell
ın, Ralet, Bonnin, & Thibault, 2005). b) Hydrothermal treatments POS were obtained by aqueous processing of sugar beet pulp and orange peels at yields of 29.7 and 24 g POS/100 g,

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Arabinogalactan type I

Galactan

HG

Arabinogalactan type II

Xylogalacturonan

Apiogalacturonan

Rhamnogalacturonan II

Galacturonic acid (GalA)

Methyl ester

Rhamnose (Rha)

Acetyl ester

Arabinose (Ara)

Fucose (Fuc)

Galactose (Gal)

Apiose (Api)

Xylose (Xyl)

Arabinan

Homogalacturonan

155

Rhamnogalacturonan I

Aceric acid (AceA)

Glucuronic acid (GlcA) Deoxyxyloheptulopyranosylaric acid (Dha) Ketodeoxymannooctulopyranosylonic acid (KDO)

Fig. 1. Simplified structure of pectin.

respectively (Mart
ınez, Gull
on, Schols, Alonso, & Paraj
o, 2009; Mart
ınez, Y
a~ nez, Alonso & Paraj
o, 2010). OGalA with DP in the range 4e6 (Qu
em
ener, D
esir
e, Debrauwer, & Rathahao, 2003) or below 10 (Miyazawa & Funazukuri, 2004; Miyazawa, Ohtsu, & Funakuzury, 2008) have been obtained from polygalacturonic acid (PGA). Enzymatic methods The enzymatic breakdown of pectin chains involves reactions of hydrolysis (catalyzed by hydrolases) or b-elimination (catalyzed by lyases). PGA depolymerization by endopolygalacturonases yielded oligogalacturonides of Table 1. Pectin contents of selected feedstocks. Source

Content (% wt)

Reference

Orange peel Lemon nuggets Lemon peel Lemon pulp Apple pulp

30 6 32 25 20.9

Sugar beet pulp Peach

16.2 18

Mango

21

Pumpkin

22

El-Nawawi & Shehata, 1987 Navarro & Navarro, 1985 Navarro & Navarro, 1985 Navarro & Navarro, 1985 Canteri-Schemin, Fertonani, Waszczynskyj, & Wosiacki, 2005 Yapo, Robert, et al., 2007 Pag
an, Ibarz, Llorca, Pag
an, & Barbosa-C
anovas, 2001 Rehman, Salariya, Habib, & Shah, 2004 Ptichkina, Markina, & Rumyantseva, 2008

DP < 7 (Coenen et al., 2008; Combo et al., 2012). Related studies have been performed with sodium pectate, citrus pectin (Iwasaki & Matsubara, 2000) and ginseng pectin (Yu et al., 2010). Processing high methoxyl pectins (HMP) or low methoxyl pectins (LMP) with endopolygalacturonases resulted in 95% conversion into POS (OlanoMartin, Mountzouris, Gibson, & Rastall, 2001); and lowDP OGalA were obtained from PGA by treatments with pectin-lyase (Delattre, Michaud, & Vijayalakshmi, 2008) or pectate-lyase (Hotchkiss, Hicks, Doner, & Irwin, 1991). Rhamnogalacturonoligosaccharides have been produced using rhamnogalacturonan-hydrolase (Coenen et al., 2008); whereas mixtures of activities (pectin-methylesterase, rhamnogalacturonase, galactanase and arabinase) have been applied to pectins from apple and sugar beet to produce low-DP rhamnooligosaccharides (RhaOS) substituted with arabinosyl residues (Bonnin, Dolo, Le Goff, & Thibault, 2002). Mixtures of up to 4 types of enzymes (pectin-methyl esterases, endopolygalacturonases, endoarabinases and endogalactases) were employed to obtain hydrolysis products with DP up to 18, with predominance of low molecular weight OGalA (Ralet et al., 2005; Ralet, Cr
epeau & Bonnin, 2008). Holck, Hjernø, et al. (2011) proposed a two-step process for producing OGalA and RhaOS in separate streams from sugar beet pulp pectin, whereas one-step production of POS has been reported from bergamot peel (Mandalari et al., 2007), sugar beet pulp (Mart
ınez, Gull
on, Y
a~nez, et al.,

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~ez, Alonso, & Paraj
o, 2009) or orange pulp (Mart
ınez, Y
an 2012). Combined methods This approach is based on consecutive steps of pretreatment and hydrolysis. Zykwinska et al. (2008) employed a number of byproducts from the food industry to obtain pectin and POS using processing schemes involving ethanol extraction and further enzymatic processing. AlTamimi, Palframan, Cooper, Gibson, and Rastall (2006) obtained AraOS with DP in the range 2e8 by enzymatic hydrolysis of sugar beet arabinan. Acidic processing followed by enzymatic hydrolysis (Garna, Mabon, Wathelet, & Paquot, 2004; Renard, Thibault, Mutter, Schols, & Voragen, 1995), or pretreatments with water, oxalate and alkali (Yapo, Robert, Etienne, Wathelet, & Paquot, 2007) have been used for the same purpose. S
eveno et al. (2009) obtained a complex mixture of oligomers from Arabidopsis mutant by consecutive stages of ethanol extraction, NaOH saponification, enzymatic processing and TFA hydrolysis. Purification and fractionation of POS POS purification The liquid media from POS production may contain contaminants that should be removed before utilization as food ingredients. Holck, Hjernø, et al. (2011) employed a regenerated cellulose membrane of 3 kDa Molecular Weight Cut Off (MWCO) for this purpose, whereas Iwasaki and Matsubara (2000) purified oligomers obtained from citrus pulp pectin using a 50 kDa MWCO membrane to retain the high MW compounds, and processed the resulting filtrate through a 15 kDa MWCO membrane to wash out monosaccharides and saccharose. POS fractionation POS fractionation intends the separation of a scope of products with different properties (for example, charge, molecular weight, and substitution pattern), which determine their potential applications. The most usual fractionation technologies are Ion Exchange Chromatography and Gel Filtration (GF) Chromatography. Renard et al. (1997) employed ion exchange columns for separating neutral from charged oligomers, whereas Ralet et al. (2005) employed DEAE-Sepharose to fractionate OGalA mixtures according their Mw and charge. In another work, unsaturated and saturated pectic oligogalacturonides were separated by Guillaumie et al. (2006) using the resin Source 15Q. The fractionation of oligomers has been successfully achieved using GF (Yu et al., 2010). Van Laere, Hartemink, Bosveld, Schols, and Voragen (2000) employed Bio-Gel P2 for the separation of AraGalOS and AraOS with DP up to 9 and 5, respectively; whereas Yu et al. (2010) obtained five purified POS fractions by eluting hydrolyzates through three columns in series containing Sephadex G-25, Sephadex G-75 and Sepharose CL-6B.

POS characterization The structural characterization of POS is difficult due to the complex chemical composition of pectins, and to the possible chemical alteration during POS manufacture. Using HPAEC-PAD, Al-Tamimi et al. (2006) identified AraOS with DP 2e8. Chromatographic fractionation (Chang, Hsu, & Chen, 2010; Ralet et al., 2005; 2008; Yapo, Lerouge, Thibault, & Ralet, 2007) has been employed as a refining step before chromatographic analysis. Complex mixtures may require additional pre-processing, for example by GF (Kurita, Fujiwara, & Yamazaki, 2008; Ralet et al., 2005; Yapo, Lerouge, et al., 2007). The identification and quantitation of the POS structural units may require a combination of separation and/or hydrolysis techniques together with specific analytical methods. In this field, Matrix-Assisted Laser Desorption Ionization e MS (Guillaumie et al., 2006; Holck, Hjernø, et al. (2011)) and Electrospray Ionization e MS (Ralet et al., 2005) present advantages derived from their high sensibility and ability to handle complex POS mixtures. NMR has been employed in the characterization of OGalA (Ralet, Lerouge, & Qu
em
ener, 2009) and rhamnogalacturonan oligomers (Colquhoun, de Ruiter, Schols, & Voragen, 1990; Renard et al., 1997). Fluorescent labelling of OGalA and RhaGalAOS was used by Ishii, Ichita, Matsue, Ono, and Maeda (2002) before NMR and ESI-MS identification. The composition of complex oligomer mixtures has also been assessed by CE e MS (Coenen et al., 2008) and CE with UV detection (Str€ om & Williams, 2004). Prebiotic potential of POS The prebiotic effects of POS depend on their chemical and physicochemical features. To assess the prebiotic activity of a given prebiotic candidate, the assimilation kinetics, the distribution of metabolic products and the effects caused on bacterial populations are key aspects to be considered. Table 2 summarizes the biological and prebiotic effects of POS observed in in vivo and in vitro studies. In vitro fermentation of POS by individual strains Van Laere et al. (2000) studied the fermentation of various oligosaccharides (including AraGalOS, AraOS, OGalA, and RhaGalAOS) by selected bacterial strains. RhaGalAOS were selectively fermented only by Bacteroides spp; whereas Clostridium spp. (which were able to ferment most substrates in some extent) showed little affinity for this type of compounds. AraGalOS and AraOS (but not RhaGalAOS or OGalA) were utilized by Bifidobacterium. The influence of the degree of methylation (DM) on the in vitro fermentability of POS by individual bacteria has been assessed (Olano-Martin, Gibson, & Rastall, 2002). Bifidobacterium angulatum, B. infantis and Bifidobacterium adolescentis were able to utilize POS, particularly low methylated substrates. Most Bifidobacterium were able to utilize a POS-rich extract of DP 3e7 obtained from bergamot peel, which

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157

Table 2. Biological and prebiotic effects of POS observed in in vivo and in vitro assays. Product

Assay

Biological and prebiotic effects

References

POS from high methylated citrus pectin POS from low methylated apple pectin POS from carrots

In vitro

Ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7

Olano-Martin, Willians, et al., 2003

In vitro

Guggenbichler et al., 1997

POS

In vitro

GalOS and FOS with or without POS POS from haw pectin

In vivo

Ability to block bacterial adherence of E. coli to uroepithelial cells Stimulation of apoptosis of human colonic adenocarcinoma cells Decreased parameters of allergic asthma Decreased serum levels of total cholesterol and triglycerides Inhibited accumulation of body fat Assimilation by Bacteroides spp. Assimilation by Bifidobacterium spp. Bifidobacterium angulatum, B. infantis and B. adolescentis were able to utilize POS, particularly low methylated substrates Increase in the number of bifidobacteria and lactobacilli and decrease of the clostridial population Selectively utilized by Bifidobacterium adolescentis, B. longum and Bacteroides vulgatus suggesting that the beneficial effects of apple ingestion are partly due to apple pectin Increased bifidobacteria and lactobacilli and generation of acetate, butyrate and propionate in the media Stimulation of bifidobacteria growth, with effects depending on molecular weight Bacteroides numbers varied depending on the substrate Clostridia decreased on all substrates Increase bifidobacteria and Eubacterium rectale numbers with coupled increases of butyrate concentrations Selective stimulation of bifidobacteria by feruloylated and nonferuloylated long-chain AraOS Non-stimulated growth of Clostridium difficile Different effects of DP4 and DP5 oligos on the ratio between Bacteroidetes and Firmicutes, indicating that slight differences in structures may result in different biological properties

Olano Mart
ın et al., 2002

In vivo

AraGalOS from soy arabinogalactan AraOS from sugar beet arabinan OGalA from polygalacturonic acid RhaGalAOS from apple rhamnogalacturonan POS from high methylated citrus pectin POS from low methylated apple pectin

In vitro

POS form bergamot peel

In vitro

AraOS from apple pectin

In vitro

POS from sugar beet and Valencia oranges

In vitro

AraOS from sugar beet pulp

In vitro

POS from orange peel

In vitro

AraOS from sugar beet pulp (with or without feruloyl substituents, DP 2e14)

In vitro

POS from sugar beet pectin

In vitro

In vitro

Olano-Martin, Rimbach, et al., 2003

Vos et al., 2007 Li et al., 2010

Van Laere et al., 2000

Mandalari et al., 2007

Suzuki et al., 2004

Hotchkiss et al., 2004, 2007

Al-Tamimi et al., 2006

Manderson et al., 2005

Holck, Lorentzen, et al., 2011

Holck, Hjernø, et al. 2011

(continued on next page)

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Table 2 (continued ) Product

Assay

Biological and prebiotic effects

References

POS mixture from apple pomace

In vitro

Gull
on et al., 2011

POS from apple pectin

In vitro

Mixtures of POS, GalOS and FOS Mixtures of POS, GalOS and FOS

In vivo

Mixtures of POS, GalOS and FOS

In vivo

Mixtures of POS, GalOS and FOS

In vivo

Increased populations of bifidobacteria and lactobacilli and decreased populations of bacteroides and clostridia Increased concentrations of short chain fatty acids Increased numbers of bifidobacteria and lactobacilli (in higher extent than pectin) Increased concentrations of acetic, lactic, and propionic acid (in higher extent than pectin) Decreased numbers of numbers of bacteroides and clostridia (the parent pectin showed the opposite effect) Increased counts of bifidobacteria and lactobacilli Increased numbers of bifidobacteria Increased proportions of bifidobacteria in the mixture GalOS/FOS/POS respect to the mixture GalOS/FOS Decreased proportions of Bacteroides and Clostridium coccoides Trend towards a lower incidence of serious endogenous infection and serious infectious episodes Stimulation of bifidobacteria growth in HIV-infected individuals Reduction in fecal pathogenic load in HIV-infected individuals

In vivo

scarcely supported the growth of Bacteroides and clostridia (Mandalari et al., 2007). Suzuki, Tanaka, Amano, Asakura, and Muramatsu (2004) assessed the in vitro fermentation of apple-derived arabinooligosaccharides by Bifidobacterium longum and Bacteroides vulgatus, which were able to utilize compounds with DP > 3 selectively, providing a possible explanation of the beneficial effects derived from apple ingestion. Olano-Martin et al. (2002) studied the in vitro fermentation properties of pectins and POS by human gut bacteria using fecal cultures, and reported a negative influence of the degree of esterification (DE) on cell growth, and increased digestibility of POS respect to pectins. In vitro fermentation of POS and pectins by fecal inocula Intestinal fermentation can be affected by synergistic, antagonistic and/or competitive effects, since complex metabolic processes are carried out by different groups of bacteria, in a way that the metabolic products generated by a given group can be assimilated by others which cannot use the original substrate directly (Gibson & Roberfroid, 1995). In vitro and in vivo studies established the colonic fermentation patterns of pectins with different structures, as well as their effects on metabolism and health (Gull
on et al., 2009). Hotchkiss et al. (2004) considered the in vitro fermentability of HM oligosaccharides from citrus and LM

Chen et al., 2013

Fanaro et al., 2005 Magne et al., 2008

Westerbeek et al., 2010

Gori et al., 2011

compounds from apple and orange peel using mixed fecal batch cultures, and concluded that all the substrates enhanced the growth of bifidobacteria and lactobacilli, while the proliferation of pathogens was limited. In a related work, Hotchkiss et al. (2007) used POS from Valencia oranges as a fermentation substrate in experiments with human fecal inocula, and reported increased bifidobacteria counts and generation of acetate, butyrate and propionate in the media. Sugar beet pulp arabinan and eight AraOS fractions were assayed by Al-Tamimi et al. (2006) as carbon sources in experiments with human gut bacteria. Different fermentation patterns (resulting in different microbial populations and metabolite concentration profiles) were observed for AraOS of different MW: lowMW oligosaccharides were more selective for Bifidobacterium than arabinan or high MW fractions, whereas the SCFA molar ratios were in agreement with data published for other arabinose-containing substrates (Fardet, Guillon, Hoebler, & Barry, 1996). The fermentation of POS from orange peel by mixed fecal bacterial cultures has been reported to result mainly in the production of acetic and butyric acids, and in increased numbers of bifidobacteria and Eubacterium rectale (Manderson et al., 2005). Fermentation of a POS-rich extract from bergamot peel with fecal inocula resulted in increased counts of bifidobacteria, lactobacilli and eubacteria,

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together with decreased clostridial population. Recently, arabinooligosaccharides from sugar beet pulp (with or without feruloyl substituents, DP 2e14) were assayed in vitro for fermentability using human fecal inocula (Holck, Lorentzen, et al., 2011). The oligomer size was more influential on the selective bacterial stimulation than the degree of substitution by feruloyl groups. An assessment of the effects caused by HG-derived POS of DP 4e5 on the relative proportions of the dominant bacterial phyla in the human intestine (measured by the Bacteroidetes/Firmicutes ratio) showed that compounds with just slightly different structures may cause significantly different biological effects (Holck, Hjernø, et al. (2011)). The fermentation of a refined POS mixture from apple pomace with human faeces (Gull
on, Gull
on, Sanz, Alonso, & Paraj
o, 2011) resulted in increased populations of Bifidobacterium, Clostridium coccoides, Eubacterium rectale and Bacteroides-Prevotella. These latter produce propionate, a compound to which healthy effects have been ascribed, including ability for decreasing the hepatic synthesis of cholesterol (Amaral, Morgan, Stephen, & Whiting, 1992) and modifying the glucose metabolism, limiting the postprandial and insuline responses (Leclere et al., 1994). In a related study, POS from apple pomace have been reported to increase the number of bifidobacteria and lactobacilli, while decreasing the counts of Bacteroides and clostridia (an effect not observed when pectin was used as a fermentation substrate) (Chen et al., 2013). Experimental data obtained with humans Limited information exists on experimental studies assessing the prebiotic effects caused by POS in humans. The administration of acidic POS as a component of infant formulae resulted in increased counts of bifidobacteria and lactobacilli (Fanaro et al., 2005; Magne et al., 2008). Enteral delivery of neutral and acidic oligosaccharides to preterm infants did not reduce the risk of serious infectious morbidity significantly, but the results showed a trend towards a lower incidence, especially for infections with endogenous bacteria (Westerbeek et al., 2010). Ingestion of a mixture of prebiotic oligosaccharides (containing short chain galactooligosaccharides, long chain fructooligosaccharides and acidic pectic oligosaccharides) by volunteers in the earlier stages of HIV-1 infection resulted in the modulation of the gut microbiota by increasing the bifidobacteria numbers and by decreasing the counts of pathogens belonging to the Clostridium lituseburense/Clostridium histolyticum group (Gori et al., 2011). Conclusions and future trends The identification, production and commercialization of new prebiotics with enhanced properties offer new research and business opportunities. Among them, pectin-derived oligosaccharides may play a significant role, owing to their putative prebiotic activity and to their ability to exert a number of other healthy effects. There are sound scientific

159

evidences of the biological activity of POS, but new technical and scientific information is needed before industrial development. Particularly, cost-effective processes for the manufacture of purified POS with tailored structure have to be developed on the basis of new knowledge about reaction and separation technologies, and reliable data information on the structure-function interrelationships is needed. For this purpose, in vitro studies performed with target products of different purity, structure and MW (for example, using an artificial colon) could provide key information on the kinetics of substrate assimilation in the various colon sections and on the major metabolic products, narrowing the experimental focus for additional in vivo assays, which are needed to confirm the resulting health benefits. Acknowledgements Authors are grateful to the Spanish Ministry of Science and Innovation (Project ref: CTQ2008-05322/PPQ) and to Xunta de Galicia (INBIOMED project) for the financial support of this work. Both projects were partially funded by the FEDER Program of the European Union (“Unha maneira de facer Europa”). Remedios Y
a~nez thanks Xunta de Galicia for her “Isidro Parga Pondal” contract, Bel
en G
omez thanks the Spanish Ministry of Education, Culture and Sports for her FPU research grant and Beatriz Gull
on thanks the FCT (Fundac¸~ao para a Ci^encia e Tecnologia) for her postdoctoral fellowship (reference SFRH/BPD/ 79941/2011). References Al-Tamimi, M. A. H. M., Palframan, R. J., Cooper, J. M., Gibson, G. R., & Rastall, R. A. (2006). In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora. Journal of Applied Microbiology, 100(2), 407e414. Amaral, L., Morgan, D., Stephen, A. M., & Whiting, S. (1992). Effect of propionate on lipid-metabolism in healthy-human subjects. FASEB Journal, 6, A1655. Bonnin, E., Dolo, E., Le Goff, A., & Thibault, J.-F. (2002). Characterisation of pectin subunits released by an optimised combination of enzymes. Carbohydrate Research, 337(18), 1687e1696. Burana-Osot, J., Soonthornchareonnon, N., Hosoyama, S., Linhardt, R., & Toida, T. (2010). Partial depolymerisation of pectin by a photochemical reaction. Carbohydrate Research, 345(9), 1205e1210. Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G. (2005). Extraction of pectin from apple pomace. Brazilian Archives of Biology and Technology, 48(2), 259e266. Chang, S. C., Hsu, B. Y., & Chen, B. H. (2010). Structural characterization of polysaccharides from Zizyphus jujuba and evaluation of antioxidant activity. International Journal of Biological Macromolecules, 47(4), 445e453. Chen, J., Liang, R. H., Liu, W., Li, T., Liu, C. M., Wu, S., et al. (2013). Pectic-oligosaccharides prepared by dynamic high-pressure microfluidization and their in vitro fermentation properties. Carbohydrate Polymers, 91(1), 175e182. Coenen, G. J., Kabel, M. A., Schols, H. A., & Voragen, A. G. J. (2008). CE-MSn of complex pectin-derived oligomers. Electrophoresis, 29(10), 2101e2111.

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