gati

caraen5, 6/n, 5

Tecnolgicas, Av. Washington soares, 1321, 60811-905 Fortaleza, Cear, BrazilUniversidade de Fortaleza, Centro de Cincias da Sad

. All rights reserved.

Grisel, 2008).The major plant sources of galactomannans for commercial ap-

plications in food and non-food products are Cyamopsis tetragono-loba (guar gum), Caesalpinia spinosa (tara gum) and Ceratonia siliqua(locust bean gum) (Cerqueira et al., 2009, 2011a). In the food

primarily used asal., 2011; Reddy,

has been used as ant, Ren, & Ross-ential deterrent toar-containing foodosity (Cui, Ikeda, &btained by partialions of this dietaryages, yogurts, milk

drinks, whipped creams, soups and enteral solutions (Kapoor &Juneja, 2009). Despite the worldwide use of guar gum in the foodindustry, frequent supply shortages and the high cost of thisingredient caused by variations in crop production have causedmanufacturers to seek alternative and sustainable sources of seedgums (Byrne, 2012; Mathur, 2011; Mathur & Mathur, 2005).Although information about potential guar gum substitutes isfound in the literature (Cerqueira et al., 2011a; Cunha, Vieira,Arriaga, de Paula, & Feitosa, 2009; Hu, Kong, Yang, & Pan, 2011;

* Corresponding author. Tel.: 55 88 31127562; fax: 55 88 31127455.

Contents lists availab

Food Hydr

ls

Food Hydrocolloids 35 (2014) 512e521E-mail address: karina.dos-santos@embrapa.br (K.M.O. dos Santos). 2013 Elsevier Ltd

1. Introduction

Galactomannans are used as a form of carbohydrate storage inplants, consisting of a main chain of (1 / 4) linked b-D-man-nopyranosyl residues with a single unit of a-D-galactopyranosylside-chain residues. The molecular weight, mannose/galactose ra-tio (M/G) and galactose units distribution over the mannose back-bone are properties that depend on the galactomannan source andinuence the technological applications of the polysaccharides(Jiang, Jian, Cristhian, Zang, & Sun, 2011; Tapie, Malhiac, Hucher, &

industry, guar, tara and locust bean gums arethickening and stabilising agents (Oliveira etMohan, Satla, & Gaikwad, 2011). Guar gum alsodietary bre supplement (Ellis, Wang, RaymeMurphy, 2001); however, it is considered a potpalatability since sensorial characteristics of guproducts tend to be poor due to high levels of viscEskin, 2007). A low viscosity ingredient was ohydrolysis of the guar gum to expand the applicatbre source in food products, especially in beverproduction and storage. PHGM presented suitable properties to be added as an alternative dietary bresource in a wide range of food products, particularly in beverages.a r t i c l e i n f o

Article history:Received 3 April 2013Accepted 16 July 2013

Keywords:Caesalpinia pulcherrimaDietary breGalactomannanGuar substituteEnzymatic hydrolysisAbsolute viscosity0268-005X/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2013.07.015e, Av. Washington Soares, 1321, 60811-905 Fortaleza, Cear, Brazil

a b s t r a c t

A galactomannan extracted from the endosperm of Caesalpinia pulcherrima seeds was submitted topartial enzymatic hydrolysis to overcome the limitation of use of this polysaccharide as a dietary breingredient, since the intact galactomannan may promote a considerable increase in the viscosity of foodproducts, even when added at low concentrations. The total dietary bre recovered from the partiallyhydrolysed galactomannan (PHGM) was 78.27%. The molar mass of PHGM and the absolute viscosity ofaqueous solutions were reduced in relation to the intact galactomannan. Nevertheless, their structuralcharacteristics were very similar when evaluated by thermogravimetric analysis, infrared spectroscopy,and also by 1H and 13C nuclear magnetic resonance. A lack of growth promotion in the food-grade strainstested using PHGM during a preliminary in vitro fermentation assay suggests that the metabolism ofthese microorganisms most likely would not affect the galactomannan content in products during theird Instituto Federal de Educao Cincia e TecnoeUniversidade de Fortaleza, Centro de CinciasfcUniversidade Federal do Cear, Departamento de Qumica Orgnica e Inorgnica, P.O. Box 6021, 60455-760 Fortaleza, Cear, Brazillogia do Cear, Campus de Sobral, Av. Dr. Guarani, 317, 62040-370 Sobral, Cear, BrazilCharacterisation of partially hydrolysedCaesalpinia pulcherrima seeds as a poten

Flvia C.A. Buriti a,b, Karina M.O. dos Santos a,*, VenDaniele M.A. Teixeira S d, Hvila O. Salles a, GilcenJudith P.A. Feitosa c, Ana C.O. Monteiro Moreira f, Ra Empresa Brasileira de Pesquisa Agropecuria, Embrapa Caprinos e Ovinos, P.O. Box 14bUniversidade Estadual da Paraba, Departamento de Farmcia, Rua Juvncio Arruda, s

journal homepage: www.eAll rights reserved.lactomannan fromal dietary bre

ios G. Sombra c, Jeanny S. Maciel c,Oliveira e, Regina C.M. de Paula c,

ato A. Moreira f, Antonio S. Egito a

2010-970 Sobral, Cear, Brazil8100-001 Campina Grande, Paraba, Brazil

le at ScienceDirect

ocolloids

evier .com/locate/ foodhyd

drocde Souza, Lucyszyn, Ferraz, & Sierakowsky, 2010), there is a lack ofstudies dealing with the characterisation of alternativegalactomannan-based ingredients to partially hydrolysed guar gum.

Caesalpinia pulcherrima (EN: Dwarf Poinciana, Pride ofBarbados; PT: amboyant-mirim, amboyanzinho) is a plant fromthe family Fabaceae e Leguminosae and is largely found in Brazil.Its seeds accumulate considerable amounts of galactomannan withpotential applications in the food industry, as hydrocolloid, texturemodier and source of dietary bre. The possibility of growingC. pulcherrima for commercial and industrial uses highlights op-portunities for economic and social developments in the semi-aridareas of the Caatinga, which is the main biome of the BrazilianNortheastern region, because this plant is well-adapted to thisparticular environment. The main characteristics of the gal-actomannan extracted from C. pulcherrima seeds were described instudies that focused on its potential applications in the industry(Andrade, Azero, Luciano, & Gonalves, 1999; Azero & Andrade,1999, 2002; Braga et al., 2011; Cerqueira et al., 2009, 2011b).Nonetheless, similar to intact guar gum, the galactomannan ofC. pulcherrima has a high viscosity and gelling capacity at lowconcentrations, which impede its application as a dietary bresource in semi-solid and liquid products. Partial hydrolysis of thispolysaccharide would overcome these limitations and allow itsaddition in a wide range of products at amounts high enough to beused as a dietary bre ingredient.

The present study evaluated the production of partially hydro-lysed galactomannan from C. pulcherrima using enzymatic hydro-lysis. Structural and physicochemical characterisations of intact andhydrolysed galactomannan were carried out. Due to the possibilityof fermentation of this carbohydrate by some food-grade culturescommonly used in industry and by some harmful bacteria repre-sentative of gut microbiota, in vitro fermentation of hydrolysedgalactomannan from C. pulcherrima was also investigated.

2. Material and methods

2.1. Galactomannan extraction

The pods of C. pulcherrima were collected in the cities of For-taleza, Quixeramobim and Sobral (Cear State, Brazil), betweenSeptember 2009 and August 2010. A voucher specimen ofC. pulcherrima seeds has been deposited at Herbarium PriscoBezerra e EAC (Federal University of Cear, Fortaleza, Brazil) underthe number 44718.

The polysaccharide extraction was based on the proceduresdescribed by Cerqueira et al. (2009). Seeds were removed from thepods, cleaned and placed in a blender to be mechanically broken.Afterwards, the endospermwas manually separated from the germand the hull. The endosperm obtained was suspended in 92.8%ethanol (w/w) in a proportion of 1:3 (seeds:ethanol) at 70 C for15 min to inactivate the enzymes and eliminate the low-molecular-weight compounds. Ethanol was removed and distilled water wasadded in a 1:5 (endosperm:water) proportion and the suspensionwas left overnight. The next day, the amount of water was increasedto an endosperm:water ratio of 1:10 and mixed in a blender for5 min. Next, the viscous solution was ltered through a nylon netand precipitated by adding 92.8% ethanol (w/w) at ratio of 1:2. Theprecipitate was successively washed with acetone, dried with hotair and milled. The sample obtained in this step was labelled GM.

2.2. Enzymatic hydrolysis of galactomannan

The GM powder was dissolved in distilled water (3 L per batch)to a concentration of 15 g/L under constant stirring for 3 h at room

F.C.A. Buriti et al. / Food Hytemperature using a mechanical mixer Fisatom (model 713, SoPaulo, Brazil) starting at 1000 rpm with subsequent increase up to2000 rpm to form a uniform gel. For the enzymatic hydrolysis, acommercial cellulase from Aspergillus niger (1.24 U/mg, Sigma,Buchs, Switzerland) was dissolved in distilled water to achieve aconcentration of 6.4 U/mL. The enzyme dispersion was added topolysaccharide suspension in a proportion of 12.8 U of cellulase to1 g of galactomannan. The gel was stirred for 2 h at room tem-perature with a successive decrease from 2000 rpm to 1000 rpm.These conditions of hydrolysis were established in a preliminarystudy by evaluating the relative viscosity and the dietary brecontent of suspensions containing GM throughout the time afterthe cellulase addition (Buriti et al., 2011). The hydrolysed suspen-sion was transferred to borosilicate asks with caps and immedi-ately autoclaved at 121 C for 20 min. The suspension was cooled,passed through a sieve (0.25mmopening) and dried in amini spraydrier Bchi (model B-290, Flawil, Switzerland) using an inlet tem-perature of 160 C, an outlet temperature of 103e107 C, air ow of538 L/h and a pump speed of 233 mL/h. A total of 11 batches wereprepared and the sample obtained in this step was labelled PHGM.The yield of PHGM in this step was 48.7 4.5 (%, w/w).

2.3. Galactomannan samples characterisation

2.3.1. Moisture contentThe moisture content was determined for the GM and PHGM

samples by drying 1 g of the samples at 105 C to a constant weight.

2.3.2. Total dietary breThe total dietary bre was determined for the GM and PHGM

samples using the AOAC 985.29 method (Prosky et al., 1985).

2.3.3. Thermogravimetric analysisThermogravimetric analysis (TGA) of the GM and PHGM sam-

ples was performed using a TA Instruments analyser (model Q50,New Castle, DE, USA). Approximately 10 mg of the sample wasplaced in an aluminium pan and heated over a temperature rangeof 25e900 C at 10 C/min under a synthetic air atmosphere.

2.3.4. Gel permeation chromatographyTheweight averagemolarmass (Mw) and number averagemolar

mass (Mn) of the GM and PHGM samples were determined by gelpermeation chromatography (GPC) with a Shimadzu instrument(model LC-10AD, Kyoto, Japan) at room temperature using anUltrahydrogel linear column (7.8 300 mm, Waters, Milford, MA,USA), a ow rate of 0.5 mL/min, a polysaccharide concentration of1 mg/mL, water as the solvent and NaNO3 0.1 M as the eluent. Adifferential refractometer was used as the detector. The elutionvolume was corrected using ethylene glycol as an internal markerat 11.25 mL. The GPC was calibrated using pullulan samples (Sho-dex, Showa Denko, Kawasaki, Japan) as standards.

2.3.5. Infrared spectral analysisThe Fourier transform infrared spectra (FT-IR) of the GM and

PHGM samples were recorded with a Shimadzu IR spectropho-tometer (model 8300, Kyoto, Japan) in the range of 400e4000 cm1. The samples were analysed as KBr pellet.

2.3.6. Nuclear magnetic resonance spectroscopyThe 1H and 13C nuclear magnetic resonance (NMR) spectra of

10 mg/mL solutions of the GM and PHGM samples dissolved inD2O were recorded at 70 C on a Fourier transform BrukerAvance spectrometer (model DRX 500, Rheinstetten, Germany)with an inverse multinuclear gradient probe-head equippedwith z-shielded gradient coils and Silicon Graphics. Sodium 2,2-

olloids 35 (2014) 512e521 513dimethylsilapentane-5-sulphonate (DSS) was used as an internal

residual mass were similar for the GM and PHGM samples. Thehydrolysis process reduced the dietary bre content in the PHGMsample to 78.3% (w/w) compared to 87.5% (w/w) in the GM sample.Nevertheless, after hydrolysis the total dietary bre recovered inthe PHGM sample was still considered high, as it was equivalent tothe dietary bre content of 76% (w/w) found in partially hydrolysedguar gum Sunber (Kapoor & Juneja, 2009). It is important to

Table 1Main characteristics of intact and partially hydrolysed galactomannans fromC. pulcherrima (GM and PHGM, respectively).

GM PHGM

Moisture e drying at 105 C (%, w/w) 11.11 11.38TGA residual mass (%, w/w) 0.3531 0.3184Total dietary bre (%, w/w) 87.77 78.27Mannose:galactose ratio 3.65:1 3.24:1Mn (g/mol) 2.48 106 9.89 104Mw (g/mol) 8.03 106 2.47 105Polydispersity index (Mw/Mn) 3.23 2.50

drocolloids 35 (2014) 512e521standard (0.00 ppm for 1H). A distortionless enhancement bypolarisation transfer (DEPT 135) spectrum was recorded to deter-mine the hydrogenation of each carbon. The acquisition and delaytimes were 1.0 s. Correlation spectroscopy, 1H, 13C-HSQC wereperformed using the parameters supplied in the Bruker manual.

2.3.7. Rheological measurementsThe effect of shear rate on the viscosity of the GM and PHGM

aqueous solutions at concentrations of 0.3, 0.5 and 1% (w/v) wasevaluated using a TA Instrument Rheometer (model AR-550, NewCastle, DE, USA). Tests were performed at 25 C.

2.3.8. In vitro fermentation assayThe effect of PHGM on the growth of different bacteria

compared to the absence and presence of other carbohydratesources was evaluated in a preliminary assay using an in vitromodel adapted from Ryu, Kim, Park, Lee, and Lee (2007) with amodiedMRSmedium and phenol red as indicator. One food-gradestrain commonly used as starter culture in fermented dairy prod-ucts, Streptococcus thermophilus TA-40 (Danisco, Sassenage,France), ve benecial food-grade strains, Lactobacillus acidophilusLA-5 (Chr. Hansen, Hrsholm, Denmark), L. acidophilus NCFM(Danisco, Madison, WI, USA), Lactobacillus rhamnosus Lr-32 (Dan-isco, Madison,WI, USA), Bidobacterium animalis subsp. lactis BB-12(Chr. Hansen, Hrsholm, Denmark) and B. animalis subsp. lactis Bi-07 (Danisco, Madison, WI, USA), as well as two harmful bacteriarepresentative of gut microbiota, Escherichia coli INCQS 00033(Fundao Oswaldo Cruz, Rio de Janeiro, Brazil) and Clostridiumperfringens INCQS 00130 (Fundao Oswaldo Cruz, Rio de Janeiro,Brazil), were tested. The broth medium consisted of peptone (10 g,Himedia, Mumbai, India), meat extract (8 g, Himedia, Mumbai,India), yeast extract (4 g, Acumedia, Lansing, MI, USA), Tween80 (1 mL, Cromoline, Diadema, Brazil), ammonium acetate (2 g),MgSO4$7H2O (0.18 g), MnSO4$H2O (0.05 g), Na2SO4 (2 g), K2SO4(1.25 g), Na2CO3 (0.2 g), CaCl2$2H2O (0.11 g), L()-cysteineHCl (0.5 g), phenol red (0.18 g) and distilled water (1 L). Each pre-cultured culture was diluted in peptone water and 0.1 mL aliquots(6e7 log colony forming units [CFU]) were inoculated into 5 mL ofthe broth media without or with 0.5% (w/v) PHGM and/or othercarbohydrates. The carbohydrates evaluated were inulin (OraftiHP-Gel, Beneo, Oreye, Belgium), oligofructose (FOS, Orafti P95,Beneo, Oreye, Belgium), lactose (Vetec, Rio de Janeiro, Brazil) andglucose (Quimibras, Rio de Janeiro, Brazil). Cell density was deter-mined by measuring the absorbance at 660 nm using a Shimadzuspectrophotometer (model UV1200, Kyoto, Japan) before (0 h) andafter 6 h, 24 h and 48 h of anaerobic incubation (Anaerobic SystemAnaerogen, Oxoid, Basingstoke, UK) at 37 C. The assays wereperformed in triplicate and the absorbance results were convertedinto microbial concentrations (log CFU/mL) using growth curvesfrom each microorganism. Statistical analysis was performed forthe in vitro fermentation assay data using SAS (Statistical AnalysisSystems) software version 9.2 (SAS Institute Inc., Cary, NC, USA).Repeated measures analysis of variance (RM-ANOVA) was used todetermine signicant differences among samples (P < 0.05), fol-lowed by the Tukey post-hoc test to identify contrasts. Homoge-neity of variance among samples was checked by the Cochran andBartlett tests (P < 0.05).

3. Results and discussion

3.1. Composition and structural characterisation of the intact andhydrolysed galactomannans

Table 1 shows the main characteristics obtained for the intact

F.C.A. Buriti et al. / Food Hy514and hydrolysed galactomannans. The moisture content and theemphasise that, although with this reduction in the dietary brecontent, partially hydrolysed galactomannans usually give lowerviscosity, and therefore they could be added in higher amounts infood products for improvement of total dietary bre whencompared with the intact galactomannans (Yoon, Chu, & Juneja,2008), as it will be discussed later.

The FT-IR spectra were very similar for the GM and PHGMsamples (Fig. 1), showing characteristic absorption bands of thegalactomannans as reported in the literature (Cerqueira et al.,2011b; Figueir, Ges, Moreira, & Sombra, 2004; Hu et al., 2011;Mudgil, Barak, & Khatkar, 2012b; Shobha, Vishu Kumar,Tharanathan, Koka, & Gaonkar, 2005). Bands approximately3400 cm1 are attributed to OeH stretching vibration of the poly-saccharides and the region approximately 2900 cm1 is represen-tative of eCeH stretching modes (Cerqueira et al., 2011b; Hu et al.,2011; Shobha et al., 2005). The absorption of approximately1645 cm1 is due to the bound water (Hu et al., 2011; Shobha et al.,2005). The region approximately 1380 cm1 and 1430 cm1 cor-responds toeCH2 deformation modes and the broad band between800 cm1 and 1200 cm1 results from the highly coupled CeCeO,CeOH and CeOeC stretching modes of the polymer backbone(Mudgil et al., 2012b; Shobha et al., 2005). The peak at 1151 cm1 isattributed to bending vibrational modes of the CeO present in thepyranose ring and the bands at 813 cm1 and 872 cm1 indicate theFig. 1. FT-IR spectra for intact and partially hydrolysed galactomannans fromC. pulcherrima (GM and PHGM, respectively): GM ( ) and PHGM ( ).

presence of a-linked D-galactopyranose units and b-linked D-man-nopyranose units, respectively (Cerqueira et al., 2011b; Figueiret al., 2004). In agreement with the results of the present study,Mudgil et al. (2012b) did not nd structural modications in the FT-IR spectra of the guar gum after its partial hydrolysis with acellulase from the A. niger, when compared to the intact guar gum.

The mannose/galactose (M/G) ratios obtained from the inte-grated peak areas from the 1H NMR spectrum for the anomericprotons of mannose and galactose units at d 4.74 and d 5.02,respectively (Fig. 2(a) and (b)), were 3.65:1 for the GM sample and3.24:1 for the PHGM sample (Table 1). The reduced values of M/Gratio for PHGM compared to the intact polysaccharide (GM) indi-cate that galactose was not cleaved during the enzymatic hydro-lysis. These results were expected, since the enzymatic hydrolysiswas performed with a cellulase from A. niger. In this case, thecleavage of the linkages should occur only between the mannoseunits. The value of M/G ratio for GMwas similar to the ratio of 3.6:1determined by Braga et al. (2011), however smaller values werereported by Andrade et al. (1999) and Cerqueira et al. (2009), 2.83:1and 2.88:1 respectively. The resonances of protons and carbons inthe 1H and 13C NMR spectra for the GM and PHGM obtained fromC. pulcherrima were assigned based on the literature for gal-actomannan (Bociek, Izzard, Morrison, & Weti, 1981; Chaubey &Kapoor, 2001; Cunha et al., 2009) and the 1H, 13C-HSQC correla-tion analysis (Fig. 2(c) and (d)). A summary of the assigned 1H and

13C NMR shifts is shown in Table 2. A similar 1He13C-HSQC spec-trum was obtained for the GM and the PHGM samples. Three sig-nals were veried in the anomeric region of the 13C NMR spectra (d90e110) andwere assigned as C-1 of a-D-galactose at d 99.46 for theGM and PHGM samples, C-1 of b-D-mannose at d 100.75 andd 100.76 for the GM and the PHGM, respectively, and C-1 ofsubstituted b-D-mannose at d 100.56 for the GM and PHGM sam-ples. In the 1He13C-HSQC spectrum from the GM sample, it wasobserved that there were only two correlations from the non-substituted units b-mannopyranosyl (d 4.72/100.7) and the a-gal-actopyranosyl (d 5.01/99.5) (Fig. 2(c)). The three C-6 signals atopposite amplitudes in the DEPT 135 spectrum (data not shown)exhibited correlations with their protons at d 3.91/61.2, d 3.75/61.7,and d 3.81/67.3, due to the H-6/C-6 correlations of the non-substituted units b-mannopyranosyl, a-galactopyranosyl and thetwo diastereotopic protons from the O-substituted b-mannopyr-anosyl, respectively. The correlations from the PHGM sample weresimilar (Fig. 2(d)).

The split of C-4 in the spectra of the intact and partiallyhydrolysed galactomannans was used to determine the probabili-ties of a-D-galactose substitution on the (1 / 4)-b-D-mannopyr-anose based on the study of Dea andMorrison (1975). Three signalswere assigned in the 13C NMR spectrum region of the C-4 in themannose residues for both the GM and PHGM samples (Fig. 3(a)and (b)). The rst signal at d 77.5 is attributed to two continuous

F.C.A. Buriti et al. / Food Hydrocolloids 35 (2014) 512e521 5151 1 13Fig. 2. Anomeric region H NMR ((a) and (b)) and He C-HSQC spectra ((c) and (d)) forrespectively): GM (a) and (c); PHGM (b) and (d).intact and partially hydrolysed galactomannans from C. pulcherrima (GM and PHGM,

substituted b-D-mannose units (denoted as I), the second at d 77.3 isattributed to dyads in which only one of the two b-D-mannoseunits is substituted (denoted as II) and the third at d 77.0 is due tothe unsubstituted b-D-mannose units that are adjacent to other

unsubstituted unit of the same monosaccharide (denoted as III)(Cunha et al., 2009; de Souza et al., 2010). If the cleavage occurs inthemannose units without substitution, the C-4 signal due to dyadswith unsubstituted b-D-mannose units (III) will decrease and,consequently the ratio of dyads in which only one of the two b-D-mannose units is substituted (II) or the ratio of two continuoussubstituted b-D-mannose units (I) will increase. In GM and PHGMspectra, the ratios of signal I/III and II/III were, respectively, 0.16 and1.04 for GM and 0.23 and 1.20 for PHGM indicating that thecleavage occurred in the unsubstituted b-D-mannose units. Theseresults indicate that the galactosyl groups in the backbone of PHGMwere not affected by the enzymatic hydrolysis and thermal treat-ment. Similar results were veried in the commercial partiallyhydrolysed guar gum (Yoon et al., 2008).

3.2. Gel permeation chromatography (GPC)

Fig. 4 shows the GPC chromatograms of intact and hydrolysedgalactomannan of C. pulcherrima. It was observed that the partialhydrolysis occurred in a homogeneous form, as only one peak isobserved in Fig. 4 for PHGM. In the present study, the numberaverage molar mass (Mn) and the weight average molar mass (Mw)obtained for the GM sample were 2.48 106 g/mol, and8.03 106 g/mol, respectively (Table 1). The Mw value was higherthan the intrinsic viscosity molar mass of 1.75 106 g/mol and2.10106 g/mol, as reported by Cerqueira et al. (2009) and Andradeet al. (1999) for intact galactomannan from C. pulcherrima.Furthermore, the Mn and Mw values for the PHGM sample,9.89 104 g/mol and 2.47 105 g/mol, respectively, were 25e30-fold lower when compared to the GM sample (Table 1). Thesevalues were higher than those reported in the literature for the

Table 21H NMR and 13C NMR shifts for intact and partially hydrolysed galactomannans fromC. pulcherrima (GM and PHGM, respectively).

Position 13C (d, ppm) 1H (d, ppm)

GM PHGM GM PHGM

a-D-GalactopyranosylC-1/H-1 99.46 99.46 5.02 5.02C-2/H-2 69.07 69.01 3.84 3.82C-3/H-3 70.60 70.11 4.11 4.10C-4/H-4 69.93 69.93 4.00 4.01C-5/H-5 71.82 71.79 3.89 3.88C-6/H-6 61.77 61.75 3.75 3.74

4-Linked-b-D-mannopyranosylC-1/H-1 100.75 100.76 4.73 4.72C-2/H-2 70.60 70.62 4.11 4.11C-3/H-3 72.07 72.08 3.79 3.79C-4/H-4 77.30 77.10 3.85 3.82C-5/H-5 75.70 75.69 3.55 3.55C-6/H-6 61.19 61.22 3.89 3.89

4,6-Linked-b-D-mannopyranosylC-1/H-1 100.57 100.57 4.74 4.74C-4/H-4a 77.34 77.34 3.82 3.82C-4/H-4b 77.29 77.29C-5/H-5 74.04 74.03 3.74 3.73C-6/H-6 67.24 67.29 3.81 3.80

a Samples from two continuous O-substituted b-D-mannopyranosyl units.b Samples from dyads in which only one of the two b-D-mannopyranosyl units is

substituted.

F.C.A. Buriti et al. / Food Hydrocolloids 35 (2014) 512e521516Fig. 3. 13C NMR spectral region of C-4 (mannose) for intact and partially hydrolysed galactomannans from C. pulcherrima (GM and PHGM, respectively): GM (a) and PHGM (b).

associated with water evaporation. The following events are relatedto the thermal composition of the samples and occurred at 302 Cand at 300 C for the GM and PHGM samples, respectively, and at480 C for both the GM and the PHGM samples. After the moistureloss, a constant weight was observed up to approximately 260 C.The highest weight loss was approximately 80% (w/w) of the dryweight and occurred between 290 C and 350 C. Cerqueira et al.(2011b) veried the two events during the TG experiments withgalactomannans from C. pulcherrima, Gleditsia triacanthos andAdenanthera pavonina. According to these authors, a weight loss ofapproximately 45% (w/w) was observed in the second event for thethree polysaccharides studied. For the galactomannan fromC. pulcherrima, the peak of the derivate at the weight loss curve(DTG) was 321.73 C, which is close to the temperature of thesecond event for the GM and PHGM samples in the present study.These results indicate that the PHGM sample would tolerate mosttemperatures used in the thermal treatment of food products,specically pasteurisation and sterilisation. However, the possibleinteractions of PHGM with other food components during thermal

Fig. 4. GPC curves for intact and partially hydrolysed galactomannans fromC. pulcherrima (GM and PHGM, respectively) at concentration of 1 mg/mL: GM ( )

F.C.A. Buriti et al. / Food Hydrocolloids 35 (2014) 512e521 517partially hydrolysed guar gum Sunbre, whose Mw is 2.0 104 g/mol and the range is 103e105 g/mol (Ohashi et al., 2012; Okuboet al., 1994; Yoon et al., 2008).

The polydispersity index of 3.23 for the GM sample (Table 1) washigher than the values reported for the galactomannans extractedfrom plant sources found in the Brazilian ora of 1.2 and 1.3 forMimosa scabrella (Vendruscolo et al., 2009), 1.55 for Caesalpiniaferrea (de Souza et al., 2010), 2.06 for Dimorphandra gardneriana(Cunha et al., 2009), and 2.84 for Parkinsonia aculeata (Garros-Rosa,Reicher, Petkowicz, Sierakowski, & Moreira, 2006). The hydrolysisprocess reduced the polydispersity index to 2.50 in the PHGMsample (Table 1).

3.3. Thermogravimetric analysis

The thermogravimetric (TG) curves were quite similar for theGM and PHGM samples (Fig. 5). Three mass-loss events were

and PHGM ( ).observed for both samples. The rst event was at 58 C and at 59 Cfor the GM and PHGM samples, respectively, which might be

Fig. 5. Weight loss curves for intact and partially hydrolysed galactomannans fromC. pulcherrima (GM and PHGM, respectively) at a heating rate of 10 C min1 undersynthetic air: GM ( ) and PHGM ( ).treatment may affect the products characteristics and should beinvestigated further.

3.4. Rheological properties

The effects of the shear rate on absolute viscosity in the aqueoussolutions of the GM and PHGM samples at 0.3%, 0.5%, and 1% (w/v)are shown in Fig. 6. At all concentrations studied, the GM andPHGM solutions presented a pseudo-plastic behaviour at a lowshear rate. The absolute viscosity of the GM solution at 1% (w/v)achieved 802 mPa s at a shear rate of 100 s1. The viscosity wouldincrease greatly if a liquid food product such as juices, fermentedmilks or milk beverages was added with the GM at this proportionor higher, since products considered to be source of dietary breusually contain at least 1.5 g bre per 100 kcal (Codex Alimentarius,2009). According to Rezaei, Khomeiri, Kashaninejad, and Aalami(2011), the addition of only 0.3% (w/w) of intact guar gum infrozen yogurt caused the viscosity to increase to 3305 mPa s, morethan twice the value obtained in control samples without guar gum(1522 mPa s). However, in the present study the absolute viscosityof the different aqueous solutions in the PHGM sample was 10e100-fold lower than those obtained for the GM sample at the sameconcentrations and shear rate. At a shear rate of 400 s1, the GMand PHGM samples at a concentration of 1% (w/v) showed absoluteviscosities of 286 mPa s and 6mPa s, respectively. Likewise, Mudgil,

Fig. 6. Effect of shear rate on viscosity of intact and partially hydrolysed gal-actomannans from C. pulcherrima (GM and PHGM, respectively) at concentration (w/v)

of 0.3% (6), 0.5% (B) and 1% (,). Closed symbols represent GM and open symbolsrepresent PHGM.

Barak, and Khatkar (2012a) observed that the aqueous solutions ofthe intact guar gum (889,742 g/mol) and the partially hydrolysedguar gum (7936 g/mol) at 1% (w/v) showed absolute viscosity ofapproximately 100 mPa s and 4 mPa s, respectively, at a shear rateof 400 s1. Taking into account the use of partially hydrolysedgalactomannans in liquid food products as a dietary bre source,Brennan and Tudorica (2008) obtained skimmed yogurts added of2% and 6% (w/w) partially hydrolysed guar gum (PHGG) withapparent viscosities of approximately 2500 mPa s and 3500 mPa s,respectively, at a shear rate of 10 s1, compared to approximately1500 mPa s in skimmed yogurt without addition of PHGG. Ac-cording to the results of absolute viscosity obtained in the presentstudy and their comparisonwith the data reported in the literature,the PHGM sample might be considered suitable as an alternativeingredient to PHGG to be applied in liquid food products such asyogurts and milk fermented beverages to improve their dietarybre content without excessive increase in their viscosity.

3.5. In vitro fermentation assay

The microorganisms evaluated in this study (S. thermophilusTA-40, L. acidophilus e LA-5 and NCFM strains, L. rhamnosus Lr-32,B. animalis subsp. lactis e BB-12 and Bi-07 strains, E. coli INCQS00033, and C. perfringens INCQS 00130) were not able to metabolisethe PHGM in the modied MRS medium at 37 C, under anaerobicconditions for 48 h (Figs. 7 and 8) because no signicant increase inbacterial growth compared to the control samples (with no car-bohydrate addition) was observed (P > 0.05). The growth ofL. acidophilus NCFM, E. coli, and C. perfringens in the presence of allcarbohydrates evaluated (PHGM, inulin, FOS, lactose and glucose),during the different sampling periods (0, 6, 24 and 48 h), did notdiffer signicantly from the control (P > 0.05). A signicant growthof the B. animalis (BB-12 and Bi-07 strains) and the L. rhamnosusspecies, compared to the control was only observed in the presenceof lactose and glucose (P < 0.05). However, there was no signicant

M (cfruc

F.C.A. Buriti et al. / Food Hydrocolloids 35 (2014) 512e521518Fig. 7. Populations of S. thermophilus TA-40 (a), L. acidophilus LA-5 (b), L. acidophilus NCFmedia with partially hydrolysed galactomannan of C. pulcherrima (PHGM), inulin, oligo

A,B,Cincubation under anaerobic conditions at 37 C. Capital letters denote signicant differsampling period. a,bLowercase letters denote signicant differences between the growth of), and L. rhamnosus Lr-32 (d) in the control medium (without carbohydrates) and in thetose (FOS), lactose or glucose before ( ) and after 6 h ( ), 24 h ( ) and 48 h ( ) of

ences between the growth of a same microorganism in the different media in a samea same microorganism in a same media in the different sampling periods.

drocF.C.A. Buriti et al. / Food Hydifference between the populations of B. animalis in the mediacontaining inulin, FOS, lactose and glucose after 24 h and 48 h ofassay (P> 0.05). Media containing FOS, lactose and glucose resultedin a signicant growth of both S. thermophilus and L. acidophilus LA-5 when compared to the control media after 48 h of assay.Considering the use of the PHGM in food products containing thestrains of Streptococcus, Lactobacillus and Bidobacterium tested inthe in vitro fermentation assay of the present study, our preliminaryresults indicate that the metabolism of these bacteria most likelywould not affect the dietary bre content of this ingredient duringthe fermentation and shelf life of products, particularly in foodsstored under refrigerated conditions in which the bacterial meta-bolism is decreased. Moreover, according with these results, thestrains tested would not be able to degrade directly gal-actomannans with Mw higher than 105 g/mol in the gut.

In agreement with the results of the present study using PHGM,Okubo et al. (1994) did not observe growth of L. acidophilus (IF-164,ATCC-4356 and Om strains), Lactobacillus casei (ATCC-7469 and

Fig. 8. Populations of B. animalis BB-12 (a), B. animalis Bi-07 (b), E. coli INCQS 00033 (c), andthe media with partially hydrolysed galactomannan of C. pulcherrima (PHGM), inulin, oligofrincubation under anaerobic conditions at 37 C. A,B,CCapital letters denote signicant differsampling period. a,bLowercase letters denote signicant differences between the growth ofolloids 35 (2014) 512e521 519IFO-3425), C. perfringens (C-01 and ATCC-13124 strains), and E. coli(O-601 and M602 strains) in Peptone Yeast Extract Fildes Solution(PYF) broth containing 0.5% (w/v) of the commercial PHGGSunbre when incubated anaerobically at 37 C for 48 h. Addi-tionally, 16 Bidobacterium strains (belonging to the species Bi-dobacterium bidum, Bidobacterium infantis, Bidobacterium breve,Bidobacterium adolescentis and Bidobacterium longum) were notable to grow in the presence of the PHGG using the same in vitroconditions. For this in vitro experiment with the PHGG, the authorsreported a moderate growth for the Bacteroides ovatus, Clostridiumcoccoides, Clostridium butyricum and Peptostreptococcus productusspecies. However, the authors achieved a signicant increase in thepopulation of Bidobacterium spp. in faeces of healthy humans whoconsumed beverages containing 7% (w/v) PHGG (100 mL, threetimes a day, totalling 21 g PHGG per day) over a period of 14 days.The authors reported that the population of Lactobacillus spp. infaeces also increased, although not to the same extent as veriedfor Bidobacterium spp. Furthermore, the numbers of Clostridium

C. perfringens INCQS 00130 (d) in the control medium (without carbohydrates) and inuctose (FOS), lactose or glucose before ( ) and after 6 h ( ), 24 h ( ) and 48 h ( ) ofences between the growth of a same microorganism in the different media in a samea same microorganism in a same media in the different sampling periods.

Carbohydrate Polymers, 82, 641e647.Dea, I. C. M., & Morrison, A. (1975). Chemistry and interactions on seeds

drocspp., C. perfringens, Enterobacteriaceae and Streptococcaceae infaeces, showed a tendency to decrease.

Changes in the bacterial and fatty acid composition in thepresence of PHGG were also demonstrated in studies involvingin vitro fermentation of this carbohydrate with faecal inoculum(Ohashi et al., 2012; Stewart & Slavin, 2006). However, with respectto the lack of ability of pure cultures of bidobacteria to ferment thepartially hydrolysed galactomannan, as veried in the presentstudy, Ohashi et al. (2012) suggested that PHGG would not directlystimulate these microorganisms in the same manner as wellestablished bidogenic carbohydrates, such as inulin-type fructans.The increased number of bidobacteria in faeces after the intake ofPHGG is likely due to their ability to ferment the oligosaccharidesproduced after the degradation of the partially hydrolysed gal-actomannan by other bacteria in the large intestine. According tothe authors, the Roseburia/Eubacterium rectale group also plays animportant role in the fermentation of the PHGG through the pro-duction of the butyric acid. There is some possibility that the PHGMobtained in the present study might have the same fermentationpattern as the PHGG in the gut since theMw of PHGM (2.47 105 g/mol) is in the range of molar mass values for which guar gumfermentation was demonstrated in the cited studies: 2.0 104 g/mol (Okubo et al., 1994) and from 1.5104 g/mol to 1.1106 g/mol(Stewart & Slavin, 2006).

4. Conclusions

The molar mass of the partially hydrolysed galactomannan ofC. pulcherrima and the absolute viscosity of aqueous solutions werereduced when compared to the intact galactomannan. However,the structural characteristics of the polysaccharide evaluated bythermogravimetric analysis, infrared spectroscopy, and also 1H and13C nuclear magnetic resonance were unaffected after the hydro-lysis process. These properties are comparable to the characteristicsdescribed in the literature for the galactomannan obtained from thepartial hydrolysis of the guar gum. The partially hydrolysed gal-actomannan of C. pulcherrima has not promoted the growth of thebacterial strains evaluated in the preliminary in vitro fermentationassay; these results indicate that this carbohydrate might be addedas a galactomannan ingredient to increase the dietary bre contentof products prepared with the food-grade strains tested, since itwould not be degraded by these microorganisms during processingand storage. The partially hydrolysed galactomannan obtained inthis study appears to be a potential alternative to the partiallyhydrolysed guar gum, enlarging the availability of novel food in-gredients, since food manufacturers are increasingly looking forguar gum substitutes with ready accessibility. Based on the prop-erties achieved in this study, it would be advisable to use thepartially hydrolysed galactomannan of the C. pulcherrima as a di-etary bre source, especially in liquid food products such as yogurtsand fermented milk beverages.

Acknowledgements

This study was sponsored by the Conselho Nacional deDesenvolvimento Cientco e Tecnolgico (CNPq), FundaoCearense de Apoio ao Desenvolvimento Cientco e Tecnolgico(FUNCAP) and Empresa Brasileira de Pesquisa Agropecuria(EMBRAPA). F.C.A. Buriti gratefully acknowledges the fellowshipand the nancial support from CNPq (Procs. 68.0013/2008-3 and35.0161/2009-7) and FUNCAP (DCR 010/09). K.M.O. dos Santosgratefully acknowledges the nancial support from EMBRAPA(Macroprograma-3 No. 03.09.06.026.00.00). The authors wish tothank Prof. Pedro Matias de Vasconcelos for supplying the

F.C.A. Buriti et al. / Food Hy520C. pulcherrima seeds and Dr. Sidina Cordeiro de Freitas, as well asgalactomannans.Advances inCarbohydrateChemistryandBiochemistry, 31, 241e312.Ellis, P. R., Wang, Q., Rayment, P., Ren, Y., & Ross-Murphy, S. B. (2001). Guar gum:

agricultural and botanical aspects, physicochemical and nutritional properties,and its use in the development of functional foods. In S. S. Cho, & M. L. Dreher(Eds.), Handbook of dietary bre (pp. 613e657). New York: Marcel Dekker.

Figueir, S. D., Ges, J. C., Moreira, R. A., & Sombra, A. S. B. (2004). On the physico-chemical and dielectric properties of glutaraldehyde crosslinked galactomannan-collagen lms. Carbohydrate Polymers, 56, 313e320.

Garros-Rosa, I., Reicher, F., Petkowicz, C. L. O., Sierakowski, M. R., & Moreira, R. A.(2006). Characterization of the galactomannans from Parkinsonia aculeata seedsand their application on afnity chromatography. Polmeros, 16, 99e103.

Hu, C., Kong, Q., Yang, D., & Pan, Y. (2011). Isolation and structural characterizationEmbrapa Agroindstria de Alimentos for the dietary bre anal-ysis, Centro Nordestino de Aplicao e Uso de Ressonncia Mag-ntica Nuclear (CENAUREMN) for the NMR spectra, and Beneo eOrafti, Clariant S.A., Danisco Brasil Ltda. and the Instituto Nacionalde Qualidade em Sade e Fundao Oswaldo Cruz (INCQS eFIOCRUZ) for providing part of the materials used in this study.We also wish to thank Joo Batista Paula Ibiapina, Jorge Silvestre,Jos Tabosa dos Santos, and Mrcio Freire Ponciano for theirtechnical assistance.

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F.C.A. Buriti et al. / Food Hydrocolloids 35 (2014) 512e521 521

Characterisation of partially hydrolysed galactomannan from Caesalpinia pulcherrima seeds as a potential dietary fibre1 Introduction2 Material and methods2.1 Galactomannan extraction2.2 Enzymatic hydrolysis of galactomannan2.3 Galactomannan samples characterisation2.3.1 Moisture content2.3.2 Total dietary fibre2.3.3 Thermogravimetric analysis2.3.4 Gel permeation chromatography2.3.5 Infrared spectral analysis2.3.6 Nuclear magnetic resonance spectroscopy2.3.7 Rheological measurements2.3.8 In vitro fermentation assay

3 Results and discussion3.1 Composition and structural characterisation of the intact and hydrolysed galactomannans3.2 Gel permeation chromatography (GPC)3.3 Thermogravimetric analysis3.4 Rheological properties3.5 In vitro fermentation assay

4 ConclusionsAcknowledgementsReferences