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ORIGINAL ARTICLE

Partially Hydrolyzed Guar Gums Reduce Dietary Fatty Acidand Sterol Absorption in Guinea Pigs Independent of Viscosity

Jonathan Santas • Jordi Espadaler •

Jordi Cune • Magda Rafecas

Received: 15 December 2011 / Accepted: 15 May 2012! AOCS 2012

Abstract This study investigated the effect of two par-tially hydrolyzed guar gums (PHGG) on fatty acid and

sterol excretion. PHGG were obtained by chemical

hydrolysis of guar gum (GG) with H2O:EtOH (1:1) at100 "C for 1 h (PHGG1) or 2 h (PHGG2). The viscosity of

the PHGG in a 1 % (w/v) aqueous solution corresponded to

that of a pseudoplastic fluid and was higher for PHGG1than PHGG2. Guinea pigs (n = 8 per group) were fed high

fat diets (17/100 g) that contained 12/100 g of cellulose,

PHGG1, or PHGG2 for 4 weeks. Despite the differences inviscosity, the two PHGG exerted similar physiological

effects. Compared to the control cellulose group, the body

weight gain was lower in animals fed PHGG, although noeffect on food consumption was observed. PHGG increased

the excretion of fatty acids and neutral sterols, but not bile

acids. Consumption of PHGG did not alter the fecal fattyacid profile, while intestinal bioconversion of sterols ten-

ded to increase in response to PHGG2. A reduction in the

viscosity within the range tested did not correlate withlosses in the hypocholesterolemic capacity of PHGG as

both were effective in reducing plasma cholesterol. Thus,we conclude that the chemical hydrolysis of guar gum

renders the gum suitable for inclusion in food products

without significantly altering its beneficial health effects.

Keywords Dietary fiber ! Partially hydrolyzed guar gum !Fatty acids ! Cholesterol ! Bile acids

AbbreviationsBA Bile acid(s)

CA Cholic acid

CDCA Chenodeoxycholic acidCE Cellulose

DCA Deoxycholic acid

FA Fatty acid(s)FAME Fatty acid methyl ester(s)

FID Flame ionization detector

GG Guar gumLCA Lithocholic acid

LDL Low density lipoprotein

MUFA Monounsaturated fatty acid(s)NS Neutral sterol(s)

PHGG Partially hydrolyzed guar gum

PHGG1 Partially hydrolyzed guar gum for 1 hPHGG2 Partially hydrolyzed guar gum for 2 h

PUFA Polyunsaturated fatty acid(s)

SEM Standard error of the meanSFA Saturated fatty acid(s)

TSFA Total saturated fatty acidsTUFA Total unsaturated fatty acids

UDCA Ursodeoxycholic acid

UFA Unsaturated fatty acid(s)

Introduction

Guar gum (GG) is a natural polysaccharide that consists of

a (1 ? 4)-a-D-mannopyranose backbone with single units

of linked a-D-galactopyranose. Given its hypolipidemic andhypoglycemic effects, this gum is considered one of the

most promising water-soluble dietary fibers [1, 2]. There is

J. Santas ! M. Rafecas (&)Department of Nutrition and Food Science, XaRTA-INSA,University of Barcelona, 08028 Barcelona, Spaine-mail: magdarafecas@ub.edu

J. Santas ! J. Espadaler ! J. CuneAB-Biotics, S.A., Parc Tecnologic del Valles,08290 Cerdanyola del Valles, Spain

123

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DOI 10.1007/s11745-012-3682-1

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strong evidence that GG decreases the incidence of factor

risks associated with cardiovascular diseases, such as highlevels of plasma triglycerides and cholesterol, mainly LDL-

cholesterol [2, 3]. The hypolipidemic effects of this gum

are attributed to its capacity to decrease the absorption ofdietary fat and cholesterol, reduce bile acid reabsorption in

the small intestine, and produce short chain fatty acids by

partial fermentation in the cecum [4, 5].GG is widely known for its water-soluble characteristics

and viscosity [4, 6]. In manufactured or processed foods,gums are commonly used as thickeners and emulsifiers.

However, the high viscosity of GG may limit its use at

physiologically effective concentrations in some foodproducts [7]. Partial hydrolysis has recently been reported

to improve the inclusion of this gum in food products and

increase consumer acceptance [8]. For instance, Sunfiber#

is a PHGG product that is commercially available and has

GRAS status (Generally Recognized As Safe) in the USA

[7]. The viscosity of enzymatically hydrolyzed GG can beas low as 10 mPa s in a 5 % aqueous solution, while that of

commercially available GG is about 2,000–3,000 mPa s in

a 1 % aqueous solution [7]. Nevertheless, it has beenreported that the health benefits of partially hydrolyzed GG

(PHGG) are similar to those of intact GG [7, 9]. Therefore,

it has been proposed that the health benefits of PHGG donot appear to be exclusively dependent on its molecular

weight or viscosity and that hypolipidemic mechanisms

other than viscosity may be involved [8].Various studies have reported the capacity of dietary

fiber to reduce fat absorption. However, dietary fat exerts a

range of effects on lipid metabolism depending on its steroland fatty acid composition [10]. In general, trans fatty

acids and most saturated fatty acids (SFA) are considered

hyperlipidemic. For instance, lauric (C12:0), myristic(C14:0) and palmitic (C16:0) acids increase the plasma

concentration of LDL-cholesterol mainly by decreasing the

activity of its receptors in the liver [11]. In contrast to SFA,monounsaturated (MUFA) and polyunsaturated fatty acids

(PUFA) decrease LDL-cholesterol by a series of mecha-

nisms, such as enhancing the hepatic uptake of LDL par-ticles or the activity of 7a-hydroxylase [12]. To our

knowledge, only a few studies have addressed the effect of

functional ingredients on the absorption of individual fattyacids. Interestingly, some of these ingredients exert selec-

tive fat-binding capacity, thereby affecting plasma and

liver lipid profiles [13, 14]. Given all these considerations,the study of the effect of dietary fiber on each fatty acid is

relevant.

Here, we examined the effect of chemically hydrolyzedguar gum on lipid excretion in order to provide new

insights into its hypolipidemic mechanism. The effect of

PHGG on fat excretion was studied longitudinally and anexhaustive analysis of fecal sterols and fatty acids was

conducted to assess the effect of fiber on fat excretion over

time. For this purpose, we used the guinea pig as it iswidely accepted as a more reliable model of human lipid

metabolism than other more commonly used animals, such

as mice or rats, and because, in contrast to hamsters, guineapigs do not ferment fiber in their stomachs [15].

Materials and Methods

Materials

Fatty acid methyl ester (FAME) standards were of 99 % orhigher purity. Oleic acid (C18:1n-9), linolenic acid

(C18:3n-3) and heneicosanoate acid (C21:0) were pur-

chased from Larodan Fine Chemical AB (Malmo, Swe-den). The remaining FAME standards, bile acids, Sylon

BTZ (3:2:3, BSA ? TMCS ? TMSI) and anhydrous pyr-

idine were from Sigma Chemical Co. (St. Louis, MO,USA). Sterol standards were supplied by Steraloids

(Newport, Rhode Island, USA).

Sodium methylate 0.5 N was prepared by dissolving17 g of sodium in 1 L of dry methanol. N-hexane and

boron trifluoride (20 % in anhydrous methanol) were pur-

chased from Merck (Darmstadt, Germany). Sodium chlo-ride and anhydrous sodium sulfate were from Panreac

(Barcelona, Spain) whereas chloroform, cyclohexane,

methanol, ethanol and diethyl ether were purchased fromScharlau Chemie S.A. (Sentmenat, Spain). All solvents and

reagents were of analytical grade or higher and double

distilled water was used throughout the study.

Production of PHGG

PHGG was obtained by chemical hydrolysis of natural GG

from Cyamopsis tetragonoloba. The hydrolysis was con-

ducted in a reactor vessel with H2O:EtOH (1:1) at 100 "C,4 bar and constant magnetic stirring at 700 rpm. GG was

added into the pre-warmed reactor at a concentration of

15 % (w/v) and hydrolyzed for either 1 h (PHGG1) or 2 h(PHGG2). After hydrolysis, the reactor vessel was allowed

to cool to 50 "C and samples were passed through a filter

plate of porosity No. 2 (40–100 lm, Vidrafoc, S.A., Bar-celona, Spain). The residue was subsequently washed twice

with 100 mL of EtOH 95 % and allowed to dry for 5 h at

40 "C in a vacuum oven.

Determination of Viscosity and Rheological Properties

of PHGG

The viscosity of 1 % aqueous dispersions of PHGG was

determined using a First RM Rheometer from Lamy Rhe-ometers (Champagne au Mont d’Or, France). Homogeneous

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polymer dispersions were prepared by sprinkling a given

amount of product into deionised water at 40 "C with vig-orous magnetic stirring (1,000 rpm). After 1 h of incubation,

the preparations were sonicated at 40 "C for 10 min and

finally allowed to stand at room temperature for 4 h. Theviscoelastic properties of the PHGG dispersions were deter-

mined in the same day at 25 "C at shear rates ranging from 1

to 250 s-1.

Diets

Three isocaloric diets were obtained by adding experi-

mental fiber sources (12/100 g of diet) to a non-fiber basaldiet prepared by Teklan Harlan (Madison, WI, USA)

(Table 1). The fiber used for the control group was cellu-

lose (CE) (Teklan Harlan, Madison, WI, USA) whilePHGG1 and PHGG2 were used for the experimental

groups. The fat mix content of diets was olive oil/palm-

kernel oil/safflower oil (1:2:1.8). Detailed fatty acid andsterol composition of the diets are present in Table 2.

Animals

Female Dunkin-Hartley guinea pigs were supplied by

Harlan Interfauna Iberica (Barcelona, Spain). Animalsweighing 300–400 g were randomly assigned to the treat-

ment groups (8 animals/group) after 1 week of acclimation,when all animals were fed with control CE diet. Two

guinea pigs were kept per stainless steel cage, which was

wire-bottomed to avoid coprophagia. Animals were housedin a room with a controlled light-cycle (light from 0800 to

2000 hours) at 22 ± 1 "C for 4 weeks of treatment. Food

and water were consumed ad libitum. Every 48 h, feceswere collected, animals weighed and food consumption

controlled. Feces were manually cleaned from sawdust

contamination, weighed, homogenized, freeze-dried and

Table 1 Composition of diets fed to guinea pigs

Component CE(g/100 g)

PHGG1(g/100 g)

PHGG2(g/100 g)

Energya

(%)

Protein 18.1 18.1 18.1 18.0

Fat mixb 16.9 16.9 16.9 37.9

Sugarsc 44.3 44.3 44.3 44.1

Mineralmixd

4.6 4.6 4.6

Vitaminmixc

1.0 1.0 1.0

Cellulose 12.0

Guar gum1 h

12.0

Guar gum2 h

12.0

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis)a Percentage of energy provided for each nutrient in the experimentaldiets, being the same for each experimental dietb Fat mix contained olive oil/palm kernel oil/safflower oil (1:2:1.8)c Added as cornstarch/sucrose (1:1.43)d Mineral and vitamin mix were adjusted to meet National ResearchCouncil requirements for guinea pigs. Mineral mix composition(g/100 g): calcium phosphate 38.7, potassium acetate 29.9, calciumcarbonate 9.2, magnesium oxide 9.2, potassium chloride 7.4, sodiumchloride 3.7, ferric citrate 1.1, manganese sulfate, monohydrate 0.6,zinc carbonate 0.203, cupric sulfate 0.055, potassium iodate 0.002,chromium potassium sulfate, dodecahydrate 0.018, sodium selenite,pentahydrate 0.002, ammonium paramolybdate, tetrahydrate 0.006.Vitamin mix composition (g/100 g): p-aminobenzoic acid 1.10,vitamin-C (ascorbic acid) 10.17, biotin 0.004, vitamin B12 (0.1 % inmannitol) 0.30, calcium pantothenate 0.66, choline dihydrogen citrate34.97, folic acid 0.02, inositol 1.10, vitamin K3 (menadione) 0.50,pyridoxine HCl 0.99, niacin 0.22, riboflavin 0.22, thiamin HCl 0.22,vitamin A: palmitate (500,000 IU g-1) 0.40, vitamin D3: cholecal-ciferol (500,000 IU g-1) 0.04, vitamin E: DL-alpha tocopheryl acetate(500 IU g-1) 2.42, corn starch 46.67

Table 2 Fatty acid and sterol composition of diets fed to guinea pigs

CE (mg/g) PHGG1 (mg/g) PHGG2 (mg/g)

Fatty acids

C12:0 28.55 ± 1.48 26.99 ± 0.41 28.49 ± 0.53

C14:0 10.20 ± 0.50 9.77 ± 0.11 10.11 ± 0.18

C16:0 14.25 ± 0.70 13.64 ± 0.13 14.19 ± 0.24

C17:0 0.12 ± 0.00 0.11 ± 0.00 0.11 ± 0.00

C18:0 14.20 ± 0.72 13.56 ± 0.09 14.32 ± 0.26

C20:0 0.52 ± 0.02 0.50 ± 0.00 0.51 ± 0.01

C22:0 0.43 ± 0.02 0.41 ± 0.01 0.42 ± 0.01

C24:0 0.28 ± 0.02 0.25 ± 0.02 0.25 ± 0.01

TSFA 68.54 ± 3.44 65.24 ± 0.76 68.42 ± 1.13

C18:1n-9 19.93 ± 0.92 18.69 ± 0.12 19.19 ± 0.41

C18:1n-7 1.40 ± 0.06 1.33 ± 0.01 1.36 ± 0.03

MUFA 21.33 ± 0.97 20.02 ± 0.13 20.55 ± 0.43

C18:2n-6 64.89 ± 3.11 61.41 ± 0.54 63.58 ± 1.30

C18:3n-3 2.73 ± 0.13 2.58 ± 0.03 2.69 ± 0.05

PUFA 67.62 ± 3.24 63.99 ± 0.57 66.26 ± 1.36

TUFA 88.95 ± 4.20 84.01 ± 0.70 86.82 ± 1.78

TSFA/TUFA 0.77 ± 0.00 0.78 ± 0.00 0.79 ± 0.01

n-6/n-3 23.74 ± 0.03 23.80 ± 0.05 23.68 ± 0.03

Sterols (mg/100 g)

Cholesterol 38.57 ± 0.02 38.35 ± 0.48 39.51 ± 0.76

Campesterol 8.85 ± 0.03 8.63 ± 0.24 7.86 ± 0.20

b-Sitosterol 21.18 ± 0.37 20.71 ± 0.43 21.37 ± 0.52

Sitostanol 6.11 ± 0.69 5.33 ± 0.43 4.20 ± 0.45

Results expressed as mean ± SEM for quadruplicated analysis. Nostatistical differences were found among treatments (P[ 0.1)

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis), TSFA totalsaturated fatty acids, MUFA monounsaturated fatty acids, PUFApolyunsaturated fatty acids, TUFA total unsaturated fatty acids

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stored at -20 "C. After isoflurane anesthesia, non-fasted

guinea pigs were killed by heart puncture and blood sam-ples were collected in heparinized tubes. Plasma was sep-

arated after centrifugation at 1,5009g for 15 min at 4 "Cand stored at -80 "C until analysis. All procedures wereapproved by the Animal Care and Use Committee of the

University of Barcelona.

Determination of Fatty Acid Content in Feces and Diets

Fatty acids (FA) of diets and feces were determined by gas

chromatography. FAME were obtained directly from

lyophilized samples by in situ basic–acidic derivatizationas previously described [16]. Heneicosanoate acid (C21:0)

was used as the internal standard.

Determination of Neutral Sterols and Bile Acids

Neutral sterols (NS) and bile acids (BA) were extracted aspreviously described by Keller et al. [17]. 5a-Cholestaneand 23-nor-cholic acid were used as internal standards for

NS and BA, respectively. Extracts were evaporated at30 "C under nitrogen flux, dried in a vacuum desiccator

overnight and stored at -20 "C in a nitrogen atmosphere

until silanization and injection. Silanization was carried outby dissolving the samples in an appropriate amount of

anhydrous pyridine and adding an equal volume of Sylon

BTZ. Samples were injected after silanization for 30 min atroom temperature.

GC Conditions

The FAME were analyzed using an Agilent 4890D gas

chromatograph, equipped with a flame ionization detector(FID). A split–splitless injector was used with a split ratio

of 1:30 and the injection volume was 1 lL. The injector

and detector temperatures were kept at 270 and 300 "C,respectively. FAME were separated in a fused-silica cap-

illary column (60 m 9 0.25 mm i.d., 0.2 lm) from Supe-

lco (Bellefonte, PA, USA). The oven temperature wasprogrammed as follows: an initial temperature of 140 "Cfor 3 min, followed by an increase of 6 "C min-1 to

180 "C, then 2 "C min-1 to 200 "C and 7 "C min-1 to240 "C and it was finally left to stand for 5 min at 240 "C.Helium was used as carrier gas and nitrogen as makeup gas

to increase the sensitivity of the detector.NS and BA were analyzed using a Perkin Elmer Auto-

system gas chromatograph equipped with a FID. One

microliter of sample and a split ratio of 1:12.5 were usedfor injection. The injector and detector temperatures were

kept at 290 and 350 "C, respectively. NS and BA were

separated with a 5 % polysilarylene—95 % poly-dimethylsiloxane capillary column (60 m 9 0.25 mm i.d.,

0.25 lm) from Phenomenex (Torrance, CA, USA). Oven

temperature was programmed with an initial temperature of245 "C during 30 s, a rate of 5 "C until 2908 and it was

finally left to stand at this temperature for 30 min.

Chromatographic peaks were identified by comparingthe retention times with those of known standards and by

co-chromatography. FAME, NS and BA were quantified by

the internal standard addition method and using calibrationcurves with R2[ 0.99.

Determination of Plasma Lipids

Total cholesterol, HDL-chol, LDL-chol and triglycerideswere enzymatically determined with a Modular DPE Hit-

achi auto-analyzer from Roche Diagnostics (Mannheim,

Germany).

Statistical Analysis

Data were analyzed by SPSS v.17 for Windows. Significant

differences between treatments were determined by one-way

ANOVA and the exact nature of the differences betweengroups by Duncan’s multirange test. Differences between

initial and final values for each treatment were determined by

the Student’s t test after assessing equality of variances byLeven’s test. Results are expressed as means ± standard

error (SEM). A confidence coefficient of 95 % was used.

Results

Viscosity Measurements

The flow curves of 1 % PHGG aqueous solutions at shearrates ranging from 1 to 250 s-1 are shown in Fig. 1. Vis-

cosity of solutions at these rates ranged from 2,884 to 184

and from 1,720 to 110 mPa s for PHGG1 and PHGG2,respectively. The flow curves corresponded to pseudo-

plastic fluids as the viscosity was dependent on shear rate,

being lower at higher rates. The viscosity of PHGG dis-persions was not time-dependent and showed neither

thixotropic nor rheopectic behavior.

Body Weight and Food Consumption

All animals gained weight during the study, although thegain was significantly lower in the PHGG groups than the

control (Table 3). However, food consumption was not

affected by dietary fiber. This observation implies that foodefficiency was reduced by PHGG ingestion. Food effi-

ciency was slightly lower in animals fed PHGG1 than those

on PHGG2, but differences were not statisticallysignificant.

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Fatty Acid Excretion

Fecal dry weight was not significantly altered by PHGG

compared to CE during the study (Table 3). In contrast, FA

concentration in feces of the PHGG groups was increasedcompared to the control group (Fig. 2). SFA and UFA

concentration in feces were highly increased by PHGG

during the first week of treatment. After this time, theconcentration in feces tended to stabilize, although a con-

tinuous slight increment in SFA concentration in feces ofall groups was observed.

Table 4 shows the concentration of fecal FA at the end

of the experiment. SFA concentration in feces was higherthan that of UFA. The results reveal a higher excretion of

SFA, especially palmitic (C16:0) and stearic (C18:0) acids,

than of UFA. The concentration of all SFA was increased

by PHGG1 and PHGG2 compared to CE. Among UFA,fecal MUFA were accounted for mainly by oleic acid

(C18:1n-9), whereas linoleic acid (C18:2n-6) was the most

abundant PUFA. As observed for SFA, the consumption ofthe PHGG increased the excretion of MUFA and PUFA. As

a result, the FA profile as well as the TSFA/TUFA ratio in

feces was not altered by PHGG. The same results wereobserved for the n-6/n-3 PUFA isomer ratio. In general, the

effect of PHGG2 on FA excretion was slightly greater thanthat of PHGG1, although differences were not statistically

significant.

NS Excretion

The concentration of fecal NS is shown in Table 5. Theoverall excretion was increased in all groups after 4 weeks

on high fat diets. However, the increment in animals fed

either PHGG1 or PHGG2 was higher than that of CE,reaching final values of 16.07 ± 0.41, 17.98 ± 0.58 and

10.92 ± 0.73 mg of NS per animal and day for PHGG1,

PHGG2 and CE, respectively. PHGG consumptionincreased the excretion of cholesterol, but especially the

excretion of its major metabolic products. We calculated

the coprostanol/cholesterol and the secondary metabolitesof cholesterol/cholesterol ratios in order to assess the effect

of experimental fibers on the intestinal bioconversion of

cholesterol. No statistical differences for these parameterswere found between treatments, although the bioconver-

sion in feces of animals fed PHGG2 was slightly higher.

No effect was observed on phytosterol excretion(P[ 0.05).

BA Excretion

Fecal BA are shown in Table 6. Secondary BA were the

most abundant BA in feces and were accounted for mainlyby lithocholic acid. Total BA excretion was only slightly

Fig. 1 Rheological measurements of PHGG dispersions. Values ofshear stress (s) at various shear rates are expressed in mPa forpartially hydrolyzed guar gum (1-h hydrolysis) (filled circles) andpartially hydrolyzed guar gum (2-h hydrolysis) (filled squares) in 1 %aqueous solution. Values of viscosity are expressed in mPa s forpartially hydrolyzed guar gum (1-h hydrolysis) (open circles) andpartially hydrolyzed (2-h hydrolysis) (open squares)

Table 3 Body weight, food consumption and excretion

n CE PHGG1 PHGG2

Initial body weight (g) 8 346.3 ± 7.2 353.3 ± 5.9 352.1 ± 3.5

Final body weight (g) 8 445.8 ± 11.4a 392.1 ± 16.7b 402.5 ± 13.4b

Weight gain (g) 8 99.5 ± 6.8a 38.7 ± 13.6b 50.5 ± 13.5b

Food consumption (g animal-1 day-1) 4 27.5 ± 1.2 27.6 ± 1.1 28.2 ± 1.6

Food efficiency (g of animal 100 g consumed-1)A 8 13.1 ± 1.0a 5.0 ± 1.8b 6.2 ± 1.5b

Initial fecal dry weight (g of feces animal-1 day-1) 4 2.0 ± 0.5 2.7 ± 0.3 2.2 ± 0.7

Final fecal dry weight (g of feces animal-1 day-1) 4 1.9 ± 0.4 2.3 ± 0.7 2.7 ± 0.3

Results are expressed as means ± SEM. Results in the same row not sharing a common superscript are significantly different based on Duncan’smultirange test at P\ 0.05

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis), PHGG2 partially hydrolyzed guar gum (2-h hydrolysis)A Consumption was assumed to be equal for each animal in the same cage

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increased by PHGG1 compared to CE and PHGG2. Thisincrease led to augmented chenodeoxycholic and lithocholic

acid excretion. However, differences were not statistically

significant. The bioconversion of chenodeoxycholic acid toits secondary metabolite lithocholic acid was increased in

PHGG groups; however, this increment was statistically

significant only for PHGG2 (P\ 0.029).

Plasma Lipids

Plasma lipids at the end of the experiment are shown in

Table 7. Plasma triglycerides were not significantly

altered by PHGG compared to CE. In contrast, totalplasma cholesterol was lower in PHGG groups, mainly

due to a reduction in LDL cholesterol (P\ 0.05). As a

result, the HDL/LDL ratio was improved in animals fedPHGG diets.

Discussion

The physiological beneficial effects of GG are commonlyattributed to its water-soluble and high viscosity charac-

teristics [5, 6]. Several studies have reported that thegel-forming properties of GG reduce the rate of gastric

emptying and increase satiation. These effects may

decrease food consumption—advocated as the mainmechanism responsible for a lower weight gain in animals

fed GG [6, 18]. The viscosity of fluids is highly dependent

on various factors, such as temperature, pH, pressure, shearrate and time. Given these influences, it is difficult to

Fig. 2 Concentration of saturated and unsaturated fatty acids in fecesduring the study. Results are expressed as mean ± SEM in mg offatty acids per animal and day (n = 4). Differences between

treatments are denoted as * for P\ 0.05 and ** for P\ 0.01. CEcellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis)

Table 4 Fatty acid concentration in feces at the end of the treatment

CE PHGG1 PHGG2

C12:0 4.23 ± 0.46a 7.04 ± 0.61b 8.05 ± 0.45b

C14:0 8.76 ± 1.12a 13.31 ± 1.08b 15.66 ± 1.18b

C16:0 49.07 ± 7.99a 70.11 ± 4.29ab 84.05 ± 8.50b

C17:0 0.75 ± 0.08a 1.02 ± 0.02b 0.91 ± 0.00b

C18:0 159.59 ± 20.37a 214.99 ± 3.14b 229.28 ± 3.99b

C20:0 2.71 ± 0.22 3.21 ± 0.10 2.91 ± 0.13

C22:0 2.18 ± 0.09a 2.53 ± 0.06b 2.36 ± 0.07ab

C24:0 1.55 ± 0.08a 1.82 ± 0.05b 1.69 ± 0.05ab

TSFA 228.84 ± 28.57a 314.02 ± 3.10b 344.91 ± 8.76b

C18:1n-9 3.99 ± 0.22a 4.76 ± 0.36ab 5.70 ± 0.72b

C18:1n-7 1.12 ± 0.32 1.90 ± 0.33 2.22 ± 0.35

MUFA 5.11 ± 0.51a 6.66 ± 0.50ab 7.92 ± 0.76b

C18:2n-6 4.50 ± 0.28a 6.45 ± 0.49b 5.50 ± 0.35ab

C18:3n-3 0.34 ± 0.04a 0.47 ± 0.03b 0.54 ± 0.03b

PUFA 4.85 ± 0.25a 6.92 ± 0.48b 6.04 ± 0.39ab

TUFA 9.96 ± 0.51a 13.58 ± 0.76b 13.96 ± 1.08b

Total 238.78 ± 28.90a 327.60 ± 2.84b 358.85 ± 7.73b

TSFA/TUFA

22.88 ± 2.50 23.36 ± 1.43 25.29 ± 2.52

n-6/n-3 13.95 ± 2.36 13.94 ± 1.31 10.18 ± 0.10

Results are expressed as means ± SEM in lg of FA mg-1 of feces(n = 4). Results in the same row not sharing a common superscriptare significantly different based on Duncan’s Multirange test atP\ 0.05

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis), TSFA totalsaturated fatty acids, MUFA monounsaturated fatty acids, PUFApolyunsaturated fatty acids, TUFA total unsaturated fatty acids

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compare data from studies that have applied distinct

methodologies and conditions. It has been proposed thatoral perception of fluid viscosity correlates well with the

viscosity of the fluid at a shear rate of 10 s-1 [19]. At this

rate, the viscosity of PHGG was significantly different,registering 1,285 and 766 mPa s for PHGG1 and PHGG2,

respectively. This difference can be attributed to the higher

degree of hydrolysis of the latter. However, the viscosity ofPHGG obtained by chemical hydrolysis appears to be

considerably higher than that of enzymatically hydrolyzedGG, which has been published to be about 10 mPa s in 5 %

aqueous solution [7]. Chemical hydrolysis of GG allowed

us to compare the physiological benefits of two PHGG withdistinct degrees of viscosity. The rheological experiments

Table 5 Excretion of neutral sterols

CE PHGG1 PHGG2

Cholesterol

Initial 2.20 ± 0.23 2.80 ± 0.31 2.08 ± 0.19

Final 2.02 ± 0.31a 3.56 ± 0.54b 3.23 ± 0.49ab

Coprostanol

Initial 3.51 ± 0.39 4.52 ± 0.45 3.36 ± 0.60

Final 5.04 ± 0.91a 7.40 ± 0.44b** 9.78 ± 0.57c**

Cholestanol

Initial 1.02 ± 0.11 1.26 ± 0.14 0.94 ± 0.13

Final 1.11 ± 0.16a 1.48 ± 0.06b 1.76 ± 0.04b**

Cholestanone

Initial 0.97 ± 0.12 1.20 ± 0.11 1.01 ± 0.11

Final 1.25 ± 0.14a 1.90 ± 0.24ab* 2.01 ± 0.26b*

b-Sitosterol

Initial 1.11 ± 0.08 1.36 ± 0.11 1.09 ± 0.12

Final 1.16 ± 0.10 1.27 ± 0.10 0.83 ± 0.19

Sitostanol

Initial 0.46 ± 0.03 0.55 ± 0.06 0.44 ± 0.05

Final 0.35 ± 0.13 0.47 ± 0.02 0.37 ± 0.12

Total sterols

Initial 9.27 ± 0.80 11.68 ± 0.98 8.91 ± 1.07

Final 10.92 ± 0.73a 16.07 ± 0.41b** 17.98 ± 0.58c**

Copr./chol.A

Initial 1.64 ± 0.22 1.66 ± 0.23 1.59 ± 0.15

Final 2.86 ± 0.84 2.33 ± 0.57 3.28 ± 0.59

Secd./chol.B

Initial 2.57 ± 0.35 2.56 ± 0.33 2.53 ± 0.17

Final 3.74 ± 1.09 3.34 ± 0.69 4.48 ± 0.68

Results are expressed as means ± SEM in mg of sterol ani-mal-1 day-1 (n = 4). Results in the same row that do not share acommon superscript are significantly different based on Duncan’smultirange test at P\ 0.05. Differences between initial and finalvalues based on Student’s t test are denoted as * for P\ 0.05or** for P\ 0.01. Campesterol was found only in trace amounts

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis)A Ratio of coprostanol/cholesterolB Ratio of secondary metabolites of cholesterol (coprostanol, cho-lestanol and cholestanone)/cholesterol

Table 6 Bile acid excretion

CE PHGG1 PHGG2

Primary

CA

Initial tr tr tr

Final tr tr tr

CDCA

Initial 0.32 ± 0.12 0.44 ± 0.09 0.31 ± 0.12

Final 0.24 ± 0.07 0.39 ± 0.10 0.37 ± 0.07

Secondary

DCA

Initial nd nd nd

Final nd nd nd

LCA

Initial 2.11 ± 0.62 2.58 ± 0.52 2.14 ± 1.00

Final 1.63 ± 0.43 2.87 ± 0.90 3.15 ± 0.50

UDCA

Initial 1.05 ± 0.39 0.99 ± 0.13 1.00 ± 0.41

Final 0.53 ± 0.15 0.69 ± 0.15 0.66 ± 0.07

Total BA

Initial 3.48 ± 1.13 4.00 ± 0.72 3.45 ± 1.53

Final 2.41 ± 0.51 3.96 ± 1.12 4.18 ± 0.63

LCA/CDCA

Initial 8.04 ± 1.63 6.00 ± 0.54 6.12 ± 0.64

Final 7.16 ± 1.92 7.14 ± 0.99 8.79 ± 0.69*

Results are expressed as means ± SEM in mg of bile acid ani-mal-1 day-1 (n = 4). No statistical differences were found betweentreatments (P[ 0.05). * Differences between initial and final valuesbased on Student’s t test (P\ 0.01)

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis), CA cholicacid, CDCA chenodeoxycholic acid, DCA deoxycholic acid, LCAlithocholic acid, UDCA ursodeoxycholic acid, Total BA total bileacids, nd not detected, tr traces

Table 7 Plasma lipids

CE PHGG1 PHGG2

Triglycerides 0.63 ± 0.07 0.63 ± 0.07 0.56 ± 0.08

Total cholesterol 2.59 ± 0.68b 1.38 ± 0.20a 0.93 ± 0.11a

HDL-col 0.20 ± 0.06 0.15 ± 0.02 0.11 ± 0.01

LDL-col 2.68 ± 0.74b 1.26 ± 0.19a 0.89 ± 0.11a

HDL/LDL 0.08 ± 0.01a 0.13 ± 0.01ab 0.14 ± 0.02b

Results are expressed as means ± SEM in mmol L-1 (n = 8).Results in the same row that do not share a common superscript aresignificantly different based on Duncan’s multirange test at P\ 0.05

CE cellulose, PHGG1 partially hydrolyzed guar gum (1-h hydrolysis),PHGG2 partially hydrolyzed guar gum (2-h hydrolysis)

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of 1 % PHGG aqueous solutions revealed pseudoplastic

(shear-thinning) behavior. Previous studies have reportedsimilar findings for aqueous solutions of GG [20].

In our study, both PHGG reduced body weight gain to a

similar extent. However, this decrease was not correlatedwith changes in food consumption, which is consistent with

the findings of other studies [18]. A reduction in the

absorption of nutrients and thus the calories they providecan be partially responsible for the lower food efficiency of

diets containing PHGG.In good agreement with previous reports, fat excretion

was increased by ingestion of PHGG [21]. The longitudinal

study of PHGG consumption reveals that the effectoccurred from the beginning of the treatment and stabilized

after 1 week. Therefore, no accumulative effect was

observed after the first week.As previously reported [13], the concentrations of

medium and long chain SFA in feces, especially C16:0 and

C18:0, were higher when compared to its concentration indiets. This finding is mainly attributed to the low digest-

ibility of these FA [22]. The FA profile in feces was not

altered by PHGG consumption in a similar way to otherdietary fibers such as pectin [23] and in contrast to others

such as hydroxypropyl methylcellulose [14].

Another hypocholesterolemic property commonlyattributed to soluble dietary fiber is its capacity to increase

the excretion of BA and endogenous and exogenous cho-

lesterol. In vitro [8] and in vivo studies [24] have shownthat PHGG conserves the capacity of the native GG to

increase cholesterol excretion. In our study, NS excretion

was greater after consumption of PHGG, mainly as a resultof an increment in cholesterol metabolites such as

coprostanol and cholestanol. Thus, the bioconversion of

cholesterol tended to increase in animals ingesting PHGG2.Although it is widely accepted that GG increases BA

excretion, thus decreasing its reabsorption [24, 25], con-

troversial results have been reported for PHGG. Our resultsare consistent with the findings of Favier et al. [21], who

reported that total BA excretion was not significantly

altered by PHGG ingestion. However, as seen for NS, theconsumption of PHGG2 increased the bioconversion of

primary BA to their secondary metabolites. Similar results

on BA bioconversion were observed by Gallaher et al. [26],who reported that the lithocholic/chenodeoxycholic ratio in

feces of hamsters fed low viscosity GG was higher than

that of animals on native GG. However, that study wasperformed in hamsters, which partially ferment GG in the

pregastric pouch of the stomach [26] and have a distinct

BA profile in feces to that of guinea pigs [27]. Our resultsshow that this effect is also present in guinea pigs, a more

suitable model for the study of lipid metabolism. GG and

PHGG are partially fermented in the intestine, thus favor-ing increased endogenous bacterial population and activity

[28, 29]. For instance, the growth of intestinal bacteria such

Lactobacillus spp. and Bifidobacterium spp. is enhanced inhumans after PHGG consumption [30]. Hence, an increase

in bacterial activity may result in greater sterol biocon-

version. The lower viscosity of PHGG2 could explain itshigher pre-biotic effect as it favors interaction with intes-

tinal microbiota.

Based on these results, the increase in NS excretionappears to be one of the main mechanisms responsible for

the lower total plasma cholesterol in animals fed PHGGdiets. In addition, it is plausible that the fermentability of

PHGG can contribute to its hypocholesterolemic effect due

to the production of short chain fatty acids, mainly propi-onate, which can decrease the endogenous synthesis of

cholesterol [29, 31].

On balance, within the degree of hydrolysis tested, areduction in the viscosity of PHGG did not correlate with a

loss of hypolipidemic capacity. Current evidence indicates

that consumption of viscous fibers such as GG can alter theviscosity of digesta in the gastrointestinal tract and thereby

decrease the bioaccessibility of fat and cholesterol [6, 26,

32]. Nevertheless, it remains questionable whether altera-tions on in vitro viscosity may result in proportional

physiological responses [32, 33]. For instance, Gallaher

et al. [32] observed that although only small increments inviscosity were necessary to decrease plasma cholesterol,

further increments did not result in larger reductions of

plasma cholesterol. In addition, it has been suggested thatother mechanisms not attributed to viscosity can be com-

plementarily responsible for the hypolipidemic effect of

PHGG. The bioaccessibility of fat and cholesterol waspreviously studied by Minekus et al. [8] using a dynamic

model of the gastrointestinal tract. The authors concluded

that PHGG can also reduce lipid absorption by a depletion-flocculation mechanism, which is typical of polymers that

do not show surface activity. Since the polysaccharide does

not adsorb to the fat droplets, this can result in a regionaround the droplet where the concentration of the poly-

saccharide is lower than in the bulk solution. This phe-

nomenon could cause an osmotic pressure that would favorthe fat droplets flocculating and coalescing. As a result, it

may reduce the emulsifying effect of bile acids and

therefore the bioaccessibility of fat and cholesterol.In conclusion, both PHGG reduced body weight gain

and plasma cholesterol, regardless of their in vitro viscosity

within the ranges tested. Thus, the chemical hydrolysis canimprove the suitability of GG for use in food products

without affecting their rheology or palatability and con-

serving most of its hypolipidemic properties.

Acknowledgments J. Santas was granted a Torres Quevedofellowship. The authors thank A. Aldanondo, S. Scapucci andA. Gonciarz for their support during sample analysis.

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