Food & Function

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View Article OnlineView Journal | View Issue

aInstituto de Nutricion Animal, Estacion Exp

del Jueves s/n, 18100, Armilla, Granada, S

+34 958 572753; Tel: +34 958 572757bInstituto de Ciencia y Tecnologıa de los Alim

Madrid, Spain

Cite this: Food Funct., 2013, 4, 1016

Received 15th October 2012Accepted 13th December 2012

DOI: 10.1039/c2fo30288h

www.rsc.org/foodfunction

1016 | Food Funct., 2013, 4, 1016–10

Effects of diets supplemented with MRPs from breadcrust on the food intake and body weights in rats

Cristina Delgado-Andrade,*a Irene Roncero-Ramos,a Ana Haro,a

Francisco J. Morales,b Isabel Seiquera and Marıa Pilar Navarroa

Traditionally the effects of dietary Maillard reaction products (MRPs) on food intake and body weight have

been described in different studies, but few investigations have been conducted to analyse the main

contributors responsible. We studied the effects of long-term consumption of MRPs from bread crust

(BC) on rat growth, investigating the efficiency of diet and protein utilization. Different soluble and

insoluble fractions of BC were studied to analyse the possible contributors. Additionally, the colour of

the faecal material and the amount of fluorescent MRPs in the urine were measured in order to

demonstrate MRP excretion. Six groups of rats were fed the following diets for 88 days: control (AIN-

93G diet); bread dough (BD) and BC (containing 10% of BD or BC, respectively); low and high molecular

weight (LMW–HMW) (containing soluble compounds from BC with <5 kDa and >5 kDa, respectively);

insoluble (containing insoluble compounds from BC). Dietary MRPs tended to reduce the food intake

and body weight significantly after consumption of more complex compounds (HMW and insoluble).

The L*-value in the faeces decreased in animals fed BC and its derivatives, providing evidence of the

presence of MRPs. The fluorescence associated with MRP excretion in urine was higher when the LMW

diet was consumed, suggesting the easier absorption and clearance of the smaller compounds of BC.

Introduction

Thermal processes such as frying, roasting and baking producecolours, aromas and avours in foods, which are attractive forconsumers. One of the main factors responsible for producingthese characteristics is the Maillard reaction (MR), which takesplace between amino acids or proteins and reducing sugars oroxidized lipids, the products of which are termed Maillardreaction products (MRPs).1 This reaction can also occur in theinternal media of living beings and the products are then calledadvanced glycation end products (AGEs).2

TheWestern diet, which is rich in thermally processed foods,provides an abundant daily MRP intake,3 and bakery productsare one of the major sources of amino acid derivatives of MR inconventional diets.4

MR development begins with the appearance of a colourless,simple product; subsequently, complex, coloured compoundsare formed depending on the type of food and the thermaltreatment.5 A long time ago, the measurement of the CIELabcolour was applied to determine the participation of MRPs inthe nal appearance of foods.6 More recently, this tool hasprovided interesting information on the kinetics of bread crust

erimental del Zaidın (EEZ-CSIC), Camino

pain. E-mail: cdelgado@eez.csic.es; Fax:

entos y Nutricion (ICTAN-CSIC), 28040,

22

browning during baking7 and on the MR development in foodscooked by different culinary techniques.8

In the progress of the MR, uorescent compounds (FCs) arealso produced; therefore, monitoring of FCs formation isanother suitable non-specic tool for determining MR devel-opment.9,10 From a biological perspective, FCs are includedamong the AGEs pool formed in vivo, and their specic uo-rescence has been used to measure serum levels.11 Moreover,urinary uorescent MRP have been detected aer theconsumption of diets containing processed food, among bothhealthy subjects12 and diabetic elderly subjects.13

On the other hand, we must take into account that MRPs arenot biologically inert but have a physiopathological impact onfood intake, body weight and nutrient bioavailability.14 Experi-mental results are controversial because although MRPs arecompounds favouring the organic characteristics of foods, andthen consumption, Furniss et al.15 detected a decrease in thefood consumption aer feeding rats with a glucose–caseinheated mixture. More recently, Sarria et al.16 obtained similarresults with liquid infant formulas containing Maillard deri-vates. The effects on body weight have been also described.O’Brien and Walker17 observed a decrease in the weight of ratsthat consumed MRPs from model systems. This was subse-quently conrmed by Seiquer et al.,18 who compared the intakeof diets based on overheated or UHT milk in rats.

On the other hand, it is still unclear which kind of MRPscould be the major component responsible for these actions.

This journal is ª The Royal Society of Chemistry 2013

Paper Food & Function

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The goal of the present work was to investigate the effects oflong-term consumption of MRPs from bread crust on ratgrowth, studying the efficiency of diet and protein utilization.The colour of the faecal material and the presence of uorescentMRPs in the urine were measured in order to demonstrateMRPs excretion. Different soluble and insoluble fractions of BCwere studied to analyse the possible contributors for the effectsobserved.

Material and methodsChemicals

All chemicals used were of analytical grade and were obtainedfrom Merck (Darmstadt, Germany), unless stated otherwise.Pronase E (4 000 000 PU g�1) was also purchased from thiscompany. HPLC-grade acetonitrile was obtained from Lab-San(Dublin, Ireland). Furosine was obtained from NeosystemLaboratories (Strasbourg, France).

Preparation of the diets

The AIN-93G puried diet for laboratory rodents (Dyets Inc,Bethlehem, PA) was used as the control diet. Bread dough (BD)and crust (BC) were supplied by a Spanish manufacturer ofcereal-derived food products. The BDwas lyophilized and addedto the AIN-93G diet to reach a nal concentration of 10%. Thisdiet was termed BD and was used as an additional control. Theprocess by which the BC was removed is described in a previouspaper by Roncero-Ramos et al.,19 as is the extraction of itssoluble and insoluble fractions by enzymatic digestion withpronase. The lyophilized BC was added to the AIN-93G diet toreach a nal concentration of 10%. This was termed the BC diet.In order to determine the factors responsible for any effectsobserved in the trial, as described previously19 the soluble LMW(low molecular weight, <5 kDa), soluble HMW (high molecularweight, >5 kDa) and insoluble fractions from BC were alsoindividually added to the diet in the same proportion as theywere present in the 10% crust, this proportion being calculatedfrom the recovery of each fraction aer pronase E digestion.These diets were termed LMW, HMW, and insoluble,respectively.

The individual analysis of the different diets revealed nomodication of the overall nutrient composition in any case(AIN-93G). The mean � SD nutrient content of the diets was:moisture (%) 7.9 � 0.3; protein (g kg�1) 167.7 � 3.9; fat (g kg�1)77.8 � 1.5 and ashes (%) 2.6 � 0.2.

Biological assays

Sixty-eight weanling Wistar rats weighing 41.00 � 0.16 g (mean� SD), supplied by Charles River Laboratories Spain S.A., wereused in the study. The animals were randomly distributedinto six groups (12 animals per group, except in the insolubleone, in which the accidental death of four animals limited thenumber to 8) and each group was assigned to one of the die-tary treatments. The animals were individually housedin metabolic cages in an environmentally controlled roomunder standard conditions (temperature: 20–22 �C with a 12 h

This journal is ª The Royal Society of Chemistry 2013

light–dark cycle and 55–70% humidity). The rats had adlibitum access to their diets and demineralized water(Milli-Q Ultrapure Water System, Millipore Corp., Bedford, MA,USA).

The animals were fed the different diets for 88 days. Solidfood intake and body weight were monitored weekly duringthis period. In the last week of the experimental period (days82–88) faeces and urine from each animal were collected dailyand stored separately as a 1 week pool. The faeces wereweighed, lyophilized and homogenized. The urine wascollected in 0.5% HCl (vol/vol), ltered (Whatman Filter Paperno. 40, ashless, Whatman, England) and diluted to an appro-priate volume.

Taking into account the data obtained from food and proteinconsumption, as well as body weight gain, the food efficiencyand the protein efficiency rate (PER) were calculated for days 1to 28, 29 to 56 and 57 to 88, as follows:

Food efficiency ¼ weight gained (g)/food intake (g dry matter)

PER ¼ weight gained (g)/protein intake (g dry matter)

All management and experimental procedures carried out inthis study were in strict accordance with the current Europeanregulations (86/609 E.E.C.) regarding laboratory animals. TheBioethics Committee for Animal Experimentation at our insti-tution (EEZ-CSIC) approved the study protocol.

Diet characterization

Furosine and HMF content. Dietary furosine determinationwas performed according to Delgado-Andrade et al.20 Furosinewas quantied by the external standard method, using a cali-bration curve built from a stock solution (1 mgml�1 of furosine)in the range 10–0.05 mg l�1.

The determination of HMF was based on the methoddescribed by Ruan-Henares et al.,21 quantifying by the externalstandard method in the range 2–100 mM.

In both analyses, the HPLC system consisted of a Series 200pump, a Series 225 autosampler, a Series 200 ultraviolet-visibledetector, a Series 200 Peltier column oven and TotalChromWorkstation soware v. 6.3.1. for data acquisition and treat-ment, all obtained from Perkin-Elmer (Perkin-Elmer, Waltham,MA).

Measurement of colour. The colour of the different diets wasdetermined using a Chroma Meter CR-400 optical sensor(Konica Minolta Sensing, Inc., Osaka, Japan) according to theCIELab scale.6 The system provides the values of three colourcomponents, L* (black-white component, luminosity) togetherwith the chromaticity coordinates, a* (+red to �green compo-nent) and b* (+yellow to �blue component). The samples wereplaced in a 34 mm optical glass cell. The sample was illumi-nated with D65-articial daylight (10� standard angle) inaccordance with the manufacturer’s instructions. Each colourvalue reported was the mean of three determinations at 22–24�C. The yellowing index (YI) was calculated from the equationsYI ¼ 142.86b*/L*.

Food Funct., 2013, 4, 1016–1022 | 1017

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Measurement of colour in faeces

The colour of powdered and lyophilized faeces was determinedin triplicate using the previously described Chroma Meter CR-400 optical sensor (Konica Minolta Sensing, Inc., Osaka, Japan).From the data for three colour components (L*, a* and b*) theyellowing index (YI) was calculated as described above.

Urinary excretion of MRPs uorescent compounds

The excretion of uorescent compounds (FCs) in urine aer thewhole experimental period was determined using the 1 weekpool of urine collected during days 82–88 of the trial. Analysis ofFCs at a typical wavelength for MR compounds (347 nm exci-tation; 415 nm emission) was carried out by direct dilution ofurine to avoid any quenching effects.22 A uorescence spectro-photometer (LS 55, Perkin-Elmer, Waltham, MA) was used,together with quartz glass cuvettes (QS-1.000 Suprasil, HellmaGmbH & Co, Germany) with a light path of 1 cm. The sampleswere measured at least in duplicate, data are expressed asarbitrary units �105 per day.

Statistical analysis

All data were statistically tested by one-way analysis of thevariance (ANOVA), followed by Duncan’s test to make multiplecomparisons of means that showed a signicant variation (P <0.05). Analyses were performed using Statgraphics Plus, version5.1, 2001. Relationships between the different variables wereevaluated by computing the relevant correlation coefficient(Pearson’s linear correlation).

Results and discussionDiet characterisation

The levels of furosine and HMF in the experimental diets weredetermined in order to establish the specic presence of thesecompounds, which have been widely used to monitor the extentof MR in cereal derived products.21,22 The highest furosine andHMF contents were found in the BC diet (Table 1). Ramırez-Jimenez et al.23 also showed high HMF levels in BC comparedwith the crumb of different types of bread.

When different fractions from BC were included in theAIN-93G diet, the levels of both markers were always lower thanin the BC diet (Table 1). The baseline content of bothcompounds in the control diet was even higher than the levels

Table 1 Parameters L*, a*, b*, yellowing index (YI) and furosine and HMF conten

Diets L* a* b*

Control 91.32 � 0.37a �0.99 � 0.04a 8.74 � 0.11a

BD 89.72 � 0.44b �0.56 � 0.04b 8.90 � 0.25a

BC 84.89 � 0.17c 1.93 � 0.08c 13.27 � 0.24b

LMW 89.67 � 0.44b �0.72 � 0.05d 10.47 � 0.18c

HMW 88.46 � 0.20d �0.49 � 0.03b 9.59 � 0.14d

Insoluble 88.30 � 0.11d 0.72 � 0.08e 10.15 � 0.26c

a Values are means � SD (standard deviation), n ¼ 3. Different letters wib YI ¼ 142.86b*/L*.

1018 | Food Funct., 2013, 4, 1016–1022

detected in the BD one. Previous assays in which mice were fedthe AIN-93G diet have also demonstrated the presence ofMRPs.24 Interestingly, the HMF and furosine data found for theLMW, HMWand insoluble diets did notmatch the total amountdetermined in the BC diet, suggesting that the non-specicaction of pronase on BC could interfere with the MRPs that wereinitially present.

As a consequence of the Maillard and caramelization reac-tions during baking, the initial luminosity of the control dietdecreased due to the inclusion of bread derivatives (Table 1),following the order: control > BD ¼ LMW > HMW ¼ insoluble >BC. The implication of MRPs in this fact was supported by thehigh inverse correlation found between L* and the furosinecontent in the diets (r ¼ �0.784; P < 0.001) as well as with HMF(r ¼ �0.888; P < 0.001). Similarly, Ramırez-Jimenez et al.23

reported positive linear correlations of close to one between thespecic index and the parameter 100 � L* (darkness).

The BC diet had the greatest a* and b* values, i.e., withsignicant red and yellow components. The opposite situationoccurred in the Control diet, with the lowest values of bothparameters. The Insoluble diet showed the same tendency asthe BC diet, due to the presence of the most complex MRPs. It isduring the nal stages of the MR that complex melanoidins areformed and some of these become insoluble.25 However, theHMW and LMW diets produced negative a*-values, movingtowards the green. Regarding colour formation in MR modelsystems, Ames26 considered that the major contribution camefrom macromolecules, with little participation by LMWcompounds.

The YI, used as a more specic colour index of the MR inprocessed foods,8 reected a similar behaviour to the b*-value,indicating the higher presence of MR derivatives in the BC diet(Table 1). This parameter was closely related to dietary furosine(r ¼ 0.863; P < 0.001) and HMF (r ¼ 0.943; P < 0.001). We alsofound a strong correlation with the a*-value (r ¼ 0.893; P <0.001), which suggests that the displacement to the red zonewas directly related to the development of the MR.

Biological assay

Food intake, body weight, food efficiency and PER. Ingeneral, MR-derived products give foods their pleasant aromaand avour, improving palatability and the consumers’ appe-tite. Thus, Birlouez-Aragon et al.27 described an increase in foodintake by healthy subjects who consumed a high vs. low MRPs

t in the experimental dietsa

YIb Furosine (mg kg�1 diet) HMF (mg kg�1 diet)

13.67 � 0.19a 28.80 � 0.50a 0.44 � 0.06ac

14.18 � 0.36a 22.32 � 0.05b 0.37 � 0.04a

22.34 � 0.40b 49.47 � 0.27c 4.26 � 0.02b

16.68 � 0.34c 39.68 � 1.37d 0.47 � 0.04c

15.49 � 0.26d 39.40 � 0.99d 0.47 � 0.01c

16.42 � 0.44c 34.73 � 0.84e 0.89 � 0.01d

thin a column indicate signicant differences between diets (P < 0.05).

This journal is ª The Royal Society of Chemistry 2013

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diet. However, animal assays have produced controversialresults. Stability in food consumption has been reported aerfeeding mice diets containing high or low MRPs levels.28 Incontrast, decreases in food intake have been reported followingthe administration of browning products from a glucose–lysinemodel system heated at 150 �C for 90 minutes to rats,29 when anin-bottle-sterilized infant formula was provided.30 In the presentstudy, the consumption of diets containing BC fractionsdecreased food intake from the outset (Fig. 1A), while theinuence of BC and BD was apparent from the second month,although in the BC group the decline was attenuated and wasnot signicant in the third period. The animals in the insolublegroup recorded the lowest food intake, which could be related tothe scant digestibility of the complex MRPs present in thisfraction. As described by Koopsman31, food consumption couldbe modulated by the indigestible food volume reaching thesmall intestine.

From the same initial body weight, the weight gain did notsignicantly vary during the rst or the second period except inthe insoluble group, although the controls always had thehighest values (Fig. 1B). At the end of the assay, only the animalsfed diets containing the higher molecular weight fractions(HMW and insoluble) and the BD group displayed lower bodyweights. The BC and the LMW groups were not signicantlydifferent from the control group. These data do not match those

Fig. 1 Food intake (A) and body weights (B) of animals fed experimental dietsduring the three periods of the assay. Different letters within a period indicatesignificant differences (P < 0.05).

This journal is ª The Royal Society of Chemistry 2013

reported by Cai et al.,32 who established a trend of an increase inthe body weight of mice fed a calorie-restricted high AGEs dietcompared with the control diet without AGEs but were alsocalorie-decient. Similarly, Sebekova et al.,33 observed anincrease in body weight when rats consumed a diet containing25% BC but not when the proportion included (5%), which wascloser to that used in the present assay (10%). However, inhumans, short-term studies carried out on healthy34 or diabeticsubjects35 have documented non-effects on the body weightaer consumption of high AGE diets for a period of two weeks.This last assay documented a decrease when the period studiedwas extended to six months.

In our assay, the lower food consumption was responsiblefor the reduced body weight, a conclusion supported by thepositive correlation detected between these two parameters (r ¼0.594; P < 0.001) and because food efficiency remained stable(Fig. 2A). Similarly, Sarria et al.30 observed no modications infood efficiency.

The low PER values (Fig. 2B) could be due to the high proteincontent in the diets (17%), which was well above the standard10% adopted in the PER studies. As expected, aer the rstmonth, the period of most intense growth, the PER datadecreased and remained stable in all groups. Only during therst period did it tend to increase or signicantly increase in theBD, LMW and insoluble groups, and this was the only stage inwhich the PER and body weight were signicantly correlated(r ¼ 0.732; P < 0.001).

Measurement of colour in faeces. The introduction of breadderivatives into the Control diet decreased the L*-value in faeces(Table 2) with the BC and insoluble groups being the mostseverely affected. This suggests that the insoluble compoundswere darker and less digestible and that their presence in faecesinduced the lower L*-values. According to the studies reviewedby Somoza36 the faecal excretion of HMW melanoidins invarious model systems ranges from 26% to 87%. Dietsincluding soluble fractions of BC (LMW and HMW) produced aless marked decrease in the luminosity of faeces, probably dueto the more absorbable compounds present in these diets andto their higher luminosity. Thus, the L*-value for the diets wasdirectly correlated with that for the faeces (r ¼ 0.737; P < 0.001).Faeces from the BC group presented the highest red component(a*-value), followed by the insoluble group; on the other hand,faeces from the LMW and HMW groups displayed a lowerdegree of redness than those from the BC group, which isindicative of the effect of insoluble compounds. To some extent,the red component of the diets determined the redness of thefaeces (r ¼ 0.575; P < 0.001). The suitability of the b*-values andYI in faeces was limited due to the presence of bile pigments,and so the yellowness was mainly derived from the latter ratherthan from MRP. This fact could also explain the lack of corre-lation between diets and faeces in these two parameters.

Urinary excretion of MRP uorescent compounds. In thisstudy we used the detection of FCs at the characteristic wave-lengths of MRPs. In this way, although a particular predomi-nant compound is not determined, a varied pool of substancesis measured as an unspecic but useful tool to analyse theexcretion of uorescent MRPs.

Food Funct., 2013, 4, 1016–1022 | 1019

Fig. 2 Food efficiency (A) and protein efficiency rate (B) of animals fed experimental diets during the three periods of the assay. Different letters within a periodindicate significant differences (P < 0.05).

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The amount of FCs was lower in the BD and BC groupscompared with the control one, whereas these productsincreased in the urine from the animals of the LMW group(Fig. 3). This would suggest that uorescent low molecularweight MRPs are more easily absorbed and subsequentlydetoxied by urine. Surprisingly, aer consumption of the BDdiet, in which low amounts of FCs should be detected, theurinary elimination was similar to the BC diet. As observed byForster et al.37 for the FC pentosidine, the reason for this couldbe that the protein-bound form is among the majority of MRPsformed in bakery products, which impairs proteolytic break-down and may lead to a lower absorption rate and then elimi-nation. On the contrary, the compound had a better recoverywhen it was administered from a coffee brew, where thepredominant form was free pentosidine.

Scarce information is available in the scientic literatureabout MRP-associated uorescence excretions in urine. Theresearch team of de la Maza has made signicant efforts inexploring this subject and established that the prole ofurinary FCs in healthy young and elderly adults was similarand unaffected by their dietary pattern.38 Later, they also stated

Table 2 Parameters L*, a*, b* and yellowing index (YI) of faeces from animals fed

Diets L* a*

Control 74.90 � 0.48a 1.73 � 0.26BD 72.30 � 0.47b 1.39 � 0.12BC 69.32 � 0.36c 2.58 � 0.08LMW 72.16 � 0.27b 1.58 � 0.10HMW 72.85 � 0.33b 1.65 � 0.12Insoluble 70.28 � 0.31c 1.99 � 0.17

a Values are means � SD (standard deviation), n ¼ 3. Different letters wib YI ¼ 142.86b*/L.

1020 | Food Funct., 2013, 4, 1016–1022

the lack of correlation between the intake of FCs and itsurinary elimination.13

To sum up, under our experimental conditions, theconsumption of MRPs from whole BC within a diet does notmodify the food intake or food efficiency, and so the bodyweight remains stable, even in intense growth periods. Never-theless, isolated consumption of the most complex and leastsoluble compounds depresses food intake and subsequentlybody weight. Therefore, the effects of MRPs intake oendescribed on these parameters could be attributed to the moreadvanced and complex compounds. The major presence of FCsin urine aer consumption of the LMW fraction from BCsuggests the easier detoxication of smaller sized compounds,while the metabolic transit of those that are protein-boundseemed to be impaired. Further studies are necessary to analysethe impact of a complex pool of dietary MRPs on the kidneymechanism of clearance.

Author disclosure statement

The authors declare there is no conict of interest.

the experimental dietsa

b* YIb

ab 21.30 � 0.67abc 40.69 � 1.46ab 22.57 � 0.71b 44.64 � 1.50bc 20.72 � 0.37ac 42.68 � 0.58abab 21.10 � 0.36abc 41.77 � 0.69abab 22.02 � 0.54bc 43.20 � 1.15aba 19.85 � 0.60a 40.37 � 1.27a

thin a column indicate signicant differences between diets (P < 0.05).

This journal is ª The Royal Society of Chemistry 2013

Fig. 3 FCs excreted in the urine after consumption of the experimental diets.Different letters within a parameter indicate significant differences (P < 0.05).

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Acknowledgements

This work was supported by a project of the Spanish Ministry ofScience and Innovation. The authors thank Grupo Siro, aSpanish manufacturer of cereal-derived food products, forsupplying the bread crust samples.

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This journal is ª The Royal Society of Chemistry 2013