Development of Low-Calorie Guava Preserves with Prebiotics And

Food and Chemical Toxicology 50 (2012) 3719–3724

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Food and Chemical Toxicology

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Development of low-calorie guava preserves with prebiotics and evaluation of their effects on carcinogenesis biomarkers in rats ⇑ Camila Carvalho Menezes a, , João de Deus Souza Carneiro a,1, Soraia Vilela Borges a,2, Vera Sônia Nunes da Silva b,3, Maísa Ribeiro Pereira Lima Brigagão c,4, Luciana Azevedo d,5

a UFLA Federal University of Lavras, Department of Food Science, 37200-000, Lavras, MG, Brazil b ITAL Institute of Food Technology, CCQA Science Center and Food Quality, 13070-178, Campinas, SP, Brazil c UNIFAL Federal University of Alfenas, Department of Exact Sciences, 37130-000, Alfenas, Brazil d UNIFAL Federal University of Alfenas, Department of Nutrition, 37130-000, Alfenas, Brazil

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Article history: Faced with the search for healthy products that provide additional beneﬁts to consumers’ health, the Received 21 May 2011 main objectives of this work were to develop a low-calorie preserve containing prebiotics (lactulose Accepted 17 July 2012 and polydextrose) and to evaluate the effects of these prebiotics on oxidative stress and colon carcinogen- Available online 23 July 2012 esis in male rats treated with 1,2-dimethylhydrazine (DMH). A total of 62.5% w/w of the sucrose in conventional preserves was replaced by polydextrose, and lactulose was added at 0%, 16%, 19.5% or 23% w/w Keywords: concentrations. The acceptance of these four low-calorie guava preserve samples and the conventional Lactulose sample was equal (P > 0.05), with a score of 6.49. The level of degradation of lactulose was low Polydextrose (18.45 g 100 gÀ1 lactulose), ensuring that even at a lower concentration of this prebiotic (16% w/w), Aberrant crypt foci Oxidative stress the concentration remained above the minimum level considered functional. We found that consumption of the low-calorie preserves with prebiotics does not have an effect on the development of mucin-negative ACF and classical ACF in the initiation phase of the mutagenic process. However, the consumption of 1.5 g of the preserve/rat/day potentiated lipid peroxidation and proteic oxidation in the liver. Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. View metadata, citation and similar papers at core.ac.uk brought to you by CORE

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1. Introduction These conditions as well as the ageing process are associated with oxidative stress due to the elevation of reactive oxidative spe- Colorectal cancer is the second leading cause of cancer death cies (ROS) or insufficient ROS detoxification. Oxidative stress (OS) and the third most common type of cancer in both men and wo- results when the production of ROS exceeds the capacity of cellular men in Western countries (ACS, 2008; Jemal et al., 2007); further- antioxidant defences to remove these toxic species (Limón-Pach- more, it is an increasing cause of death in Brazil, where it eco and Gonsebatt, 2009). The prevention of free radical damage accounted for 11,322 deaths in 2007 and approximately 28,000 has often been attributed to the consumption of antioxidants pres- new cases in 2010 (Brazil, 2010). Among the causes reported, epi- ent in fruits and vegetables, such as vitamins C and E, and carote- demiological and clinical studies have suggested that environmen- noids, such as lycopene, phenolic acids, and flavonoids (Michels tal factors, such as diet and lifestyle, are the most important et al., 2005; Rouphael et al., 2010). These compounds are fre- influences on the carcinogenesis process (Scharlau et al., 2009). quently concentrated to produce dietary supplements for human use. However, the additive and synergistic effects of other food constituents are frequently ignored (Hodek et al., 2009). ⇑ Corresponding author. Departamento de Ciência dos Alimentos, Universidade Guava is a characteristic ingredient of many products, such as Federal de Lavras, Campus Universitário, Lavras, Brazil. Tel.: +55 35 38291031; fax: preserves and jellies, which are enjoyed by consumers and achieve +55 35 38291401. annual retail sales in the U.S.A. of approximately $650 million E-mail addresses: [email protected]fla.br (C.C. Menezes), joa (International Jelly and Preserve Association, 2008). However, [email protected]fla.br (J. de Deus Souza Carneiro), [email protected]fla.br (S.V. Borges), [email protected] (V.S.N. da Silva), [email protected] (Maísa there is still a shortage of low-calorie preserves containing com- Ribeiro Pereira Lima Brigagão), [email protected] (L. Azevedo). pounds that have functional properties (Gonzalez et al., 2011) that 1 Tel.: +55 35 3829 1013. guarantee the formation of a product with conventional character- 2 Tel.: +55 35 3829 1394. istics. Among the compounds with functional properties, those 3 Tel.: +55 19 3743 1765. that maintain the health of the colon are prebiotics, lactulose and 4 Tel.: +55 35 3299 1063. 5 Tel.: +55 35 3299 1110. polydextrose. Because these prebiotics consist of dietary fibre, they

0278-6915 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.fct.2012.07.022 3720 C.C. Menezes et al. / Food and Chemical Toxicology 50 (2012) 3719–3724 are not absorbed in the small intestine and reach the colon, which added: lactulose, sucralose, BTM, xanthan gum and locust gum. Pectin BTM was di- increases faecal matter, intestinal transit and the adsorption of car- luted (6 g of pectin BTM in 100 mL of water) at 80 °C under constant stirring, and this solution was added to the xanthan gum and locust gum mixture. The cooking cinogens; prebiotics also modify the composition of microflora due was concluded when reached 60% soluble solid. The citric acid and potassium sor- to the growth incentive for probiotic bacteria, such as Lactobacillus bate were added to the mixture at the end of the cooking process in all treatments. and Bifidobacterium (Juskiewicz and Zdunczyk, 2010; Rastall, At the end of the process, the five preserve formulations were hot-packed in trans- 2010). These alleged health-promoting properties could include parent polypropylene containers (5.5 cm height and 9 cm in diameter). prebiotic activity, which encompasses the cell protective effects of particular antioxidants that can be liberated in the colon after 2.2. Acceptance test fermentation by gut flora (Ferguson et al., 2003, 2005). Colonic bacteria then ferment lactulose and polydextrose, primarily resulting The acceptance test was conducted with 104 consumers in individual stalls in in the formation of short-chain fatty acids (SCFA), such as acetate, the Sensorial Analysis Laboratory of the Food Science Department of the Federal propionate, and butyrate. Those SCFA, especially butyrate, are University of Lavras, where the guava preserve samples were appraised based on recognised for their potential to act through secondary chemopre- their global acceptance using a nine-point structured hedonic scale; the scores var- ied between the hedonic terms ‘‘I extremely disliked (score 1)’’ and ‘‘I extremely vention by slowing growth and activating apoptosis in colon can- liked (score 9)’’ (Acosta et al., 2008). On average, the tasters received 15 g of each cer cells. Additionally, SCFA can also act in primarily a of sample in disposable plastic cups codified with three digit numbers, which were preventative manner through the activation of various drug meta- monadically served following the presentation order according to the experimental bolising enzymes. Therefore, this can reduce the burden of carcin- design for the treatment evaluations obtained by Wakeling and Macfie (1995). ogens and decrease the number of mutations, thereby reducing cancer risk (Roy et al., 2009; Scharlau et al., 2009). Furthermore, 2.3. Physiochemical analyses those acids are associated with a decrease in colonic pH; this inhib- its the formation of secondary biliary acids generated above pH 6.5, The determination of moisture, protein, ethereal extract, ash, total dietary fibre, which are associated with the carcinogenic process (Juskiewicz carbohydrates and caloric value levels was conducted according to guidelines and Zdunczyk, 2010). established by the Association of Official Analytical Chemists (AOAC, 2000). The lactulose, glucose, fructose and sucrose levels were determined using a high Thus, the objectives of this work were to develop a new product, performance liquid chromatography - HPLC (Varian Analytical Instruments, Walnut a low-calorie preserve with prebiotics, to meet new consumer de- Creek CA, USA: Model 350, with a refractive index detector - RI) apparatus equipped mands and to evaluate their effects on colon tumorigenesis in mice with an isocratic pump, a RheodyneÒ 7725i injector (Rohner Park, CA, USA) (20 lL treated with 1,2-dimethylhydrazine (DMH). DMH is a potent colon sample loop). A C18-column (LiChrospherÒ 100 NH2, 250 Â 4 mm, 5 lm) and a pre- carcinogen (CCC) that undergoes oxidative metabolism resulting in column (Lichrospher 100 NH2 4 Â 4 mm, 5 lm) (Merck KGaA, Darmstadt, Germany) were used to acquire the data, which were processed with Borwin HPLC software. the electrophilic diazonium ion, which is known to elicit oxidative The column temperature was 40 °C, and the mobile phase was composed of a mix- stress. ture of water and acetonitrile (80:20 v/v); the flow rate was 1 mL/min. The detection limits of the method were 0.2 g 100 gÀ1 for fructose, glucose and sucrose and 0.5 g 100 gÀ1 for lactulose. The percentage of lactulose degradation was determined 2. Materials and methods from the ratio of final to initial lactulose concentrations. Because polydextrose is an extremely stable polymer within a wide range of pH and temperature values as well 2.1. Development of low-calorie guava preserves with lactulose as processing and storage conditions (Jie et al., 2000), its concentration in the preserves was estimated from the initial polydextrose ratio and the yielding of the Guavas (Psidium guajava, L) of the ‘Pedro Sato’ cultivar were produced in Lavras, preserves. MG, Brazil. In addition, the following reagents were used in this study: sucrose, cit- The water activity was determined using Aqualab equipment (Decagon model ric acid monohydrate (VETEC, RJ, Brazil), high methoxyl citrus pectin (BTM) (VETEC, 3TE, Washington, USA) at a controlled temperature of 25.0 ± 0.3 °C. RJ, Brazil), potassium sorbate (VETEC, RJ, Brazil), polydextrose (DaniscoÒ, SP, Brazil), xanthan and locust gum (DaniscoÒ, SP, Brazil), low methoxyl citrus pectin SF369 ex- tra (DaniscoÒ, SP, Brazil), sucralose (NutramaxÒ, SP, Brazil) and 50% w/w lactulose syrup (HenrifarmaÒ, SP, Brazil). 2.4. Animals and treatments Five preserve formulations were developed consisting of one with conventional and four of low caloric value, which was accomplished through the addition of dif- The University Ethical Committee for Animal Research approved the protocols ferent quantities of lactulose (0%, 16%, 19.5% and 23% w/w). Conventional guava used in this study (protocol No. 288/2010). preserves (CGP) were prepared according to the procedures described by Menezes et al. (2009) to be used as standards for the comparison with the low-calorie guava preserves (LCGP) (Table 1). The fruits were washed in running water and then sanitised in a 200 mg LÀ1 so- dium hypochlorite solution for 15 min; the pulp was removed using an electric dep- ulper (Macanuda, SC, Brazil) with a 6 mm mesh sieve. The conventional preserve was processed in an open pot (Macanuda, SC, Brazil), and the cooking was inter- rupted when reached 70 % soluble solid. For the formulation of the low-calorie preserves, guava was added to the open pot followed by the addition of polydextrose. When this mixture reached 45% soluble solids, the following components were

Table 1 Formulations of conventional guava (CGP) and low-calorie (LCGP) preserves without lactulose.

Ingredients CGP (percent)* LCGP without lactulose (percent)* Sucrose 40 15 Polydextrose – 20 High methoxyl pectin 0.5 – Low methoxyl pectin – 0.6 Locust bean gum – 0.05 Xanthan gum – 0.05 Sucralose – 0.01 Potassium sorbate 0.05 0.05 Citric acid 0.3 0.2 Fig. 1. Experimental design: (G1) DMH; (G2) control; (G3) DMH + 0.5 preserve/ animal; (G4) DMH + 1.0 g preserve/animal; (G5) DMH + 1.5 g preserve/animal; (G6) * Amounts proportional to the guava pulp used. EDTA + 1.5 g preserve/animal. C.C. Menezes et al. / Food and Chemical Toxicology 50 (2012) 3719–3724 3721

Table 2 Nutritional composition, water activity and global acceptance of the guava preserves.

Response variables Treatments* CGP LCGP LCGP + 16% lactulose LCGP + 19.5% lactulose LCGP + 23% lactulose Moisture (g 100 gÀ1) 24.49d 34.98c 37.25b 38.00a 38.04a Protein (g 100 gÀ1) 0.24a 0.13b 0.12b 0.14b 0.14b Fat (g 100 gÀ1) 1.01a 0.39b 0.35b 0.56b 0.59b Carbohydrate (g 100 gÀ1) 69.05a 25.51b 20.45c 19.85c 20.30c Sucrose (g 100 gÀ1) 21.87a 15.70b 9.60c 8.29d 8.54d Fructose (g 100 gÀ1) 8.40a 7.00b 4.76c 4.07d 3.83e Glucose (g 100 gÀ1) 7.20a 6.50b 4.89c 4.32d 4.18d Lactulose (g 100 gÀ1) – – 14.55c 16.53b 17.89a Ash (g 100 gÀ1) 0.58a 0.46b 0.44b 0.34c 0.24c Total dietary ﬁbre (g 100 gÀ1) 4.63b 5.72a 4.20b 3.47c 3.55c Polydextrose (g 100 gÀ1)** – 19.11 21.13 22.64 32.26 Calorie (kJ 100 gÀ1)** 286.2a 138.4b 108.0c 106.1c 107.4c Water activity 0.762a 0.872b 0.884b 0.882b 0.874b Global acceptance 6.55a 6.37a 6.04a 6.39a 6.12a

CGP = conventional guava preserve. LCGP = low-calorie guava preserve. * Mean values followed by the same letter in the same line are equal among themselves by the Tukey test (P < 0.05). ** Determined by calculation.

Male 4-week-old Wistar rats were obtained from CEMIB (UNICAMP Campinas, The MDA and protein carbonyl levels were standardised for total protein con- SP, Brazil). The animals were kept in polypropylene cages (three or four animals/ tent (Bradford, 1976). cage) covered with metallic grids at 22 ± 3 °C, 55 ± 5% humidity and a 12-h light– dark cycle. They were fed commercial Nuvilab CR-1Ò (Nuvital Nutriente S/A, Colom- bo, PR, Brazil) and water ad libitum during the experiment. 2.7. Statistical analysis The animals were randomly allocated into five groups (n = 10) (Fig. 1): groups 1, 3, 4 and 5 were administered four s.c. injections of DMH (40 mg/kg b.w.) twice a Analysis of variance was used to evaluate the significance of the sugar interfer- week for two weeks (second and third experimental week), and groups 2 and 6 re- ence by substitutions with sucralose and polydextrose; in addition, the acceptance ceived similar injections of EDTA solution (DMH vehicle, 37 mg/100 mL distilled and the chemical parameters of the preserves were analysed using variance analysis water) (Dias et al., 2006, 2010). Food and water consumption and animal weight for lactulose addition at three concentrations. Variance analysis was also used for were measured twice a week throughout the experimental period. At the end of the evaluation of the weight of animals, body mass gain, food and water consump- the experiment, all animals were anaesthetised with ketamine (0.08 mg/kg b.w.) tion and oxidative stress analyses. If significant (P < 0.05), the Tukey test was used and xylazine (0.04 mg/kg b.w.) and were sacrificed by withdrawing blood from to determine the difference between treatments. The total number of ACF, the num- the heart. At necropsy, the liver and colon were removed from all animals. ber of aberrant crypts per focus, and the crypt/ACF ratio in the different groups were analysed by Kruskal–Wallis test (Dias et al., 2010). The statistical analysis was performed using R software, version 2.10 (R Development Core Team, 2009). 2.5. Colon processing, classical ACF and mucin-negative ACF analysis

At necropsy, the entire colon was removed, opened longitudinally and rinsed in saline solution. Because the incidences and/or multiplicities of murine colon ACF, 3. Results and discussion adenomas and adenocarcinomas are consistently higher in the middle and distal colon than in the proximal colon of animals treated with DMH (Dias et al., 2010; Jack- 3.1. Development of low-calorie preserves with prebiotics son et al., 2003), one-seventh of the colon length was excised from the proximal region for analysis of oxidative stress in this tissue. The other segments were fixed The reduction of sucrose content from 40% w/w to 15% w/w was in a flattened manner in 10% v/v buffered formalin for 24 h. For classical ACF, the colon was stained with 1% v/v methylene for 1 min, and the number of ACF/colon enough to create low-calorie preserves (Table 2); thus, there was a was determined; in addition, the number of aberrant crypts in each ACF was deter- decrease of 62.91% w/w and 51.65% w/w for carbohydrates and mined under light microscopy at 40Â magnification. After de-staining with 70% v/v caloric value, respectively, which placed the preserves in the ethanol with gentle shaking at room temperature, the colons were re-stained with a low-calorie category according to current legislation (Brazil, 0.025% w/v toluidine blue (TB, pH 2.5) in a 3% v/v acetic acid solution to detect mucin-negative ACF (Dias et al., 2010). 1998; CFR, 2007; FDA, 2008). Similar results were found by Nachti- gall et al. (2004) when replacing 50% w/w of the sucrose used in the production of black mulberry jelly with high intensity sweet- 2.6. Oxidative stress in liver, plasma and colon eners. The lactulose addition also contributed to the reduction in

For this analysis, we used the group that consumed 1.5 g preserves/rat/day for caloric value; however, among the different concentrations stud- 28 days (G6) compared with the control group (G2). ied, there was no significant difference (P P 0.05) (Table 2). One-seventh of the colon length was excised from the proximal region, the con- The acceptance of these 4 low-calorie guava preserve samples tents were removed, and the intestine was cleansed in ice–cold saline solution; and the conventional sample was equal (P > 0.05), with a mean then, the sample was homogenised in 1.5 mL of saline solution using a glass Teflon score of 6.49, (which fell between the hedonic terms ‘‘I liked homogeniser with 20 strokes (Itagaki et al., 2009). The liver was perfused with a 0.9% w/v NaCl solution. After being neutralised in the reaction mixture (20 lL), slightly’’ and ‘‘I liked moderately’’). Such an occurrence may be the samples were then separated on a 250 mm Â 4.6 mm i.d. VC-ODS RP18 column attributed to the fact that there are few significant differences be- with 50:50 (v/v) 25 mM phosphate buffer (pH 6.5) with methanol as the mobile tween sucralose and sucrose when added to foods, which only ap- À1 phase and a flow rate of 0.8 mL min . Fluorometric detection was performed at pear in relation to the sweetness equivalence (Wiet and Miller, wavelengths of kex 532 nm and kem 553 nm using a model RF-10AXL detector cou- pled to a HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) to ensure the 1997). Furthermore, polydextrose does not contribute to the fla- sensitivity needed for measuring the low concentrations of the MDA(TBA)2 adduct vour of foods (Jie et al., 2000). present in the plasma samples. The adduct was quantified using TEP as a standard, The presence of glucose in the preserves (Table 2) may be be- and it was processed in exactly the same way as the serum samples (Brown and cause a fraction of the sucrose suffered an inversion process due Kelly, 1996). to the acidity of the medium established by the presence of citric The analysis of protein oxidative damage in the liver was performed by measuring the concentration of protein carbonyl levels using a spectrophotometer at acid and by exposure to high temperatures (85 °C) during the cook- 370 nm (BIOCHROM LIBRA 522, Berlin, Germany) (Eymard et al., 2009). ing process. 3722 C.C. Menezes et al. / Food and Chemical Toxicology 50 (2012) 3719–3724

Table 3 Initial and ﬁnal body weights, body mass gain, and food and water consumption in the induction effect of experimental groups.

Group/ Initial body Final body Body weight Consumptions % Sweet treatment weight (g) weight (g) gain (g) from diet Water Food Food LCGP with 16% kJ from LCGP (g/rat/day) (g/rat/day) (kJ/rat/day)* lactulose (g/rat/day) (kJ/rat/day)** (G1) RC + DMH 118.7 ± 13.3a 242.7 ± 15.0bc 124.0 ± 9.7b 38.8 ± 14.1b 15.0 ± 4.0b 55.8 – – – (G2) RC + EDTA 118.9 ± 11.1a 266.7 ± 20.3ª 140.1 ± 18.1a 39.1 ± 8.4b 15.8 ± 3.4ab 58.8 – – – (G3) 0.5 g preserve + DMH 116.7 ± 12.9a 233.6 ± 18.9c 116.9 ± 10.1b 30.6 ± 8.2d 13.1 ± 3.75c 48.7 0.5 ± 0.11 0.54 ± 0.12 1.10 (G4) 1.0 g preserve + DMH 121.0 ± 15.1a 242.3 ± 14.7c 121.3 ± 10.1b 32.3 ± 11.6cd 13.0 ± 3.81c 48.4 1.0 ± 0.22 1.08 ± 0.24 2.18 (G5) 1.5 g preserve + DMH 119.8 ± 15.5a 246.8 ± 16.4bc 127.0 ± 7.6b 35.0 ± 8.3c 13.1 ± 4.1c 48.7 1.3 ± 0.29 1.40 ± 0.31 2.79 (G6) 1.5 g preserve + EDTA 120.2 ± 11.8a 265.6 ± 18.8ab 145.4 ± 17.3a 45.0 ± 10.8a 16.36 ± 3.7a 60.9 1.4 ± 0.33 1.50 ± 0.36 2.40

RC, basal diet (rat chow); PRESERVE, low-calorie guava preserve with 16% lactulose; DMH, 1,2-dimethylhydrazine (4 Â 40 mg kgÀ1 b.w, s.c.); EDTA, DMH vehicle. Those marked with different letters in the same column are signiﬁcantly different by Tukey test (P < 0.05). Values represent the mean ± SD. * Food calorie = 372.09 ± 2.01 kJ.100 gÀ1. ** LCGP calorie = 108.00 kJ.100 gÀ1.

Despite the differences in lactulose concentrations in the final the caloric value for the diet. We therefore provided what would products (Table 2), there was no significant difference (P > 0.05) correspond to 8% of the caloric value of the animals’ diets (3.6 kJ/ among its degradation percentages in the preserves rat/day or 1.0 g/rat/day). However, the sweetened food possessed (18.45 g 100 gÀ1 lactulose). Although reports exist that its nutri- low caloric value (108 kJ 100 gÀ1), and it permitted doses of 0.5, tious properties are not easily affected by food processing (Mcin- 1.0 and 1.5 g/rat/day, supplying just 0.54, 1.08 and 1.62 kJ/animal, tosh, 1996), the average degradation of 18.45 g 100 gÀ1 lactulose respectively, with a large amount of fibre. In contrast, many foods indicated that the production conditions of this product are, to that contain added sugars often supply calories but few or no some extent, unfavourable for the stability of the disaccharide. essential nutrients and no dietary fibre (USDA, 2010). Lactulose and polydextrose, classified as fibre because they are With regard to changes in body weight, the LCGP with lactulose not digested in the human small intestine (Juskiewicz and Zdunc- (G6) did not result in body mass gain (P < 0.01) compared to the zyk, 2010; Rastall, 2010), were not identified by enzymatic analysis negative control (G2) (Table 3). All the rats survived until the for the quantification of total dietary fibre (Zhang et al., 2010). end of the experiment, as was found in studies conducted by Dias Therefore, even if the formulations of LCGP with lactulose result et al. (2010) and Boyle et al. (2008), in which they evaluated the in lower fibre levels, we still may conclude that the addition of lac- effect of prebiotics on ACF incidence. tulose and polydextrose enriched the fibre content of the preserves Food and water consumption was significantly lower (P < 0.01) (Brazil, 1998). in the preserve-treated and initiated groups (G3, G4 and G5) com- The moisture levels of LCGP formulations increased in relation pared to the other groups (G1, G2 and G6) as shown in Table 3. to the conventional formulation (Table 2), as Khouryieh et al. All DMH-initiated animals developed classical ACF (Fig. 2) and (2005) determined on elaborating sugar-free jelly using xanthan mucin-negative ACF in their colons (Table 4), demonstrating the and locust gums. These combined gums possess a high water induction potential of the carcinogen during the period of the retention capacity (Lurueña-Martínez et al., 2004) during the elab- experiment. Therefore, the adopted study allowed sufficient time oration of fruit jellies and preserves. Furthermore, the sucrose to evaluate the effect of the preserves on the initiation phase of reduction in the formulations caused an increase in water activity the colon carcinogenesis process. Despite reports that some of (Table 2), even with the addition of polydextrose, which is a highly the components of the preserves such as lactulose, polydextrose hygroscopic polymeric agent that provides body and texture to and lycopene, which are naturally present in guava, have protec- foods. Such alterations are not desirable because they increase free tive effects on oxidative stress (Franco et al., 2008; Reuter et al., water levels infood, which is used for deteriorative reactions 2010) and colon carcinogenesis (Dias et al., 2010; Erhardt et al., (Menezes et al., 2009). 2003), the preserves had no significant (P > 0.05) inhibitory effect on ACF number in the colon or on crypt multiplicity. 3.2. Effect of the preserves in vivo We observed low multiplicity of the crypts in all experimental groups, which is typical of the initial step of carcinogenesis. At later For the in vivo analysis, LCGP was used with 16% w/w of lactu- times, the colon may contain a large number of crypts present in lose (doses: 0.5, 1 and 1.5 g/rat) because it showed good accep- each focus (crypt multiplicity) (Bolognani et al., 2001; Bird and tance. In addition, even with the degradation of the prebiotics Good, 2000), corresponding to an effect on the promotion step of during the processing of the preserve, they still remained above colon tumorigenesis (Lima et al., 2005; Zhang et al., 2004). the minimum levels considered functional and recommended by Regarding oxidative stress, there was no observable effect of li- legislation for guava preserves (Brazil, 2008). The effects of this pid peroxidation in plasma or the colon related to the administra- product were investigated in male Wistar rats with and without tion of 1.5 g preserve/rat/day for 28 days, which were equal to DMH-induced colon carcinogenesis, using biomarkers for aberrant those spontaneously generated. The mean values of these MDA À1 crypt and oxidative stress parameters. The amounts were based on analyses were 27.85 ± 9.65 nmol protein mg for the plasma and À1 the Dietary Guidelines for Americans 2010 (USDA, 2010), which 15.0 ± 7.71 nmol protein mg for the colon. However, the con- shows that the proportion of added sugars in foods contributes sumption of guava preserve potentiated (P 6 0.05) lipid peroxida- À1 an average of 16% w/w of the total calories of sweets in American tion (372.0 ± 90.9 nmol MDA protein mg ) and protein oxidation À1 diets, and from this total, 6% corresponded to the consumption of (6.8 ± 0.3 nmol protein mg ) when compared to the negative con- À1 candy. In this study, the average calorie ingestion profile of the ani- trol (217.1 ± 29.3 nmol MDA protein mg and 5.9 ± 0.0 nmol pro- À1 mals was 45 kJ/rat/day, and the guava preserves were the only tein carbonil protein mg ). This result may be related to the source of added sugar in the foods. The caloric value of the conven- preserve processing, creating a realistic food matrix, where the dif- tional preserve was 386.2 kJ 100 gÀ1 (Table 2); therefore, ferent bioactive compounds may interact with each other resulting consumption of 1.0 g/rat/day, or 3.6 kJ/rat/day, represented 8% of in a wide range of action possibilities (Charalampopoulos and C.C. Menezes et al. / Food and Chemical Toxicology 50 (2012) 3719–3724 3723

Fig. 2. (A) Topographic view of the area with non-altered colonic crypts; (B) one classical ACF-methylene blue stained with three aberrant crypts (arrow); (C) Topographic view of two mucin-positive ACF-toluidine blue stained with two and one aberrant crypts; (D) Topographic view of one mucin-negative ACF-toluidine blue stained with one aberrant crypt. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Table 4 Effects of low-calorie guava preserve with prebiotics on the development of classical aberrant crypt foci (ACF) and mucin-negative ACF induced by DMH in the rat colon.

Group/treatment N Number of aberrant crypts per ACF Total number AC/ACF Mucin-negative ACF 1 crypt 2 crypt 3 crypt >3 crypts AC ACF 1 crypt (% >1 crypt (% depletion) depletion) (G1) RC + DMH 10 27.1 ± 16.8a 10.6 ± 7.0a 3.1 ± 2.4a 0.9 ± 1.2a 61.4 ± 35.7a 41.7 ± 24.6a 1.5 ± 0.2a 1.0 ± 2.0a 0.4 ± 1.0a (G2) RC + EDTA 10 0b 0b 0b 0b 0b 0b 0b 0a 0a (G3) 0.5 g 10 14.3 ± 13.9a 4.1 ± 3.1a 0.7 ± 0.7a 0.1 ± 0.3a 23.4 ± 19.3a 19.7 ± 16.2a 1.2 ± 0.2a 1.2 ± 2.1a 0.6 ± 1.3a preserve + DMH (G4) 1.0 g 10 23.3 ± 17.0a 5.8 ± 3.4a 1.7 ± 1.7a 0.1 ± 0.3a 40.4 ± 26.4a 30.9 ± 20.6a 1.3 ± 0.2a 0.7 ± 1.6a 0a preserve + DMH (G5) 1.5 g 10 51.2 ± 41.0a 9.2 ± 7.2a 1.4 ± 1.5a 0.3 ± 0.7a 75.0 ± 54.0a 62.1 ± 46.30a 1.2 ± 0.2a 1.9 ± 1.1a 1.6 ± 2.8a preserve + DMH (G6) 1.5 g 10 0b 0b 0b 0b 0b 0b 0.0 ± 0.0b 0a 0a preserve + EDTA

N, number of animals; AC, aberrant crypt; ACF, aberrant crypt f°Ci. RC, basal diet (rat chow); PRESERVE, low-calorie guava preserve with 16% lactulose; DMH, 1,2-dimethylhydrazine (4 Â 40 mg kgÀ1 b.wt., s.c.); EDTA, DMH vehicle. a,b Those marked with different letters on the same column are signiﬁcantly different by Kruskal–Wallis test (P 6 0.05). Values represent the mean ± SD.

Rastall, 2012). These data reinforce the conclusion that significant Conflict of Interest consumption of these compounds alone or synergistically with other compounds in the diet may not have a protective function, The authors declare that there were no conflicts of interest. but instead have adverse effects (Bjelakovic et al., 2007). Acknowledgements 4. Conclusion The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for undergradu- The present study determined that it is possible to produce a ate research and doctoral scholarships, and the authors would also low-calorie preserve with 62.5% w/w sucrose reduction that con- like to thank the Fundação de Amparo à Pesquisa do Estado de tains the prebiotics polydextrose and lactulose and has a high fibre Minas Gerais (FAPEMIG/00849-08) for funding the overall project. content, while still maintaining good sensorial acceptance. We found that consumption of the low-calorie preserves with prebiotics does not have an effect on the development of mucin- References negative ACF and classical ACF in the initiation phase of the mutagenic process. However, the consumption of 1.5 g of the preserve/ Acosta, O., Víquez, F., Cuebero, E., 2008. Optimisation of low calorie mixed fruit jelly by response surface methodology. Food Qual. Preference 19, 79–85. rat/day potentiated lipid peroxidation and proteic oxidation in the ACS (American Cancer Society) 2008. Cancer facts and figures. http:// liver. www.cancer.org. (Accessed January 20, 2011). 3724 C.C. Menezes et al. / Food and Chemical Toxicology 50 (2012) 3719–3724

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