Food Chemistry 182 (2015) 268–274

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Effects of gluten-free breads, with varying functional supplements, on the biochemical parameters and antioxidant status of rat serum Michał S´wieca a,⇑, Julita Reguła b, Joanna Suliburska b, Urszula Złotek a, Urszula Gawlik-Dziki a a b

Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland ´ , Poland Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, Wojska Polskiego Str. 31, 60-624 Poznan

a r t i c l e

i n f o

Article history: Received 24 October 2014 Received in revised form 11 February 2015 Accepted 4 March 2015 Available online 11 March 2015 Keywords: Gluten free bread Antioxidant status in vivo Bread enrichment Bioavailability in vitro Phenolics

a b s t r a c t This paper examines the effects of gluten-free bread enriched with functional ingredients (milk powder, poppy, sunflower and pumpkin seeds, egg yolk, carum, hazel nuts and amaranth) on the morphological and biochemical parameters and antioxidant status of rats serum. Rats were provided test diets – glutenfree breads and water ad libitum. After 14 days, the animals were weighed and killed. A hazel nutamaranth bread diet significantly increased the level of thrombocytes when compared to control bread. A mixed bread diet significantly decreased cholesterol levels in rats. All fortified breads decreased triglyceride levels and alanine transaminase activity and caused an increase in antiradical activity of the serum. In rats fed with poppy-milk bread, milk-seed bread and mixed bread, a marked decrease in superoxide dismutase activity was found. Enriched breads reduced the levels of triglyceride and improved the antiradical properties of serum, although the physiological relevance of this needs to be confirmed by human studies. Ó 2015 Published by Elsevier Ltd.

1. Introduction Bread is one of the most popular staple foods in the world. As a result of its nutritive value, low price, and simplicity of usage, it has become the basis of all civilizations’ diets. Bread consumption provides energy (mainly from starch) and delivers dietary fiber, proteins and a wide range of vitamins and minerals (Nanditha & Prabhasankar, 2009). Bread is mainly produced from wheat and rye flours containing, inter alia, prolamins, and a protein fraction (determined as gluten) responsible for adequate structure and quality. However, there is a group of people for whom consumption of prolamins – gliadins of wheat, secalins of rye, hordeins of barley and avenins of oats – causes disorders, such as: coeliac disease, allergy, and non-celiac gluten sensitivity. In addition, their consumption leads to damage to the mucosa of the small intestine. It contributes to impaired absorption and, as a consequence, to malnutrition and the occurrence of many systemic complications (such as anemia, rickets, osteoporosis, deficit of body weight) (Hamer, 2005). A necessary precondition in the preparation of food for this group of consumers is to develop a product in such a way that the harmful factor is eliminated, while adequate value is preserved. A problem arises from the fact that, frequently, such an

⇑ Corresponding author. Tel.: +48 81 4623327; fax: +48 81 4623324. E-mail address: [email protected] (M. S´wieca). http://dx.doi.org/10.1016/j.foodchem.2015.03.014 0308-8146/Ó 2015 Published by Elsevier Ltd.

approach leads to a reduction in the nutritive value of the product. In the case of gluten-free foodstuffs, elimination of a raw material causing gluten intolerance in the final product (wheat, rye, barley or oats), leads to a considerable reduction of the levels of dietary fiber, vitamins B and minerals (magnesium, zinc, iron, copper). According to the needs of consumers, optional ingredients can be added to produce special and novel bread with enhanced nutritional and nutraceutical quality and organoleptic characteristics (Balestra, Cocci, Pinnavaia, & Romani, 2011; Gambus´, Gambus´, & Sabat, 2002; Gambus´ et al., 2009; Gawlik-Dziki et al., 2013; Mariotti, Lucisano, Ambrogina Pagani, & Ng, 2009; Nanditha & Prabhasankar, 2009; S´wieca, Gawlik-Dziki, Dziki, Baraniak, & Czyz, 2013). Also, in the case of gluten-free bread, enrichment is closely linked with the incorporation of functional ingredients that may influence life quality and wellness (Dwyer et al., 2014). The introduction of new gluten-free products to the market requires wide-ranging studies of the safety of diets and potential positive and/or negative effects of functional food components. The gluten-free bread in this study was supplemented with some commonly used ingredients that are characterized by well documented bioactivity in vitro. These are excellent sources of selected macro- and microelements (e.g., milk, poppy – Ca; carum – Fe, Cu; amaranth – Fe, Mg) (Gelderblom et al., 2013; Suliburska, Krejpcio, Reguła, & Grochowicz, 2013). Additionally, seeds and nuts used for bread enrichment are known to contain large amounts of

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bioactive components, such as phenolics, essential oils, unsaturated fatty acids, fiber, vitamins (Bozan & Temelli, 2008). Most of them exhibit prohealth properties, including antioxidant, anti-inflammatory, anticancer activities, that are usually positively correlated with phenolic levels (Liu, 2013). There have been many investigations on reducing LDL cholesterol and triglyceride concentrations and/or on improving antioxidant status in rats fed diets high in polyphenols (Hsu & Yen, 2007; Mildner-Szkudlarz & Bajerska, 2013). However, very little attention has been focussed on the cereal products which are consumed daily and which may be involved in the prevention of diet-related diseases. Therefore, the present study was designed to investigate the safety and potential use of selected functional supplements (in commonly found combinations) for fortification of gluten-free bread. The effect of these fortified breads on the blood morphological and biochemical parameters and antioxidant status of Wistar rats was assessed as the main objective of this study. 2. Materials and methods 2.1. Chemicals Ferrozine (PubChem CID:23662871), ABTS (PubChem CID:5815211 2), a-amylase, pancreatin, pepsin, bile extract, Folin– Ciocalteu reagent, ammoniumthiocyanate (PubChem CID:15666) and potassium hexacyanoferrate (III) were purchased from Sigma–Aldrich (Poznan, Poland). All other chemicals were of analytical grade. 2.2. Bread preparation Breads were produced from rice flour, potato flour and corn starch (Glutenex Company, Poland), rapeseed oil (Kruszwica, Poland), salt (Solino, Poland), fresh yeast (Saccharomyces cerevisiae) (Lesaffre, Poland), and saccharose (Pfeifer & Langen, Poland). To enrich the product, natural components commonly available on the market were used: milk powder (SM Mlekovita, Poland), poppy seeds (VOG, Poland), sunflower seeds (VOG, Poland), flax seeds (Bio Planet, Poland), pumpkin seeds (VOG, Poland), egg yolk (Ovovita, Poland), carum (KOTÁNYI, Poland), hazel nuts (VOG, Poland), amaranth (Bio Planet, Poland) and combinations (PMB-poppy-milk bread; CB-carum bread; HAB-hazel nuts-amaranth bread;

Table 1 Breads composition. Ingredients (%)

Rice flour Corn starch Potato flour Yeast Sacharose NaCl Rapeseed oil Milk powder Amaranth flour Flax Sunflower seeds Pumpkin seeds Hazel nuts Poppy Egg yolk Carum Water

Breads WB

PMB

CB

HAB

MSB

ECB

MB

20.7 10.4 20.7 2.6 3.1 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 41.5

19.7 9.9 14.3 2.5 3.0 0.5 0.5 3.0 0.0 0.0 0.0 0.0 0.0 7.4 0.0 0.0 39.4

17.2 8.6 10.8 2.2 2.6 0.4 0.4 0.0 2.6 2.2 2.2 2.2 1.7 0.0 0.4 4.3 42.2

17.7 8.8 11.1 2.2 2.7 0.4 0.4 0.0 2.7 2.2 2.2 2.2 2.7 2.2 0.0 0.0 42.5

17.2 8.6 10.8 2.2 2.6 0.4 0.4 2.6 2.6 2.2 1.7 2.2 1.7 2.6 0.0 0.0 42.2

17.4 8.7 10.9 2.2 2.6 0.4 0.4 0.0 2.6 2.2 2.2 2.2 2.2 0.0 0.9 2.6 42.6

16.6 8.3 10.4 2.1 2.5 0.4 0.4 2.5 2.5 2.1 2.1 2.1 2.1 4.1 0.4 0.0 41.5

WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nutsamaranth bread; MSB-milk-seeds bread; ECB – egg yolk-carum bread; MB – mixed bread.

269

MSB-milk-seeds bread; ECB-egg yolk-carum bread; MB-mixed bread) (Table 1). White bread was used as the control bread – WB without supplements. Breads were baked using two-phase dough fermentation with a starter at 180 °C for 45 min. After baking, breads were dried and homogenized in an electric grinder. 2.3. Animals and diets Studies were conducted for 14 days on 42 growing (around 6week-old) male rats of the albino Wistar strain with a mean body weight of 232.5 g ± 26.6. The experiment was performed with the agreement of the local bioethics committee (approval No. 888/11). Animals were kept in individual metabolic cages in a room with natural lighting, at a temperature of 19–22 °C and relative humidity of 55–60%. The food consumption was recorded daily and the animals were weighed once a week. In the adaptation period, the animals consumed a pelleted maintenance feed for laboratory animals: Labofeed B. After a 3-day adaptation period, the animals were fed test diets (breads – Table 1) and water ad libitum. At the completion of the feeding period (2 weeks) on the last day of the experiment, after 12 h of fasting, the animals were anesthetized with a sodium thiopental injection (40 mg/kg body weight) and killed by cardiac puncture. 2.4. Gastrointestinal digestion in vitro For preparation of a potentially bioavailable fraction of bread extracts, simulated digestion was performed (S´wieca, Baraniak, & Gawlik-Dziki, 2013). Simulated saliva solution was prepared by dissolving 2.38 g of Na2HPO4, 0.19 g of KH2PO4, 8 g of NaCl, and 100 mg of mucin in 1 L of distilled water. The solution was adjusted to pH = 6.75 and a-amylase (E.C. 3.2.1.1.) was added to obtain 200 U per ml of enzyme activity. For the gastric digestion, a solution of 300 U/ml of pepsin (from porcine stomach mucosa, pepsin A, (EC3.4.23.1) in 0.03 M NaCl, pH = 1.2, was prepared. Further, simulated intestinal juice was prepared by dissolving 0.05 g of pancreatin (activity equivalent 4  USP) and 0.3 g of bile extract in 35 ml of 0.1 M NaHCO3. The bread samples were subjected to simulated gastrointestinal digestion as follows: 1 g of powdered sample was homogenized in a stomacher laboratory blender for 1 min to simulate mastication with the presence of 15 ml of simulated salivary fluid, and, subsequently, the samples were shaken for 10 min at 37 °C. The samples were adjusted to pH = 1.2 using 5 M HCl, and, subsequently, 15 ml of simulated gastric fluid were added. The samples were shaken for 60 min at 37 °C. After digestion with the gastric fluid, the samples were adjusted to pH = 6 with 0.1 M of NaHCO3 and then 15 ml of a mixture of bile extract and pancreatin were added. The extracts were adjusted to pH = 7 with 1 M NaOH and finally 5 ml of 120 mM NaCl and 5 ml of mM KCl were added to each sample. The prepared samples were subjected to in vitro digestion for 120 min, at 37 °C in the darkness. After that, samples were centrifuged and supernatants were used for further analysis. 2.5. Analysis of blood morphological and biochemical parameters The blood was collected by cardiac puncture in tubes with heparin sodium to obtain whole blood for morphological tests and in serum-separated tubes for biochemical parameters. The coagulated blood was left to clot at room temperature for 30 min, and then it was centrifuged for 15 min at 3600g. The following morphological parameters were determined: WBC – white blood cells, MCV – mean corpuscular volume, MCH – mean corpuscular hemoglobin, MCHC – mean corpuscular hemoglobin concentration, LYM – lymphocytes; PDW – platelet distribution width, RDW – red blood cell distribution width, MPV – mean platelet volume, as well

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2.6. Total phenolics content Total phenols contents in bread were estimated according to the Folin–Ciocalteau method (Singleton, Orthofer, & LamuelaRaventós, 1998). The amount of total phenolics was expressed as gallic acid equivalents (GAE). 2.7. Antioxidant capacity 2.7.1. Free radicals-scavenging ability (ABTS) The abilities to quench free radicals by bread and rat serum were determined, using an improved ABTS decolorization assay (Re et al., 1999). The ability of the extracts to quench the ABTS free radical was determined by the following equation:

scavenging % ¼ ½ðAC  AA Þ=AC   100 where, AC = absorbance of control, AA = absorbance of sample. Antiradical activity was expressed as Trolox equivalent. 2.7.2. Ferric reducing power (FRAP) Reducing powers of bread and rat serum were determined by the method of Oyaizu (1986). Analyzed sample (2.5 ml) was mixed with phosphate buffer (2.5 ml, 200 mM, pH 6.6) and potassium hexacyanoferrate (III) [K3[Fe(CN6)] (2.5 ml, 1%). The mixture was incubated at 50 °C for 20 min. Reactions were stopped with 0.5 ml of 10% TCA and centrifuging for 10 min at 6500g. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and 0.5 ml of 0.1% FeCl3 and the absorbance was measured at 700 nm. Reducing power was expressed as Trolox equivalents. 2.7.3. Catalase activity assay (CAT) CAT activity was assayed by the method of Claiborne (1985) with some modification. The assay mixture consisted of 1.95 ml of phosphate buffer (0.05 M, pH 7.0), 1.0 ml H2O2 (0.019 M) and 0.05 ml of serum. The decomposition of H2O2 was determined directly by the extinction at 240 nm per unit time (3 min) and this was used as a measure of catalase activity. The catalase activity was expressed in U, where 1 U was defined as the amount of enzyme present that decomposed 1 lmol of H2O2 per min (method conditions). 2.7.4. Superoxide dismutase assay (SOD) SOD activity was determined using a kinetic mode (Zhou & Prognon, 2006). 2.7 ml of reagent mixture containing 0.07 mM NBT, 1.1 mM DETAPAC and 0.17 mM xanthine in 50 mM phosphate buffer (pH 7.8) was mixed with 100 ll of serum. The reaction was initiated by adding 100 ll of xanthine oxidase solution under agitation. One minute after addition of xanthine oxidase, the agitation was stopped and the absorbance change at 560 nm was monitored at 25 °C, against air for 5 min. The rate of change of absorbance variation DA560/min of an uninhibited assay (in absence of SOD) should be between 0.015 and 0.025; if not, the

XO concentration is adjusted. The SOD activity was expressed in U, where 1 U was defined as the amount of enzyme present that produced a change in absorbance of 0.001 per min (method conditions). 2.8. Statistical analysis Statistical tests were performed using Statistica 6.0 software (StatSoft, Inc., Tulsa, USA). All data were tested for normal distribution by use of the Shapiro–Wilk test. Normally distributed values were compared using ANOVA. Means of analyzed traits in the groups were compared using one-way analysis of variance, while intergroup differences were assessed by the post hoc Turkey test at the significance level P < 0.05. 3. Results Supplements used for bread fortification in this study are good sources of polyphenols; thus, the levels of potentially bioaccessible phenolics and their antioxidant activities were determined (Fig. 1). Fortification of bread significantly increased phenolic levels. The highest amounts of potentially bioaccessible phenolics were found for CB, HAB and MSB (increases of 89%, 95% and 96% with respect to WB). Most importantly, these changes were translated into antioxidant activity (R = 0.86). The antioxidant capacities were significantly higher than those determined for control bread (WB). The best results were obtained for ECB (an increase of 61% with respect to WB); however, for CB, MB and HAB the elevation also exceeded 50% (Fig. 1). To confirm the usefulness of the studied breads in the creation of the antioxidant status in vivo, these studies were performed in an animal model with Wistar rats (Table 2). Daily diet intakes were comparable between groups. The studied breads exhibited different influences on the body mass of rats. Despite the fact that changes were not significant for WB, CB and MSB, a reduction of mean body mass after the experiment was determined. By contrast, PMB, ECB and MB diets caused a slight increase in mean body mass after the experiment. Generally, the studied gluten-free breads did not significantly influence the morphological parameters of rat blood (Table 3). Significant changes were only found in the case of thrombocytes, where HAB bread increased their level by 13% with respect to the control (WB). Significantly lower glucose levels were observed

35

R2=0.86

BC

C

BC d

d

25

15

14 12

cd c

A 20

BC

B

B

30

c

b

10 8

a

6

10

4

5

2

Antiradical capcity [umol TE/ g bread]

as biochemical indices: glucose, albumins, triglycerides, ALT – alanine transaminase, AST – aspartate transaminase, total cholesterol, HDL – high density lipoprotein cholesterol. Values for the morphological indices were determined using a Sysmex K-1000 hematological analyzer (TAO Medical Electronics Co., Kobe, Japan) according to the standard procedures. The concentration of glucose in the blood serum was estimated by the glucose oxidase method (Carroll, Smith, & Babson, 1970). Total cholesterol and triglyceride levels in serum were measured using commercial kits (Randox Laboratory Ltd., UK). Albumin was measured by the immunoassay method, using a rat kit. The activities of liver enzymes, such as ALT and AST, were determined according to Dembin´ska-Kiec´ and Nastalski (2002).

Total phenolics [mg GAE/ g bread]

270

0

0 WB

PMB

CB

Total phenolics

HAB Breads

MSB

ECB

MB

Antiradical capacity

Fig. 1. Phenolics content and antiradical capacity of a potential fraction of studied breads. Means (n = 6, ±standard deviation) followed by different lower case letters are significantly different at P < 0.05. WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nut-amaranth bread; MSB – milk-seed bread; ECB – egg yolk-carum bread; MB – mixed bread; TE – Trolox equivalent; GAE – gallic acid equivalent.

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271

Table 2 Diet intake and mean body mass in rats. Breads

Mean body mass before experiment (g) Mean body mass after experiment (g) Daily diet intake (g/day/rat)

WB

PMB

CB

HAB

MSB

ECB

MB

231 ± 42 217 ± 39 12.4 ± 3.31

233 ± 34 234 ± 31 14.5 ± 3.62

236 ± 20 224 ± 21 11.9 ± 2.42

237 ± 18 237 ± 19 14.6 ± 3.51

225 ± 26 220 ± 24 12.1 ± 2.76

227 ± 29 229 ± 31 13.3 ± 2.80

236 ± 34 243 ± 25 14.7 ± 2.87

WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nuts-amaranth bread; MSB-milk-seeds bread; ECB – egg yolk-carum bread; MB – mixed bread. Means (n = 6, ±standard deviation).

Table 3 Effects of bread diets on the blood morphological parameters. Breads

WBC (103/ll) Erythrocytes (106/ll) Hemoglobin (g/dl) Hematocrit (%) MCV (fl) MCH (pg) MCHC (g/dl) Thrombocytes (103/ll) LYM (%) LYM (103/ll)

WB

PMB

CB

HAB

MSB

ECB

MB

3.12 ± 0.37 7.14 ± 0.27 14.3 ± 0.36 41.5 ± 1.15 56.8 ± 0.77 19.9 ± 0.59 34.9 ± 1.01 798 ± 63ab 91.3 ± 2.22 3.07 ± 0.23

2.96 ± 0.85 7.18 ± 0.11 14.1 ± 0.42 41.3 ± 1.33 57.0 ± 0.92 19.4 ± 0.53 33.7 ± 0.51 761 ± 47a 92.6 ± 2.34 3.20 ± 0.93

3.18 ± 0.53 7.28 ± 0.42 13.9 ± 0.47 40.9 ± 2.31 56.9 ± 2.57 19.6 ± 0.49 33.9 ± 1.08 826 ± 113ab 92.9 ± 1.85 3.45 ± 0.83

3.35 ± 0.42 7.37 ± 0.16 14.3 ± 0.31 42.9 ± 0.73 58.2 ± 1.34 19.2 ± 0.23 33.6 ± 0.88 901 ± 44b 94.1 ± 1.30 2.96 ± 0.50

3.60 ± 0.54 7.13 ± 0.11 13.9 ± 0.41 41.9 ± 1.41 57.6 ± 1.05 19.5 ± 0.49 33.6 ± 0.81 815 ± 76ab 86.2 ± 8.95 3.03 ± 0.60

2.84 ± 0.32 7.20 ± 0.17 13.8 ± 0.31 41.2 ± 1.32 57.2 ± 1.53 19.1 ± 0.52 33.4 ± 0.54 836 ± 75ab 92.8 ± 2.54 2.85 ± 0.62

3.66 ± 0.66 7.26 ± 0.30 13.8 ± 0.28 41.9 ± 0.65 56.7 ± 1.64 18.9 ± 0.50 33.2 ± 0.64 856 ± 67ab 93.0 ± 0.82 3.60 ± 0.49

Means (n = 6, ±standard deviation) followed by different small letters within columns are significantly different at P < 0.05. WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nuts-amaranth bread; MSB – milk-seeds bread; ECB – egg yolk-carum bread; MB – mixed bread. WBC – white blood cells; MCV – mean corpuscular volume; MCH – mean corpuscular hemoglobin; MCHC – mean corpuscular hemoglobin concentration; LYM – lymphocytes.

in rats fed with ECB than in those fed with CB (Table 4). There were no significant changes in albumin levels or AST activity, but it should be mentioned that breads containing the studied functional supplements decreased the activity of ALT (by 50% with respect to WB). The most dynamic changes were observed in the lipid profiles of animals. With respect to WB, in animals fed with CB, an increase in total cholesterol levels was observed (by 12%). On the other hand, the MB bread caused a significant reduction of the studied parameter (by 22%). Similar changes were observed in the HDL fraction of cholesterol. Most importantly, rats fed with enriched breads exhibited a significant reduction of triglycerides levels. The lowest levels were determined for HAB and MB (reduction of 70% in regard to WB). The antioxidant status of rats fed with enriched breads was determined, based on the reducing and antiradical abilities of serum, and changes in the activity of two enzymes involved in enzymatic antioxidant defence (Fig. 2). Incorporation of functional ingredients in control gluten-free breads significantly improved the antiradical capacity of rats. The best results were found for ECB, HAB, MSB and MB, where, with respect to WB, an increase of about 50% was determined. Considering the changes of reducing potential effected by diets, it should be noted that all studied

feeding variants caused an increase; however, statistically significant results were obtained only in the case of CB, PMB and MB (enhancement of the reducing abilities by 9%, 16% and 14%, respectively). Serum superoxide dismutase activity was significantly decreased in rats fed with PMB, MSB and MB, by 81%, 29% and 55%, respectively, in comparison with WB. Serum catalase activity was significantly elevated by ECB, PMB and MSB diets (2.6, 1.9- and 3.3-fold with respect to WB). Contrary to these results, in CB, HAB and MB diets, a significant reduction in the studied activity was observed.

4. Discussion With respect to standard bakery products (those based on wheat flour), gluten-free bread contains smaller amounts of minerals, vitamins and dietary fiber (Alvarez-Jubete, Arendt, & Gallagher, 2010). The gluten-free supplements used in these studies for bread fortification are known to be good sources of many nutrients (protein, lipids), macro- and microelements, vitamins and biologically active compounds, e.g., phenolics (Bozan & Temelli, 2008; Griel & Kris-Etherton, 2006). Although nuts and seeds are typically high

Table 4 Effects of bread diets on the serum biochemical parameters in rats. Diet

Glucose (mg/dl)

Albumins (g/dl)

Cholesterol (mg/dl)

HDL (mg/dl)

Triglycerides (mg/dl)

AST (U/l)

ALT (U/l)

WB PMB CB HAB MSB ECB MB

129 ± 9.29ab 143 ± 12.6b 148 ± 25.9ab 144 ± 22.1ab 125 ± 20.3ab 112 ± 7.7a 112 ± 9.9a

3.40 ± 0.13 3.57 ± 0.08 3.65 ± 0.33 3.52 ± 0.12 3.48 ± 0.12 3.48 ± 0.10 3.52 ± 0.08

87.0 ± 4.00bc 87.8 ± 3.95bc 97.8 ± 8.98c 76.6 ± 9.58ab 74.4 ± 9.29ab 89.4 ± 6.35bc 68.0 ± 7.71a

30.2 ± 1.93ab 31.4 ± 5.06ab 33.2 ± 2.78b 27.2 ± 4.26ab 27.3 ± 2.29a 30.6 ± 2.05ab 25.0 ± 2.48a

51.8 ± 6.85c 24.0 ± 4.53a 39.3 ± 5.62bc 16.6 ± 3.51a 31.4 ± 4.93b 20.0 ± 3.24a 15.4 ± 3.78a

66.5 ± 9.88 73.4 ± 22.40 72.4 ± 7.99 67.3 ± 20.90 68.6 ± 6.35 60.2 ± 6.02 64.0 ± 7.44

29.4 ± 6.07b 16.2 ± 1.79a 16.7 ± 5.01a 17.8 ± 2.99a 15.4 ± 3.97a 14.7 ± 2.16a 16.0 ± 1.22a

Means (n = 6, ±standard deviation) followed by different small letters within rows (interline) are significantly different at P < 0.05. WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nuts-amaranth bread; MSB – milk-seeds bread; ECB – egg yolk-carum bread; MB – mixed bread. ALT – alanine transaminase; AST – aspartate transaminase; HDL – high density lipoprotein cholesterol.

M. S´wieca et al. / Food Chemistry 182 (2015) 268–274

272

350 c

250

bc

c

c

18

b

cd

14

a

c

12

200 150 100

10 b 8 6 a

4

50

2 0

0 WB

CB

ECB

HAB

PMB

MSB

WB

MB

CB

40

b

b

ab

PMB

MSB

MB

e

80

35

D

e

90

b ab

ab

a

HAB

100

B

45

ECB

Diet enriched with breads

Diet enriched with breads

70 Catalase activity [kU/ dl]

Reducing potential [umolTE/dl]

C

de

e de

16

SOD activity [kU/ dl]

Antirdical activuty [umol TE/ dl]

300

20

A

c

30 25 20 15

60 d

50 40 c 30

10

b

20

5

a

a

10

0 WB

CB

ECB

HAB

PMB

MSB

MB

Diets enriched with breads

0 WB

CB

ECB

HAB

PMB

MSB

MB

Diets enriched with breads

Fig. 2. Effects of bread diets on the antioxidant status in rats: (A) radical-scavenging capacity of serum; (B) reducing potential of serum; (C) super anion dismutase activity; (D) catalase activity. Means (n = 6, ±standard deviation) followed by different lower case letters are significantly different at P < 0.05. WB – white bread; PMB – poppy-milk bread; CB – carum bread; HAB – hazel nut-amaranth bread; MSB – milk-seed bread; ECB – egg yolk-carum bread; MB – mixed bread; TE – Trolox equivalent.

in dietary fat, novel studies have shown that regular consumption of these ‘‘cardio-friendly’’ foods might confer a pro-health effect (Griel & Kris-Etherton, 2006). Food fortification is an effective tool for improving functional quality of gluten-free products; however, it should be kept in mind that the final effect is strongly determined by the bioaccessibility and bioavailability of bioactive compounds. Unfortunately, there is no simple relationship between the amount of supplement introduced and product activity, which may be limited by many factors, such as interactions between bioactive compounds and the food matrix, gut microbiota action, and compound metabolism (Neilson & Ferruzzi, 2013; S´wieca, Gawlik-Dziki et al., 2013; S´wieca, Seczyk, Gawlik-Dziki, & Dziki, 2014). This study provides evidence that fortified gluten-free breads reduce serum concentrations of triglycerides and increase antioxidant status in rats. Of course, the best ways to evaluate bioactivity are in vivo studies; however, some studies have also confirmed the usefulness of in vitro digestion in the evaluation of the potential bioavailability of functional ingredients (Ahn et al., 2002; Neilson & Ferruzzi, 2013). This study also demonstrated a relationship, in terms of antioxidant activities, between results obtained after in vitro digestion and those observed with in vivo models. So far, most studies concerning in vivo evaluation of the effectiveness of the pro-health effect of the addition of polyphenols to the diet have been based on pure compounds or phenolic-rich extracts (Chen, Wu, Liu, & Shen, 2014; Farrokhfall, Khoshbaten, Zahediasl, Mehrani, & Karbalaei, 2014). Food products have also been studied, e.g., purple potato fakes (Han et al., 2013) or fruit-based functional beverages (Gunathilake, Wang, & Rupasinghe, 2013); however, such reports are rare. In our study, the whole food system was studied, where

bioactive compounds were carried by a bread matrix. It was proved that incorporation in the diet of enriched breads did not cause any negative changes in rat blood but simultaneously increased antioxidant potential. Most importantly, enrichment of gluten-free breads allowed the undesirable effect of lowering antioxidant capacity during dietary restriction to be avoided; however, in this case it is difficult to elucidate if the effect comes from the bioactive components or the higher calorie diet (Cao, Prior, Cutler, & Yu, 1997). The utilization of nutrients present in amaranth flour and flaxseed, used as ingredients in gluten-free rolls, was examined by Gambus´ et al. (2009). Similar to our studies (except the CB diet), both of these ingredients decreased the total cholesterol and HDLcholesterol content of serum in both treated groups of animals. An increase in weight gain was observed only in animals fed with enriched rolls whereas, for the control, a decrease was observed. These results fully justify the supplementation of gluten-free products (often produced from starch and/or rice flour) as a tool for improving the nutritional value of gluten-free bakery products. All basic bread components (rice flour, potato and corn starches) are low in the polyphenolic compounds mainly responsible for antioxidant potential (Ti et al., 2014). The influence of the studied breads on the antioxidant status of rats was determined, based on the changes in both enzymatic (SOD and catalase activity) and non-enzymatic (reducing and antiradical potential) systems. All enriched breads increased the total antioxidant capacity of rat serum (measured as the ability to quench ABTS radicals). A similar effect was observed after a short-term cranberry juice supplementation of a human subject. Such a diet was associated with a significant increase in plasma antioxidant capacity and a reduction in circulating oxidized LDL concentrations (Ruel, Pomerleau,

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Couture, Lamarche, & Couillard, 2005). Also, pistachio nut consumption significantly decreased triglycerides and thiobarbituric acid-reactive substance levels and significantly increased total antioxidant activity in the hyperlipidemic group (Alturfan, Emekli-Alturfan, & Uslu, 2009). It should be emphasized that additives such as sunflower, flaxseed, poppy and pumpkin are great sources of lipophilic antioxidants and unsaturated fatty acids. An important role of these components in the creation of antioxidant capacity is also well documented (Griel & Kris-Etherton, 2006; Haggag, Elsanhoty, & Ramadan, 2014). The studied breads also modified the enzymatic defence of rats. The activities of serum SOD and catalase, working together in the neutralization of superoxide radicals, are strongly determined by oxidative stress levels (Griffiths et al., 2002). The serum SOD activities of rats fed with enriched breads were comparable or lower than those determined for control gluten-free bread. A decrease in SOD activity by PMB and MB, associated with significantly elevated antiradical and reducing abilities, may suggest that components of these diets significantly reduce oxidative stress levels in rats. Similar observations have been made after consumption of standard diets, including bread crust glycated compounds (well documented antioxidants) by adult rats (Pastoriza, Roncero-Ramos, Rufián-Henares, & Delgado-Andrade, 2014). Most importantly, diets rich in Fe and Cu (ECM and MSB), in spite of an increase in antioxidant capacity, also caused a significant elevation of catalase and SOD activities, which may suggest induction of oxidative stress by these transition metals (Musacco-Sebio et al., 2014). Despite the promising results, our study has some limitations. First of all, the duration of the experiment was rather short (14 days). A longer experiment and an assay of other parameters, not only in serum, but also in other tissues, would allow broader conclusions and would help to explain mechanisms. The results in this short-term experiment confirmed the usefulness and safety of functional ingredients for the production of bakery goods for persons following a gluten-free diet. Functional supplements in gluten-free breads reduced serum concentrations of triglycerides and increased antioxidant status, although the physiological relevance of our observations should be further examined in long-term studies on experimental animals and/or humans. Acknowledgement The study was partially supported by National Science Centre (NCN) Grant OPUS No. 2011/01/B/NZ9/00130. References Ahn, H. S., Jeon, T. I., Lee, J. Y., Hwang, S. G., Lim, Y., & Park, D. K. (2002). Antioxidative activity of persimmon and grape seed extract: In vitro and in vivo. Nutrition Research, 22(11), 1265–1273. Alturfan, A. A., Emekli-Alturfan, E., & Uslu, E. (2009). Consumption of pistachio nuts beneficially affected blood lipids and total antioxidant activity in rats fed a high-cholesterol diet. Folia Biologica, 55(4), 132–136. Alvarez-Jubete, L., Arendt, E. K., & Gallagher, E. (2010). Nutritive value of pseudocereals and their increasing use as functional gluten-free ingredients. Trends in Food Science & Technology, 21(2), 106–113. Balestra, F., Cocci, E., Pinnavaia, G., & Romani, S. (2011). Evaluation of antioxidant, rheological and sensorial properties of wheat flour dough and bread containing ginger powder. LWT – Food Science and Technology, 44(3), 700–705. Bozan, B., & Temelli, F. (2008). Chemical composition and oxidative stability of flax, safflower and poppy seed and seed oils. Bioresource Technology, 99(14), 6354–6359. Cao, G., Prior, R. L., Cutler, R. G., & Yu, B. P. (1997). Effect of dietary restriction on serum antioxidant capacity in rats. Archives of Gerontology and Geriatrics, 25(3), 245–253. Carroll, J. J., Smith, N., & Babson, A. L. (1970). A colorimetric serum glucose determination using hexokinase and glucose-6-phosphate dehydrogenase. Biochemical Medicine, 4(2), 171–180. Chen, Q., Wu, X., Liu, L., & Shen, J. (2014). Polyphenol-rich extracts from oiltea camellia prevent weight gain in obese mice fed a high-fat diet and slowed the

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