Quality Characteristics and Antioxidant Properties of Breads Supplemented with Sugar Beet Molasses-Based Ingredients

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1 International Journal of Food Properties ISSN: (Print) (Online) Journal homepage: Quality Characteristics and Antioxidant Properties of Breads Supplemented with Sugar Beet Molasses-Based Ingredients Bojana Filipčev, Ljubinko Lević, Marija Bodroža-Solarov, Nevena Mišljenović & Gordana Koprivica To cite this article: Bojana Filipčev, Ljubinko Lević, Marija Bodroža-Solarov, Nevena Mišljenović & Gordana Koprivica (2010) Quality Characteristics and Antioxidant Properties of Breads Supplemented with Sugar Beet Molasses-Based Ingredients, International Journal of Food Properties, 13:5, , DOI: / To link to this article: Copyright Taylor and Francis Group, LLC Published online: 06 May Submit your article to this journal Article views: 479 Citing articles: 13 View citing articles Full Terms & Conditions of access and use can be found at

2 International Journal of Food Properties, 13: , 2010 Copyright Taylor & Francis Group, LLC ISSN: print / online DOI: / QUALITY CHARACTERISTICS AND ANTIOXIDANT PROPERTIES OF BREADS SUPPLEMENTED WITH SUGAR BEET MOLASSES-BASED INGREDIENTS Bojana Filipčev 1, Ljubinko Lević 2, Marija Bodroža-Solarov 1, Nevena Mišljenović 2, and Gordana Koprivica 2 1 Institute for Food Technology, University of Novi Sad, Novi Sad, Serbia 2 Faculty of Technology, University of Novi Sad, Novi Sad, Serbia Osmotic dehydration in sugar beet molasses as hypertonic medium was used to treat apples, plums, carrots and cabbage. Following the treatment, the fruits/vegetables were dried and ground. The obtained powders or pure beet molasses were incorporated into white wheat bread at 5 and 10% levels (flour basis). The results showed that the mineral content (K, Mg, Ca) and antioxidant potential of breads were significantly improved. The most marked was the increase in K content: 89.1% (bread with molasses)-94.1% (bread with plum), at 5% level. The highest increase in antioxidant potential was measured in the breads made with plum ( % for 5 and 10% levels, respectively). Keywords: Sugar beet molasses, Bread, Quality, Minerals, Antioxidative potential. INTRODUCTION There are a growing number of ingredients which can be regarded as health promoting. The list includes more or less known components like vitamins, minerals, phytochemicals, and fibers. Ranhotra and Gelroth [1] gave a brief discussion on ingredients most commonly used in formulating functional foods that included carotenoids, fruits, vegetables, tocopherols, minerals (calcium, potassium, magnesium, and selenium), folic acid, soybean products, fibers and components, nuts, seeds, herbs, spices, flaxseed and fish oil. So far, a number of novel functional ingredients have entered the list, for example, green tea extracts, ω-3 fatty acids, rice bran, lycopene, red seaweed extracts, various sources of flavonoids, range of probiotics and prebiotics, propolis, honey, etc. These ingredients could be used in functional food formulations in the form of isolated and/or concentrated bioactive components. According to Šušić and Sinobad, [2] sugar beet molasses represents such concentrate of many bioactive compounds, claiming that its nutritional value and functionality is superior to that of honey. Sugar-beet molasses is thick concentrated liquid syrup, by-product from the processing of sugar beet into sugar. In general, beet molasses contains approximately 50% saccharose, 1% rafinose, and 0.25% glucose and fructose by dry weight (2,3). The nonsugar content includes many important micronutrients such as minerals and vitamins. Received 19 July 2008; accepted 7 April Address correspondence to Bojana Filipčev, Institute for Food Technology, University of Novi Sad, Bul. cara Lazara 1, Novi Sad, Serbia. bojana.filipcev@fins.ns.ac.yu 1035

3 1036 FILIPČEV ET AL. Potassium, calcium, sodium, magnesium and iron are present in appreciable amounts in beet molasses (Table 1). It is important to mention that the minerals in molasses are dissolved and thus readily available for uptake. [2] Molasses also contains B vitamins and does not contain fat, fiber or cholesterol. Minor constituents of beet molasses include proteins, betain, glutamine acid, purine and pirimidine bases, organic acids, pectin, and melanoidins. [2,3] The composition of molasses is determined by numerous factors such as the quality of raw material and the processing practices. In addition to the fact that molasses is a rich source of many important nutrients, there is some evidence to imply that molasses has significant antioxidative potential. It has been recently reported that extracts from cane molasses possess significant antioxidative activity coupled with interesting physiological functions that include anti-inflammatory, vaccine adjuvant and infection resistant features as well as protective effect against DNA oxidative damage. [5 7] Sugar-beet molasses also contains compounds that exert antioxidative activity such as phenolic compounds (8) although they have not been well documented. Taken together, these results suggest that cane and presumably, beet sugar molasses contain various compounds with beneficial effects for health. Therefore, it is not odd that sugar molasses has been highly regarded by many nutritionists who recommend its daily administration to protect health. [9] Unlike sugar cane molasses, sugar beet molasses is rarely used for human consumption mainly because of its unpleasant taste and odor, although there are parts of world where people are used to consume sugar beet molasses such as some parts of Central Europe and Turkey. In Germany, concentrated non-extracted syrup from sugar beet (Zuckerrübensirup) is commonly used as a sweetener. Since sugar beet molasses alone has not been convenient for consumption for most people because of its palatability, the general idea of this research work was to include beet molasses in staple food such as white wheat bread to improve its nutritive value and functionality. It is not rare that unconventional raw materials are being introduced to standard bread recipes in order to improve nutritive quality and functionality of the final product. Pre-germinated brown rice was successfully used to make bread with more functional properties. [10] Sharif and Butt [11] showed that replacing the wheat flour with defatted rice bran gave fiber and mineral enriched pan bread with excellent sensory attributes. Deoiled tomato seed meal can be used at 10% supplementation level to make bread with improved protein quality and good sensory characteristics. [12] Fruits and vegetables have also been proven to be value-added ingredients for similar purposes. [13] Moreover, there are attempts to process fruit and vegetable by-products in order to derive novel ingredients enriched with nutritional substances. For example, byproducts of cabbage are potential sources of commercial dietary fiber powders. [14] Hence, to increase the diversity of potentially health-enhancing ingredients in bread making, powders of apples, plums, carrots and cabbage, previously osmotically dehydrated in sugar beet molasses, were included in this investigation. In previous attempts, sugar beet molasses was successfully used as a hypertonic medium in the osmotic dehydration of apples, plums, carrots and cabbage. [15 17] METHODS AND MATERIALS Raw Materials Commercially available refined wheat flour (12.57 g/100 g water content, 0.49 g/100 g d.b. (dry basis), ash content, 10.2 g/100 g d.b. protein content, obtained from

4 Table 1 Typical micronutrient content of sugar beet molasses. Nutrient (mg/100 g) Reference K Na Ca Mg Fe Niacin Riboflavin Thiamin Folic acid Sugar beet molasses (83.5% solids) Sugar beet molasses Sugar beet molasses (73 79% solids) Sugar beet molasses (80% solids) Šušić & Sinobad (1989) (2) Curtin (1983) (4) NOVUS (1996) (3) Our study

5 1038 FILIPČEV ET AL. Fidelinka Mill Subotica, Serbia), refined granulated sugar (purchased from TE-TO Sugar factory, Senta, Serbia), fresh compressed yeast (70 g/100 g moisture content, obtained from Alltech Fermin Senta, Serbia), vegetable fat ( Puratos, Belgium), salt, skimmed milk powder ( Novosadska mlekara, Dairy factory, Novi Sad, Serbia), commercial improver ( Golden Tiger purchased from Puratos, Belgium) were the basic raw materials for bread preparation. Sugar beet molasses (80 g/100 g solids) was supplied from Jugozapadna Bačka Sugar factory, Member of the Sunoko Group, Bač, Serbia. Preparation of Osmotically Dehydrated (OD) Powders of Fruits/Vegetables Idared apples, Stanley plums, carrots, and red cabbage uniform in size and quality were obtained from the local market. Prior to use, the samples were cleaned and washed. Carrots were peeled while apples and plums were analyzed with their skin on. Apple and carrot samples were cut with a cork borer in a cylindrical shape of 20-mm diameter and 20-mm height. Samples of plums and cabbage were cut into cubes of approximately 1 cm. They were placed into molasses contained beakers and lightly weighted to keep them immersed. The ratio of sample and molasses was 1:4 by weight. The beakers were covered with a plate to reduce moisture loss and placed in thermostat at constant temperature 55 C. The samples were taken out from the osmotic medium after 5 h, rinsed and lightly blotted with tissue paper to remove excess molasses. After that, the treated samples were dried at 105 C for twelve hours. Preparation of Breads The bread dough formula was: flour (100 g), sugar (3.5 g/100 g flour), compressed yeast (3 g/100 g flour), fat (2.5 g/100 g flour), salt (1.5 g/100 g flour), skimmed milk powder (1.2 g/100 g flour), commercial improver (dosed as recommended by the manufacturer), water (optimum), and molasses or powders of osmotically dehydrated fruits and vegetables (tested at 5 g/100 g flour and 10 g/100 g flour levels). Percentages are based on flour weight. Dough mixing, processing and baking were performed on laboratory-scale equipment. A straight dough process was used. Doughs were mixed to optimum consistency in a high-speed Diosna mixer (Dierks & Söhne Maschinenfabrik, Osnabrück, Germany) with low speed of 85 r min 1 for 1 min and high speed of 120 r min 1 for 7 min. Final dough temperature was 30 C. Doughs rested in bulk for a period of 45 min, and then they were hand kneaded and left to rest for 15 min. Doughs were scaled into 400 g portions, manually rounded, rolled, and put into tin pans ( cm) to final fermentation for 60 min at 30 C and 80% relative humidity. Baking was done at 230 C in a deck type oven (Termotehnika, Zagreb, Croatia) until the initial dough mass was reduced by 8%. After cooling for 1 h, breads were bagged and held at room temperature until further testing. Bread Evaluation Bread quality attributes were evaluated 24 h after baking. Loaf weight and volume (rapeseed displacement method) were determined as well as sensory evaluation of the breadcrumb and crust. Sensory evaluation was carried out by seven trained panel assessors, who had a minimum one-year experience in bread quality evaluation and they were

6 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1039 members of the group being trained to function as a permanent descriptive analysis expert assessors for a variety of foods in accordance to ISO 1994 requirements. [18] The breads were analyzed by means of descriptive tests (ISO 1985). [19] Before the sensory evaluation, the panelists were oriented in 1.5 h training sessions. During orientation, the panelists precisely defined the descriptors and how to evaluate them to quantify attribute intensity. Descriptors and their corresponding definitions are shown in Table 2. The test was carried out in a sensory laboratory that was designed in accordance with ISO [20] The panelists received encoded samples and questionnaires, as well as instructions for the evaluation of the samples in random order. Testing included three repetitions. Each bread sample was evaluated based on the following characteristics: specific volume, crumb attributes (elasticity, crumb grain structure, color), crust color, and flavor. Instrumental Textural Analysis Bread instrumental textural attributes were measured with TA.XT2 Texture Analyzer (Stable Micro Systems, Surrey, UK), using a 36 mm flat-end compression disc (probe P/36R). Bread firmness and resilience were measured according to a modified 74-10A AACC method. [21] The firmness value is the peak force of the first compression of the product. Resilience is measured as the area during the withdrawal of the first compression, divided by the area of the first compression. It describes how well a product regains its original position. It could be referred as instant springiness. Instrument settings were as follows, mode: measure force in compression, pre-test speed: 1.0 mm sec 1 ; test-speed: 1.7 mm sec 1 ; post-test speed: 1.7 mm sec 1 ; strain: 40%; trigger force: 5 g. The samples were tested one day after baking. Total sample thickness was 25 mm. The first three slices from either end were excluded from testing. Triplicate measurements for each of the two loaves were made. Table 2 Bread texture profile descriptors. Attribute Color (crust) Color intensity Color (crumb) Color intensity Texture (crumb) Elasticity (palpatory) Crumb grain structure (visually and palpatory) Flavor (oral, nasal) Description Perceiving the intensity of crust color Scale: 1 extremely pale or burned9 deepgoldenbrown Perceiving the intensity of crumb color Scale: 1 white, creamy 9 light brown Evaluation of crumb spring back after applying force produced by lightly pressing the crumb center with thumbs of both hands Scale: 1 extremely firm (very unacceptable) 9 extremely soft (very acceptable, excellent) Feeling perceived by combining the sense of touch and sight, by lightly rubbing the fingers against the cut surface and observing the thickness of pore walls Scale: 1 coarse, thick-walled grains (non-acceptable) 9 wooly, very thin-walled grains (very acceptable) The acceptance of flavor impression as a combination of odor, taste and their overall roundness Scale: 1 - dislike extremely 9 like extremely

7 1040 FILIPČEV ET AL. Color Measurements Crust and crumb color were measured using a tristimulus photocolorimeter MOM- Color 100 (Magyar Optikai Művek, Budapest, Hungary). The CIE L a b values were recorded. The L,a and b values of a white standard tile used as reference were 92.25, 1.34, and 0.69, respectively. The results represent the mean of three samples. Chemical Analyses Protein (Official Method No ), fat (Official Method No ), crude fiber content (Official Method No ), reducing sugar (Official Method No ), ash (Official Method No ) and water contents (Official Method No ) were determined by standard methods of analysis. [22] Starch content was determined by hydrochloric acid dissolution according to the ICC Standard (Method No. 123/1). [23] In the calculation of the starch content of bread, the specific optical rotation for wheat starch was used (182.7 grd ml/g dm 1 ). [24] Mineral content of breads was determined following the standard methods described by AOAC. [22] The previously wet acid-digested samples were analyzed depending on the type of elements. Mg was analyzed by atomic absorption spectrophotometer (Varian Spectra A-10, Australia) with the use of background corrector (deuterium lamp). Na and K contents were measured by Flame Atomic Emission Spectrometry on the same spectrophotometer whereas Ca was analyzed by flame photometer (Jenway-PFP7, UK). Antioxidant Activity Antioxidant activity was determined according to a method described in detail by Miller, Rigelhof, Marquart, Prakash & Kanter. [25] In brief, finely ground bread samples were weighed (30 60 mg depending on sample absorption) and added to 50 ml of 101 µmol DPPH in 50% aqueous methanol. The mixture was allowed to react in dark at 38 C with constant stirring. After four hours, the mixture was filtered and the absorption at 515 nm was recorded. Using standard curves for the reaction of Trolox with DPPH, the data was then converted to activity in terms of µmol Trolox per 100 g sample (TE). Statistical Analysis All determinations were performed in triplicate unless other vice stated. The statistical analyses were conducted using two-way ANOVA procedures. Differences in samples due to the addition of sugar beet molasses based ingredients were tested for statistical significance at p = 0.05 level. Tukey s Honestly Significant Difference was used to differentiate between the mean values. Analyses were done with Statistica 7.1 statistical software (StatSoft Inc., Tulsa, Oklahoma). RESULTS AND DISCUSSION Chemical Compositions of Powdered OD Fruits and Vegetables The chemical composition of powdered OD apples, plums, carrots, and red cabbage are reported in Table 3. The moisture content of powders was similar except for the powder made from osmotically dehydrated plum that was higher. The ash content of powders made

8 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1041 Table 3 Composition of powdered OD apples, plums, carrots, and red cabbage. Composition of powdered OD fruits/vegetables Parameter (g/100 g d.m.) Apples Plums Carrots Red cabbage Moisture content 2.91 ± 0.07 a 3.14 ± 0.04 b 2.80 ± 0.05 a 2.92 ± 0.05 a Ash content 4.09 ± 0.08 a 3.97 ± 0.06 a 6.74 ± 0.07 b 7.26 ± 0.05 c Crude fibre content 3.73 ± 0.05 b 2.05 ± 0.02 a 4.82 ± 0.04 c 6.00 ± 0.05 d Protein content 8.44 ± 0.08 a 8.26 ± 0.10 a ± 0.15 b ± 0.11 b Reducing sugar content ± 0.61 d ± 0.42 a ± 0.50 c ± 0.40 b Saccharose content ± 0.5 c ± 0.45 a ± 0.40 d ± 0.48 b K content 1.78 ± 0.04 a 2.43 ± 0.07 b 3.28 ± 0.10 c 3.82 ± 0.06 c Na content 0.21 ± a 0.30 ± b 0.65 ± c 0.92 ± d Mg content 0.02 ± 0.00 a 0.03 ± 0.00 b 0.06 ± 0.00 c 0.13 ± 0.01 d Ca content 0.10 ± a 0.16 ± b 0.35 ± c 0.41 ± d a,b,c,d Different superscripts in the same raw indicate that means were significantly different (p<0.05). from OD apples and plums was similar whereas the OD carrot and cabbage powders were significantly higher in this parameter. The OD fruit powders contained significantly less proteins as compared to the OD vegetable powders. There were significant differences in the crude fibre content, reducing sugar content and saccharose content among the powders. Significant differences were observed in the mineral content of powders, too. The OD powders of vegetables were richer in K, Na, Mg, and Ca content than the OD fruit powders. The variations in the composition of powders may be attributed to the difference in the composition of initial raw materials and the differences in tissue structure that affected the diffusion of sugars and minerals from molasses. The Effect of Molasses based Ingredients on Chemical Composition of Breads The proximate chemical compositions of specialty breads prepared with the addition of sugar beet molasses and fruits and vegetables osmotically dehydrated in sugar beet molasses at various doses (5 g and 10 g/100 g flour) were determined and are shown in Table 4. The total moisture content and crumb moisture of samples were affected by the addition of OD fruits/vegetables or molasses at the applied doses. The bread made with OD plum had highest total and crumb moisture content, which is in agreement with the moisture contents of OD powders. Significant differences in the total moisture contents were observed between the control, 5 g and 10 g/100 g flour molasses bread and 10% OD plum and cabbage bread. For the crumb moisture content, significant differences were noted between the control sample and the breads made with OD plums at both doses. The wheat bread had g/100 g d.b. (dry basis) protein. Supplementing the breads with OD apples and plums did not significantly increase the protein content as compared to the control. This could be explained by the fact that these fruits are not protein sources. However, the addition of molasses and powdered OD red cabbage and carrot significantly increased the protein content in the bread. Unlike apples and plums, red cabbage and carrot contain proteins. Since they had been added in powdered form, their contribution to the increased protein content in the breads was significant.

9 Table 4 Proximate chemical compositions of specialty breads. Specialty breads enriched with Powdered osmotically dehydrated (OD) fruits and vegetables in sugar beet molasses Parameter (g/100 g d.m.) Control bread Pure sugar beet molasses Apple Plum Carrot Red cabbage Supplementation level, g/100 g flour Total moisture content Crumb moisture content ± 0.51 a ± 0.67 a ± 0.92 a ± 0.34 a,b ± 0.62 a,b ± 0.77 a,b ± 0.42 b ± 0.64 a,b ± 0.62 a,b ± 0.61 a,b ± 0.66 b ± 1.57 a ± 0.67 a,b ± 0.19 a ± 0.64 a,b ± 1.06 a,b ± 0.74 b ± 0.37 b ± 0.54 a,b ± 0.89 a,b ± 0.23 a,b ± 1.10 a,b Ash content 1.41 ± 0.18 a 1.92 ± 0.14 b,c,d 2.04 ± 0.14 b,c,d 1.62 ± 0.19 a,b 1.79 ± 0.12 a,b,c 1.65 ± 0.11 a,b 1.92 ± 0.15 b,c,d 1.94 ± 0.15 b,c,d 2.18 ± 0.14 c,d 2.00 ± 0.13 b,c 2.32 ± 0.13 d Protein content ± 0.07 a,b ± 0.14 c,d ± 0.14 c,d ± 0.12 b,c ± 0.17 a ± 0.25 a ± 0.23 a ± 0.27 c,d ± 0.12 c,d ± 0.26 c,d ± 0.26 d Crude fibre content 1.00 ± 0.05 a 0.99 ± 0.02 a 1.01 ± 0.01 a,b 1.10 ± 0.05 a,b,c 1.18 ± 0.03 c,d 1.02 ± 0.04 a,b 1.05 ± 0.01 a,b 1.12 ± 0.04 b,c 1.24 ± 0.03 d 1.18 ± 0.01 c,d 1.36 ± 0.02 d Fat content 3.44 ± 0.18 a 3.34 ± 0.11 a 3.22 ± 0.01 a 3.40 ± 0.14 a 3.34 ± 0.09 a 3.41 ± 0.06 a 3.19 ± 0.09 a 3.41 ± 0.08 a 3.30 ± 0.04 a 3.44 ± 0.04 a 3.28 ± 0.11 a Starch content ± 0.78 d ± 1.45 a,b,c ± 1.62 a,b ± 1.62 c,d ± 1.54 a ± 1.59 a,b,c ± 1.47 a ± 1.16 b,c,d ± 1.49 a,b,c ± 1.52 a,b,c ± 1.53 a,b Reducing sugar 8.63 ± 0.17 a,b 9.02 ± 0.25 b ± 0.60 c,f 9.13 ± 0.55 b,c ± 0.25 f ± 0.22 c,d ± 0.08 c 8.34 ± 0.44 a,b ± 0.19 f 7.88 ± 0.41 a ± 0.66 d,e content a,b,c,d,f Different superscripts in the same raw indicate that means were significantly different (p < 0.05). 1042

10 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1043 The crude fiber content of control wheat bread was 1.00 g/100 g d.b. The addition of pure molasses and OD plums at both levels and lower level of OD apples had no effect on the crude fiber contents in the bread while the addition of 10 g/100 g flour OD apples and OD carrots and red cabbage at both levels significantly increased the fiber content in the bread. These variations are due to the composition of used ingredients: carrot and red cabbage are richer sources of crude fibers as compared to apples and plums. The ash content of the control wheat bread was 1.41 g/100 g d.b., which increased gradually among the breads supplemented with molasses and OD fruits/vegetables. The highest ash content was recorded in the breads that contained molasses, OD carrot, red cabbage, and plum at 10% supplementation level. The increase in the ash content is related to the high content of minerals in molasses as well as in OD fruits/vegetables. The fat content of breads did not significantly vary. The control bread exhibited the highest starch content g/100 g d.b. that gradually and significantly decreased among the supplemented breads as the supplementation levels rose, which is a consequence of starch dilution. The reducing sugar content of control bread was 8.63 g/100 g d.b. The breads containing higher supplementation level (10 g/100 g flour) of pure molasses and OD fruits/vegetables manifested significant increase in reducing sugar content. The Effect of Molasses-based Ingredients on Mineral Content of Breads Control bread had mg/100 g d.b. of potassium, mg/100 g d.b. of sodium, 17.9 mg/100 g d.b. of magnesium and mg/100 g d.b. calcium (Fig. 1). The addition of molasses and OD powders significantly affected the mineral content of specialty breads as compared to the control. The increase in the potassium content was significant: for the lower supplementation level this increase ranged from %, for the bread made with5g/100 g flour OD carrot powder and the bread made with 5 g/100 g flour OD plum powder, respectively, whereas the increase in the potassium content was more marked in the breads with 10 g/100 g flour supplementation level ranging from % for the bread made with 10 g/100 g flour OD apple powder and the bread made with 10 g/100 g flour molasses, respectively. The sodium content of specialty breads was not significantly different from the control. The bread samples made with OD cabbage powder and carrot powder were higher in the sodium content as compared to the other samples but the difference was significant only between the samples of bread made with OD cabbage and the samples made with 10 g/100 g flour OD apple and plum. The addition of molasses and OD powders significantly raised the magnesium content of breads. The increase in Mg content was lower for the breads made with OD fruit powders (by %). More pronounced increase for the Mg content was found in the breads made with OD vegetable powders and pure molasses (by %). The highest magnesium content was observed in the case of bread made with 10 g/100 g flour OD cabbage (28.3 mg/100 g d.b.) and 10 g/100 g flour molasses (25.1 mg/100 g d.b.). The calcium content significantly increased in the supplemented breads. It increased by % for the breads made with OD fruit powders. Higher increase in the Ca content was measured in the breads made with OD vegetable powders (by %) while the addition of pure molasses increased calcium by 23 49% for 5 and 10 g/100 g flour levels, respectively. The supplementation of breads with molasses and OD fruit and vegetable powders significantly improved the mineral content of wheat bread.

11 OD Cabbage 10% OD Cabbage 5% OD Carrot 10% OD Carrot 5% OD Plum 10% OD Plum 5% OD Apple 10% OD Apple 5% Molasses 10% Molasses 5% Control f,g c,d,e f c f e c,d b g d,e a OD Cabbage 10% OD Cabbage 5% OD Carrot 10% OD Carrot 5% OD Plum 10% OD Plum 5% OD Apple 10% OD Apple 5% Molasses 10% Molasses 5% Control d c,d b,c,d a,b,c,d a a,b,c a,b a,b,c a,b,c a,b,c a,b,c,d K content, mg/100 g d.b Na content, mg/100 g d.b. OD Cabbage 10% OD Cabbage 5% OD Carrot 10% OD Carrot 5% OD Plum 10% OD Plum 5% OD Apple 10% OD Apple 5% Molasses 10% Molasses 5% Control 28.3 g 23.3 e 22.7 e 20.5 c 20.7 c 19.0 b 18.9 b 18.5 a,b 25.1 f 21.6 d 17.9 a OD Cabbage 10% OD Cabbage 5% OD Carrot 10% OD Carrot 5% OD Plum 10% OD Plum 5% OD Apple 10% OD Apple 5% Molasses 10% Molasses 5% Control 68.3 j 51.6 h 63.7 i 49.2 f 46.1 e 40.1 c 40.5 c 37.2 b g 41.7 d 33.9 a Mg content, mg/100 g d.b Ca content, mg/100 g d.b. a, b, c, d, e, f Bars designated with different letters are significantly different (p<0.05). 5% and 10% - supplementation levels at 5 g/100 g flour and 10 g/100 g flour, respectively. Figure 1 Mineral content (K, Na, Mg, Ca) of breads supplemented with molasses and OD fruits/vegetables. 1044

12 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1045 The Effect of Molasses based Ingredients on Antioxidant Potential of Specialty Breads There are many methods currently in use for measuring the antioxidant activity in food. Mainly, indirect methods are used that measure the antioxidative activity of food sample, per se, i.e., the antioxidative potential of food. [26] However, the real ability of food sample for antioxidative action after ingestion and digestion is still not fully understood although there are some results to confirm that moderate amounts of natural antioxidants incorporated into a main food may influence some parameters of the human immune system. Recently, Seidel et al. [27] provided some evidence to suggest that prebiotic breads supplemented with antioxidants (green tea powder, herbs and tomato paste) enhanced the levels of carotenoids and antioxidative capacity in the blood plasma. In addition, there is a consistent body of data, based mainly on epidemiological studies, which suggests that a regularly high intake of plant-derived food is linked to reduced incidence of cancer, coronary heart disease, obesity, hypertension and other chronic diseases. Fruits and vegetables have been widely recognized to be rich sources of antioxidants. Therefore, there is hardly any doubt that any component of the diet that is capable of acting as an antioxidant could be expected to offer protection against the damaging effects of free radicals. [28] In this study, the breads made with various molasses-based ingredients were tested for the antioxidant content using DPPH, a stable radical, as the detection agent. The observed activities are shown in Fig. 2. The addition of molasses and molasses-based ingredients significantly increased the antioxidative activities in the breads depending on the type of ingredients used and the supplementation level. The control bread showed antioxidative activity at µmol TE/100 g. Miller et al. (25) reported the antioxidant activity level of white bread at 1200 µmol TE/100 g whereas the wholegrain bread had 2000 µmol TE/100 g indicating the contribution of bran and germ. Other authors reported that the free-radical scavenging activity of white wheat bread mostly ranged from 5 to OD Cabbage 10% OD Cabbage 5% OD Carrot 10% OD Carrot 5% OD Plum 10% OD Plum 5% OD Apple 10% OD Apple 5% Molasses 10% Molasses 5% Control 923.3a e c b b f d,e d c c b micromol TE/100 g a, b, c, d, e, f Bars designated with different letters are significantly different (p<0.05). 5% and 10% - supplementation level at 5 g/100 g flour and 10 g/100 g flour, respectively. Figure 2 Antioxidative activity of breads supplemented with molasses and OD fruits/vegetables.

13 1046 FILIPČEV ET AL. 15% whereas different types of enriched bread showed scavenging activities that reached even 40% in some cases. [29,30] The antioxidant activity of white bread is usually attributed to the presence of Maillard reaction products that are known to posses free-radical scavenging activities. [29,30,31] The highest increase in the antioxidative activity (approximately 82%) was observed in the bread formulated with 10 g/100 g flour OD plum powder. High antioxidative activities were also noted for breads made with 5 g/100 g flour OD plums and 10 g/100 g flour OD cabbage and apple that were increased by approximately 60 67% as compared to the control bread. The lowest increase in the antioxidative activity was found in the breads made with carrot at both supplementation levels as well as with 5 g/100 g flour molasses. The antioxidative activity of bread made with 10 g/100 g flour molasses increased by 43% when compared to the control. It can be assumed that the type of fruits/vegetables used strongly affected the antioxidative properties of breads. Plums and red cabbage are being considered as the richest sources of bioactive compounds that are ranked amongst the highest in antioxidant potencies while carrots are relatively low in antioxidants. [25,32] The apple phytochemicals and their health benefits have been most extensively studied. Apples have been found to have very strong antioxidant activity that is linked to lowering the risks of many chronic diseases such as cardiovascular diseases and cancer by many epidemiological studies. [33] Plums are also well known sources of flavonoids and phenolic acids, [34] whichhavestrong antioxidative capabilities. [35] Red cabbage contains anthocyanins that have a range of biological activities with beneficial effects: examples range from inhibition of DNA damage in cancer cells in vitro, [36] reduction of inflammatory processes [37] to protection against agerelated decline in brain function. [38] As reported in the study of Li, Pickard and Beta, [39] although anthocyanins seemed to have degraded during baking, the final product (muffins enriched with brans of purple wheat) remained high in DPPH scavenging activity. It has been confirmed in various studies that sugar molasses contains antioxidative compounds originated from the beet or cane plant (such as phenolic compounds) or generated during the production process (melanoidins, sugar anhydrides, etc.). [2,4,8,40] Guimarães et al. [7] reported that extracts of cane molasses exhibited high overall antioxidative activity showing, in addition, no pro-oxidant activity. Molasses extracts with high antioxidative activities showed strong protective effect against induced DNA damage. The Effect of Molasses-based Ingredients on Physical, Textural, and Sensory Properties of Breads Loaf volume is regarded as the most important bread quality attribute especially from the consumers standpoint. The effect of molasses and powders of OD fruits/vegetables on the specific volume of breads is shown in Fig. 3. There was a significant decrease in the specific volume of supplemented breads as compared to the control except for the sample enriched with 5 g/100 g flour OD apple powder. It was found that the type of ingredient added to bread did not significantly affect the specific volume but the supplementation level did. Color is also considered an important quality trait in bakery products as it significantly contributes to consumer s preference. The color parameters for specialty breads are reported in Fig. 4. The crust of breads made with the addition of powdered OD fruits and vegetables or molasses was darker in color (as indicated by their lower L values). These differences were even more pronounced for breadcrumb. The darkest crust color was observed in the breads made with 10 g/100 g flour molasses, OD plum, apple and

14 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS a b a,b b b b Specific volume, ml/g c c c c c 1 0 Control Molasses 5% Molasses 10% Apple 5% Apple 10% Plum 5% Plum 10% Carrot 5% Carrot 10% Red cabbage 5% Bread types a, b, c Bars designated with different letters are significantly different (p<0.05). 5% and 10% - supplementation levels at 5 g/100 g flour and 10 g/100 g flour, respectively. Figure 3 Specific volume of specialty breads. Red cabbage 10% cabbage powders. The color of bakery products is formed in the reactions of caramelization and Maillard reactions. The presence of molasses contributes to the formation of dark color because it, besides containing significant amounts of sugars available for Maillard reactions, already contains the color products. But, it is interesting to note that the breads with molasses were the lightest in crumb color among the supplemented breads which imply to the fact that color formation depends not only on the type of ingredient but on the temperature regime as well. The a values, which are indicative of the red color in breads, had a declining trend in the supplemented breads. As compared to the control, the crust and crumb of the breads had very little red color. This drop in a value was less marked in the crust of the breads made with OD red cabbage and in the crumb of bread made with 10 g/100 g flour OD carrot and could be due to the presence of pigments responsible for the red color as anthocyanins and carotenoids. However, the a values measured in the crumb do not support this assumption. The contribution of anthocyanins to the red color in bread seems to be complex in nature because it depends on the pigment s heat stability, ph, concentration, and on the temperature regime. Li et al. [39] reported that no anthocyanins were detected in purple wheat bran contained muffins. Purple wheat bran used in the study contained 1.15 mg/g of anthocyanin. Red cabbage was reported to have similar content of anthocyanins, 1.37 mg/g. [41] On the other hand, carotenoids are known to be relatively heat stable. The positive b values, which indicate yellow color, increased in the crumb of supplemented breads when compared to the control. The most marked increase in b value was observed in the bread made with 5 g/100 g flour OD carrot (29.65) presumably because of the presence of carotenoids, which are usually associated with yellow and orange shades of many fruits and vegetables. Also, an increase in yellow color was characteristic for the crust of breads supplemented with 5 g/100 g flour of OD powders and

15 Color CIELab parameters for bread crust 0 Control Molasses 5% Molasses 10% OD Apple 5% OD Apple 10% OD Plum 5% OD Plum 10% OD Carrot 5% OD Carrot 10% OD Cabbage 5% OD Cabbage 10% Control Molasses 5% Molasses 10% OD Apple 5% OD Apple 10% OD Plum 5% OD Plum 10% OD Carrot 5% OD Carrot 10% OD Cabbage 5% OD Cabbage 10% Bread type DE*ab Color CIELab parameters for bread crust a* b* L* Bread type a* b* L* Crumb Crust Control Molasses 5% Molasses 10% OD Apple 5% OD Apple 10% OD Plum 5% OD Plum 10% OD Carrot 5% OD Carrot 10% OD Cabbage 5% OD Cabbage 10% Figure 4 Color parameters (CIELab system) of specialty breads. 5% and 10% - supplementation levels at 5 g/100 g flour and 10 g/100 g flour, respectively. 1048

16 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1049 molasses but not for higher supplementation level. It is noteworthy that both crumb and crust of breads with higher supplementation level of OD powders and molasses showed adropinb values as compared to the crumb of breads made with 5 g/100 g flour of OD powders. Similar observations were made with regards to a values in almost 50% of samples. In general, supplementation of breads contributes to an increase in dark tones in both crumb and crust. The colors were well expressed (as indicated by high CIELab color difference E ab; Fig. 4). Increasing the supplementation level of added ingredients leads to an increase in bread firmness (Table 5). There is a significant difference in the bread firmness between the group of lower supplemented breads and higher supplemented breads. The fruit/vegetable type also significantly influences the bread firmness but no pattern was observed. A strong positive correlation was found between the reducing sugar content in the bread and firmness. Bread resilience (instant elasticity) has also been affected by the addition of ingredients (Table 5). The most marked decreasing trend was observed when higher doses (10 g/100 g flour) of molasses and powdered OD carrot were added to bread. The sensory evaluations of the breads are shown in Table 6. When crumb was scored for elasticity, a combined effect of how well the crumb deforms under pressure and how well it retains its position was evaluated. The elasticity scores correlated well with the firmness showing that higher supplementation levels decreased the crumb elasticity. The lowest crumb elasticity was observed in the samples with 10 g/100 g flour molasses and OD apples. Similar effect was observed with the crumb quality. The finest crumb pores were observed in the control sample, 5 g/100 g flour powdered OD apple, plum and carrot bread whereas the increase in supplementation level led to gradual but significant increase in crumb pore roughness. Either the type or the level of ingredients significantly affected the crust color. The samples containing higher supplementation levels scored higher, i.e., the samples had a more intensive color due to the presence of sugars and proteins that form color compounds. It was also observed that the samples made with OD carrot produced lighter crust color at both supplementation levels whereas other ingredients formed significantly darker color at 10 g/100 g flour level. Table 5 Textural characteristics of specialty breads. Bread type Supplementation level, g/100 g flour Firmness (Force, g) Resilience (%) Control ± a ± 1.67 c,d,e Supplemented with pure ± b,c ± b,c,d molasses ± e ± 2.68 a Supplemented with powdered Apple ± a,b ± 0.96 c,d,e OD fruits and vegetables in ± e ± 0.96 b,c sugar beet molasses Plum ± b,c ± 0.52 b,c ± d ± 0.48 b,c,d,e Carrot ± a,b,c ± 1.24 c,d,e ± d ± 0.79 a,b Red cabbage ± c ± 0.41 b,c,d ± d ± 0.91 d,e a,b,c,d,e Different superscripts in the same column indicate that means were significantly different (p < 0.05).

17 Table 6 Sensory and physical characteristics of specialty bread. Bread samples Supplementation level, g/100 g flour Crumb elasticity Crumb grain structure Crust colour Crumb colour Flavour Control 8.63 ± 0.08 f 7.50 ± 0.40 c 4.50 ± 0.74 a 2.05 ± 0.15 a 7.50 ± 0.48 c,f Molasses ± 0.32 c,d,e 4.67 ± 0.58 a,b 7.20 ± 0.45 b,c,d 4.40 ± 0.08 b 6.00 ± 0.10 a,b,c,d ± 0.58 a 3.82 ± 0.27 a 7.50 ± 0.32 b,c,d 5.58 ± 0.20 c 5.00 ± 0.00 a,b OD apple ± 0.45 e,f 6.72 ± 0.33 d,e 6.25 ± 0.48 b 7.10 ± 0.30 e 8.10 ± 0.53 f ± 0.20 a,b 5.65 ± 0.20 b,c,d 8.00 ± 0.08 c,d 8.00 ± 0.11 g,h 6.72 ± 0.47 c,d,e OD plum ± 0.48 d,e,f 6.57 ± 0.52 d,e 6.67 ± 0.32 b,c 7.00 ± 0.12 d,e 8.50 ± 0.35 f ± 0.20 b,c 5.40 ± 0.31 b,c 8.50 ± 0.30 d 8.31 ± 0.05 h 7.25 ± 0.35 d,e,f OD carrot ± 0.34 c,d,e 6.45 ± 0.47 c,d,e 6.20 ± 0.18 b 6.50 ± 0.22 d 6.32 ± 0.37 b,c,d,e ± 0.15 b,c 5.21 ± 0.37 b 6.87 ± 0.78 b,c,d 7.69 ± 0.30 f,g 5.82 ± 0.41 a,b,c OD cabbage ± 0.28 b,c,d 5.68 ± 0.25 b,c,d 7.38 ± 0.45 b,c,d 7.18 ± 0.12 e,f 5.50 ± 0.74 a,b,c ± 0.30 b,c 4.82 ± 0.31 a,b 8.25 ± 0.58 c,d 8.20 ± 0.30 g,h 4.87 ± 0.49 a a,b,c,d,e,f Different superscripts in the same column indicate that means were significantly different (p < 0.05). 1050

18 BREAD SUPPLEMENTED WITH SUGAR BEET MOLASSES INGREDIENTS 1051 The crumb color of the breads changed from white to dull brown, as the level of molasses based ingredients was increased. Among the supplemented breads, the lightest crumb was observed in the bread made with 5 g/100 g flour molasses g/100 g flour molasses while the breads made with 10 g/100 g flour plums, cabbage and apples were the darkest. Regarding the taste, none of the samples was disqualified. The highest score in this parameter was given to the breads made with 5 g/100 g flour and 10 g/100 g flour OD plum, 5 g/100 g flour OD apple and the control. The samples made with OD red cabbage (5, 10 g/100 g flour), molasses (5, 10 g/100 g flour) and OD carrots (10 g/100 g flour) were scored the lowest. CONCLUSION Incorporation of sugar beet molasses or powders of OD fruits/vegetables in molasses improved the mineral content and antioxidative properties of the breads. The mineral content varied depending on the type of ingredient used and the supplementation level. Especially marked increase was observed in the potassium content: increases ranging from % were recorded for lower supplementation level and up to 167% for higher supplementation level. The calcium and magnesium contents increased less markedly but also significantly: increase in calcium ranged between 10 52% for 5 g/100 g flour level or % for 10 g/100 g flour level whereas increase in magnesium ranged between % for 5 g/100 g flour level or % for 10 g/100 g flour level. In general, it was observed that OD vegetable powders and pure molasses provided higher increases in the bread minerals. Similar observations were made regarding the antioxidative properties of the breads. The highest antioxidant potential was recorded in the bread made with 10 g/100 g flour OD plum powder. High antioxidative potentials were also noted for breads made with 5 g/100 g flour OD plums and 10 g/100 g flour OD cabbage and apple. Bread supplementation with molasses-based ingredients affected the sensory attributes, as well. The results suggested that lower supplementation level would not interfere with the sensory acceptance of the breads. Even breads made with 10 g/100 g flour molasses-based ingredients were not disqualified though higher doses than 10 g/100 g flour would not be recommended. The present results suggest that the sugar beet molassesbased ingredients could be incorporated successfully to bread. These ingredients exerted several potentially health-promoting effects that include the increase in the mineral content of bread (potassium, magnesium, calcium) and its antioxidative potential. REFERENCES 1. Ranhotra, G.; Gelroth, J. Bakery products as functional foods (nutraceuticals). AIB Technical Bulletin 1998, XX (10), Šušić, S.; Sinobad, V. Researches aimed at advancing the sugar industry of Yugoslavia About chemical compositions of bee honey and sugar beet molasses and their biological values in human diet (Istraživanja u cilju unapre - denja industrije šećera Jugoslavije o hemijskim sastavima pčelinjeg meda i melase šećerene repe i o njihovim biološkim vrednostima za ljudsku ishranu). Hemijska Industrija 1989, 43 (Suppl. 1 2), (In Serbian). 3. NOVUS, International. Raw material compendinum,2 nd ed.; Novus Europe: Brusseles, Belgium, 1996, Curtin, L.V. Molasses-general considerations. In Molasses in animal nutrition; National Feed Ingredients Association: Des Moines, Iowa, 1983; 1 11.

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