Volume 3, Number 1, 2013

Journal of Food Science and Engineering
Volume 3, Number 1, January 2013 (Serial Number 18)
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Journal of Food Science and Engineering

Volume 3, Number 1, January 2013 (Serial Number 18)
Contents
Research Papers
1 Changes in Texture, Structure and Pectin of Peach during Pressurization, Heating or Processing of High-pressure-induced and Heat-induced Jam Hiroko Kuwada, Yuri Jibu, Keiko Nakamura, Mayumi Tabuchi, Ai Teramoto, Kayoko Ishii, Yasumi Kimura1 and Michiko Fuchigami 9 Effects of Rate, Time and Splitting of Nitrogen Fertilization on the Technological Quality of Wheat Lidiane Borges Dias de Moraes, Janete Deliberali Freo, Barbara Biduski, Moacir Cardoso Elias and Luiz Carlos Gutkoski 19 Response of Chlorophyll a, SPAD Values and Chlorophyll Fluorescence Parameters in Leaves of Apricot Affected Some Abiotic Factors Adrijana Filipovi, Milan Poljak and Dragan kobi 25 Optimization of High EPA Structured Phospholipids Synthesis from -3 Fatty Acid Enriched Oil and Soy Lecithin Teti Estiasih, Kgs Ahmadi, Erliana Ginting and Arya Ulil Albab 33 Effect of Radiant Energy Vacuum on Physical and Microbial Properties of Beef Jerky John Scott Church, Carley Marie MacIntyre, Wade Robert Archambault, Paul Edward Moote, Jason Laco Cochran, Timothy Douglas Durance and Jonathan Douglas Van Hamme 40 The Study of Supply and Demand of Organic Products in the European Union and Serbia Ljubomir Pupovac, Tomislav Sudarevi and Suzana Salai 47 Sensory and Organoleptic Characteristic, Zinc and Iron Content of Fortified Chips from Cassava Flour Siti Helmyati, Nindya Putri Pamungkas, Lily Arsanti Lestari and Narendra Yoga Hendarta
Journal of Food Science and Engineering 3 (2013) 1-8
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Changes in Texture, Structure and Pectin of Peach during Pressurization, Heating or Processing of High-pressure-induced and Heat-induced Jam
Hiroko Kuwada1, Yuri Jibu2, Keiko Nakamura2, Mayumi Tabuchi2, Ai Teramoto3, Kayoko Ishii1, Yasumi Kimura1 and Michiko Fuchigami1
1. Department of Nutrition and Life Science, Fukuyama University, Fukuyama, Hirosima-ken 729-0292, Japan 2. Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama-ken 719-1197, Japan 3. Department of Health and Nutrition, Kanto Gakuin University, Yokohama, Kanagawa-ken 236-8501, Japan Received: October 26, 2012 / Published: January 20, 2013. Abstract: The objectives of this study are to research the relationship between pectin and the softening of peach by soaking in citric acid solutions for 24 h at 35 C, pressurizing for 30 min at 500 MPa or heating for 10 min. Also, comparing high-pressure-induced jam (HP-jam) and heat-induced jam (H-jam) were evaluated. It was found that firmness of the peach decreased greatly when soaked at pH 2.0 > heated > soaked at pH 2.2 or 2.5 > pressurized, respectively. About 88% of the peach pectin was water-soluble-pectin and high-methoxyl pectin, while low-methoxyl pectin was slight. During pressurization, the pectin did not change. However, pectin degraded through hydrolysis during heating; consequently, the middle lamella separated. Also, eight kinds of peach jam (65% sugar, pH 2.0 or pH 2.2, and 50% or 60% sugar, pH 2.5) were compared. Both color and flavor of HP-jam were better than H-jam. As the pH values were lower, L-, a-, b-values of jam became higher, and the jam became pinker. Raw peach contained about 0.3%-0.4% pectin, therefore, an addition of 0.6% pectin was needed for both HP- and H-jams. However, there was no great difference in rheology or sensory evaluation between HP- and H-jams. Key words: Peach, pectin, high pressure, processing, jam, texture, structure.
1. Introduction
Some fruits can be preserved in concentrated sugar solutions. Jam, marmalade and jelly are prepared by boiling fruits and fruit juice with sugar [1]. And also, boiling should continue till certain concentration of the mass is reached. During boiling in cooking pan, two processes, which are pectin extraction and jam manufacture, are performed [2]. The extra amount of pectin is added to give these fruit products more structure and body [1]. A characteristic of pectic substances that has a bearing on their function in cell wall matrix is their
Corresponding author: Hiroko Kuwada, assistant researcher, research field: cookery science. E-mail: kuwada@fubac.fukuyama-u.ac.jp.
ability to form gels in the presence of divalent ions or, under acid conditions, high concentrations of sugar. These gelling properties are of commercial importance and have been studied extensively [3]. Some of the reviews on the subject have been presented by Kertesz [4], Doesburg [5], Rees [6] and Voragen et al. [2]. Gel formation with divalent and trivalent ions is dependent on the presence of a sufficient percentage of nonesterified carboxyl groups [3]. Gel formation in sugar-acid systems is favored by a high percentage of methoxylation, a low percentage of acetylation, a long polymer chain length and a low proportion of neutral sugar side chains [3]. The high sugar concentration creates conditions of low water activity which in turn promote chain-chain rather than chain-solvent
Changes in Texture, Structure and Pectin of Peach during Pressurization, Heating or Processing of High-pressure-induced and Heat-induced jam
interaction, whereas the acid lessens the negative charges on the carboxyl groups, thus diminishing electrostatic chain repulsion [2]. The junction zones of three-dimensional network are stabilized by hydrogen bonding between undissociated carboxyl and secondary alcohol groups and hydrophobic interactions in methoxyl groups. Both types of binding forces are fortified by sucrose [2]. Heat-induced-jam (H-jam) has some faults such as off-flavor and deterioration of food components, nutrients and especially color. However, high pressure can produce jam without heating because pressurization accelerates hydrogen bonds between pectin macromolecules. It does not greatly change food color during processing [7]. Thus, High-pressure-induced-jam (HP-jam) was patented by Meidi-ya Ltd. in Japan in 1990 (Japan patent No. H3-219844) and HP-jam, such as strawberries, blue berries and apples have been marketed. However, pectin is added to this HP-jam. Citrus peel possesses sufficient pectin and acid to form marmalade. Thus, a processing for citrus yuzu marmalade without the addition of pectin was investigated [8-10]. During pressurization, the peel did not soften because the pectin did not degrade through -elimination. Therefore, a method for softening yuzu peel without heating should be required for HP-marmalade. In previous papers [11, 12], high-methoxyl pectin was extracted by soaking in 0.01 N HCl solution (pH 2.0) at 35 C due to the removal of Ca2+, therefore, vegetables were softened. This extraction method of pectin was used for softening the peel by using citric acid instead of HCl for yuzu marmalade [9]. Consequently, the peel softened. However, during maturation, the peach flesh became soft due to the solubilization of pectin and polysaccharides by polygalaturonase [13]. Although peach is softer than yuzu peel, soaking in citric acid solution may be useful for the extraction of pectin from peach. A peach possess 0.4% pectin per fresh weight [13]. Use of high pressure in food has been studied. The effect of
hydrostatic pressure on sterilization and preservation of citrus juice [14] and the effect of high pressure on microbial quality of apple juices [15] have been studied. Additionally, sugar composition effects on textural parameters of peach jam [16] and fruit content influence on gel strength of peach jam [17] have been studied. However, few studies have examined high pressure peach jam. Thus, the objectives of this study are to research the relationship between pectin and the softening of peach by soaking in citric acid solution, pressurizing or heating, and to establish a process for HP-jam and compare it with H-jam.
2. Materials and Methods

2.1 Sample Preparation Peach (Prunus persica L., harvested in Okayama, Japan, weight: 369 g 37 g, sugar content: 10.4% 1.2%, pH 4.46 0.03) was diced into 1 cm pieces. The vacuum-packed pieces were pressurized for 30 min at 500 MPa at room temperature (about 25 C) using a Dr. Chef high pressure food processor (Kobe Steel Ltd., Kobe, Japan) [18] or boiled in hot water (850 mL) for 10 min. Also, pieces were soaked in about 2%, 1% or 0.3% citric acid solutions (pH 2.0, 2.2 or 2.5, respectively) for 24 h at 35 C. 2.2 Texture Measurement Changes in texture of peach samples during soaking, pressurizing or heating were measured by a creepmeter (Rheoner, RE-33005, Yamaden Ltd., Tokyo, Japan). The sample was punctured at 1 mm s-1 by a plunger (cylindrical shape: 8 mm in diameter, 22 mm long) using a loadcell of 2 kg. The rupture stress and rupture strain (the mean of ten measurements) were indicated. 2.3 Structure Measurement Histological structures of the peach samples, which were soaked in citric acid solution at pH 2.5, pressurized or heated, and also peach jam were
observed using a cryo-scanning electron microscope (S-4500, Hitachi Ltd., Tokyo, Japan) [19]. Samples were cut into 6 mm 1 mm 1 mm, and dehydrated with 40% and 50% ethanol. A specimen was contained in a metal holder and quickly frozen by immersing in liquid nitrogen. A frozen specimen was transferred to the cold stage of a cryo-SEM and then cut with a knife (-150 C). After etching at -85 C and further cooling, the surface was observed (at about -120 C) under low acceleration voltage (1 kV). The magnification used to observe cell walls was 20,000. 2.4 Extraction of Pectin An alcohol insoluble solid (AIS) was prepared from raw, pressurized or heated samples. Samples were cut into tiny pieces manually with a knife and blended with ethanol (4 times sample amount) using an excel auto homogenizer (Nissei Ltd., Tokyo). The precipitates were then successively washed with 80% ethanol until sugars could not be detected in the wash by the phenol sulphuric acid method [20], then 99% ethanol, followed by an acetone wash before drying at room temperature. Pectic substances were extracted from AIS by successive extraction with distilled water (20 C, 24 h), 0.01 N HCl (at pH 2.0 and 35 C for 24 h 2-5 times), 0.1 M sodium acetate buffer (at pH 4.0 and 35 C for 24 h 1-2 times), 2% sodium hexametaphosphate solution (at pH 4.0 and 90 C for 3.5 h 2-6 times), and 0.05 N HCl (at 90 C for 3.5 h 3-8 times) [11, 12]. Each extraction was repeated until no sugar was detected. These extracts were designated as WSP (Water-Soluble Pectin), PA (Pectin A: 0.01N HCl-soluble pectin), PB (Pectin B: 0.1M acetate buffer-soluble pectin), PC (Pectin C: 2% sodium hexametaphosphate-soluble pectin) and PD (Pectin D: 0.05N HCl-soluble pectin) respectively. The amount of galacturonic acid was determined by the carbazole method [21]. 2.5 Jam Preparation Eight kinds of peach jam were produced. Peach-dices were soaked in citric acid solutions (pH
2.0, 2.2 or 2.5), mixed in a ratio of 2:1 with homogenized peach, then 50% (at pH 2.5), 60% (at pH 2.5) or 65% (at pH 2.0 or 2.2) sucrose (Nacalai Tesque, Icn., Kyoto, Japan) of total weight were added. However, sugar content of peach was subtracted from the added sucrose content. They were vacuum-packed, then pressurized for 30 min at 500 MPa (HP-jam) or boiled for 10 min (H-jam), respectively. The sugar contents (brix) of raw peach and peach jam were measured by a digital refract meter (PR-100, PR-200 or PR-300, Atago Ltd., Tokyo, Japan). 2.6 Color Measurement of Jam The color (L-, a- and b-values) of HP- and H-jams was measured using a spectrophotometer (ZE-6000, NDK, Osaka, Japan). 2.7 Rheology Measurement of Jam The steady-flow viscosity, thixotropy and dynamic-viscoelasticity of jelly in all peach jams were measured at 25 C by using a Rheosol-G3000 (UBM Ltd., Kyoto, Japan). Steady-flow viscosity of eight kinds of peach jams was compared. 2.8 Sensory Evaluation of Jam Sensory evaluation of peach jam was performed using a five point scale (-2+2). The color (bad-excellent), transparency (opaque-transparent), flavor (smell) of fruit (weak-strong), texture of jam (soft-firm), sweetness and sourness (weak-strong, not like-like), mouth feel (rough-smooth), total taste and preference of jam (not like-like) were compared. Samples were evaluated by 10 female students (20-21 years old). Statistical analysis (significant differences by T-test at P < 0.05) was carried out using software (Edu-STAT, Higashiyama Shobo, Kyoto, Japan).
3. Results and Discussion

3.1 Changes in Texture of Peach during Soaking, Pressurizing or Heating Changes in rupture stress and rupture strain of
peach during soaking in citric acid solutions, pressurizing or heating are shown in Fig. 1. Firmness of the peach decreased greatly when soaked at pH 2.0 > heated > soaked at pH 2.2 or 2.5 > pressurized, respectively. 3.2 Changes in Histological Structure of Peach during Soaking, Pressurizing or Heating Cryo-scanning electron micrographs of cell walls after soaking at pH 2.5 for 24 h, pressurizing or heating are compared in Fig. 2. Middle lamella of cell walls, rich in pectic substances, was separated more from heating for 10 min than soaking at pH 2.5. However, they did not separate when pressurized. Cell wall of HP-jam was similar to the raw and pressurized cell walls. Thus, great change was not found. However, the middle lamella of the H-jam was separated. 3.3 Changes in Pectin Composition of Peach during Pressurizing and Heating Changes in pectin composition of peach during pressurizing and heating are shown in Fig. 3. The amount of pectin in peach was about 340 mg 100 g -1 and the percentage of water soluble pectin WSP, PA, PB, PC and PD of raw peach was 39.8%, 48.3%, 4.3%, 6.0% and 1.6%, respectively. While dipping tissues in water at 20 C and 0.01 N HCl (pH 2.0) at 35 C, 39.8% and 48.3% pectic substances were extracted, respectively. WSP increased by polygalacturonase during maturation [13]. A diluted HCl solution (pH 2.0) is calcium sequestering agent. Pectic acid and low
methoxyl pectin are precipitated at pH 2.0, therefore, it is difficult to extract their pectic substances at pH 2.0. HCl-soluble components were highly methyl-esterifies [11, 12]. About 88% of the peach pectin was WSP of low molecular weight and high-methoxyl-pectin (PA), while low-methoxyl pectin (PB, PC and PD) was slight. Amount and composition of the pectin did not change during pressurization. In previous paper [18], with increased pressure, PA in carrots decreased, while PB increased. The degree of esterification of carrot pectin apparently decreased during pressurization, suggesting pectin methyl esterase (PME) activity occurred. Therefore, pressurized carrots maintained their firmness during pressurization and did not undergo degradation of the pectin main chain [18]. However, pectic composition of peach did not change by pressurization. This suggests the peach did not have PME. When heated, total pectin and the percentage of PA in peach decreased. Pectin degraded during heating; consequently the middle lamella separated. The pH value of raw peach was 4.46. Since pectin does not degrade through -elimination by pressurization [18] or by heating at pH 4 [22, 23], it might degrade through hydrolysis. 3.4 Changes in Rheology of Jam The initial and final sugar percentages and pH values of jam are shown in Table 1. During processing, the pH values increased and sugar content decreased slightly.
Fig. 1 Changes in rupture stress and rupture strain of peach during soaking in citric acid solution, pressurizing at 500 MPa for 30 min or heating for 10 min in boiling water.
Changes in Texture, Structure and Pectin of Peach during Pressurization, Heating or Processing of High-pressure-induced and Heat-induced jam Table 1 of jam.
pH 2.00 2.20 2.50 2.50
The initial and final pH values and sugar contents

Final pH and sugar content of jam pH HP-jam 65.0 65.0 60.0 50.0 2.43 2.73 2.94 2.91 H-jam 2.62 2.74 2.93 2.90 65.5 63.5 58.0 44.1 Sugar (%) HP-jam H-jam 64.5 61.3 57.6 46.8
Initial pH and sugar Sugar (%)
Raw peach: pH 4.46 0.03, sugar 10.4 1.2%. HP-jam: high-pressure-induced jam; H-jam: heat-induced jam.
The steady-flow viscosity of jam is shown in Fig. 4. The viscosity of H-jam was slightly higher than HP-jam. As the pH value of citric acid solution was higher and sugar content was lower, viscosity decreased. Since the thixotropy and dynamic-viscoelasticity showed a similar tendency, thixotropy and dynamic-viscoelasticity are not shown.
Fig. 2 Cryo-scanning electron micropraphs of cell walls of peach. Soaked: soaked in citric acid for 24 hrs at pH 2.5; Pressurized: pressurized at 500 MPa for 30 min; Heated: heated for 10 min in boiling water; HP-jam: high-pressure-induced jam; H-jam: heat-induced jam.
3.5 Changes in Color of Jam L-value (+ brightness), a-value (+ red, - green), b-value (+ yellow, - blue) of jam are shown in Fig. 5. As the pH values were lower, L-, a-, b-values of jam became higher and jam became pinker. L-, a-, b-values of HP-jam were higher than H-jam. This suggests that the amount of anthozyan (pigment of peach) was maintained by pressurizing but decreased by heating. 3.6 Sensory Evaluation of Jam Sensory evaluation of jam is shown in Fig. 6. Sensory evaluation of high-pressure-induced (HP-) and heat-induced (H-) jams (pH 2.0 and pH 2.2) with 65% sugar was compared. H-jams were more transparent than HP-jams. The flavor of fruit was judged that HP-jams were stronger than H-jams. Jams of pH 2.0 were firmer than jams of pH 2.2. It seems that pectin was more extracted when soaked at pH 2.0 than pH 2.2.
Fig. 3 Changes in pectin composition of peach during pressurizing and heating.
Sweetness of jams (pH 2.2) was stronger than that of jams (pH 2.0), although the same sugar content. The jams of pH 2 were too strong in acidity, and sensory evaluation was the worst. Mouth-feel of all jams was smooth. Consequently, heat-induced jam of pH 2.2 was estimated most highly. Subsequently, high-pressure-induced jam of pH 2.2 was liked. The jam of pH 2.0 was too sour. Therefore, jams of pH 2.5 with 50% and 60% sugar were compared. The flavor of fruit of all jams was comparatively good. However, texture of all jams was judged to be very soft and not to like. Sweetness of jams with 60% sugar was stronger than that of 50% sugar and also 65%
Fig. 4 Steady-flow viscosity of high-pressure-induced and heat-induced peach jams.
sugar (pH 2.0 and pH 2.2). However, sourness of jams with 60% sugar (pH 2.5) was weak. Mouth-feel of all jams was smooth. Consequently, there was no great difference in total evaluation. However, HP-jam with 60% sugar (pH 2.5) was judged to like most. There was no significant difference in sensory evaluation between HP- and H-jams.
High-pressure-induced jam, Heat-induced jam.
4. Conclusions
Firmness of the peach decreased greatly when soaked in citric acid solution for 24 h at pH 2.0 > heated for 10 min > soaked at pH 2.2 or 2.5 > pressurized for 30 min at 500 MPa, respectively. Pectin did not change during pressurization but degraded through hydrolysis during heating; consequently, the middle lamella of heated peach separated. Eight kinds of peach jam (65% sugar, pH 2.0 or pH 2.2, and 50% or 60% sugar, pH 2.5) were compared. Color and flavor of HP-jam were better than H-jam. However, there was no great difference in rheology or sensory evaluation between HP- and H-jams. Raw peach contained about 0.3%-0.4% pectin, therefore, an addition of 0.6% pectin was needed for pressure-induced jam.
Acknowledgments
The authors thank Ms. A. Ogura and Ms. A. Yamamoto for technical assistance. A part of this work
Fig. 5 L-, a- and b-values of peach jam.
Fig. 6
Sensory evaluation of peach jam. [2] A.G.J. Voragen, W. Pilnik, J.F. Thibault, M.A.V. Axelos, C.M.G. Renard, Pectin, in: A.M. Stephen (Ed.), Food Polysaccharides and Their Applications, Marcel Dekker Inc., New York, 1995, pp. 287-339. [3] J.P. Van Buren, The chemistry of texture in fruits and vegetables, J. Texture Studies 10 (1970) 1-23. [4] Z.I. Kertesz, The Pectic Substances, Interscience Publishers, New York, 1951. [5] J.J. Doesburg, Pectic substances in fresh and preserved fruits and vegetables, I.B. V.T. Wageningen, 1965.
was supported by a Grant-in Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture in Japan.
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Effects of Rate, Time and Splitting of Nitrogen Fertilization on the Technological Quality of Wheat
Lidiane Borges Dias de Moraes , Janete Deliberali Freo , Barbara Biduski , Moacir Cardoso Elias and Luiz 2 Carlos Gutkoski
1. Department of Agroindustrial Science and Technology/DCTA-Federal University of Pelotas/UFPel, P.O. Box 354, Capo do Leo CEP 96010900, RS, Brazil 2. Food Research Center, University of Passo Fundo/UPF, BR 285, Passo Fundo CEP 99052900, RS, Brazil Received: October 12, 2012 / Published: January 20, 2013. Abstract: The bread-making quality of wheat is a highly complex trait that depends on both genetic and environmental factors. This study aims at evaluating the effects of different rates, time and splitting of nitrogen fertilization on the technological quality of wheat cultivated in the Brazilian Southern region. The samples of bread wheat (Triticum aestivum L.), Onix, Quartzo and Mirante cultivars, were obtained through the use of nitrogen (N) fertilizer applied in doses of 36, 100 and 120 kg N ha-1 at sowing, tillering and flowering. Laboratorial tests were carried out in a completely randomized design with four repetitions. The parameters analyzed were: grain yield, total protein, protein fractions, gliadins, glutenins, albumins and globulins, sulfur, gluten strength (W), dough tenacity (P), extensibility (L) and stability (S), bread specific volume and bread firmness. While the content of total and reserve proteins is significantly increased with a higher rate and splitting of N, the content of metabolic proteins remains constant. A mean increase in the quality parameters W (24.37%), L (14.86%) and P (11.59%) among cultivars was noticed after application of 120 kg N ha-1, split at sowing, tillering and flowering. Bread specific volume increased, while bread firmness decreased with a higher rate of N fertilizer. Wheat fertilization with high doses of N does not cause induction to S deficiency in the grains. Not only increasing the N fertilization rate, but also splitting the N rate had a beneficial effect on the technological quality of wheat. Key words: Triticum aestivum, rheology, gluten, nitrogen.
1 1 2 1
1. Introduction
The development of highly productive wheat cultivars adapted to different regions in the country and resistant to diseases has guided the major researches focused on the Brazilian wheat production. However, with the growing demand from the milling industry and the establishment of a new official standard for wheat classification in Brazil through the Normative Instruction 38 of the Ministry of Agriculture, Livestock and Supply (MAPA) in November 30th, 2010, the quality variable has been taken into account in the production of grains that are compatible with the market requirements.
Corresponding author: Luiz Carlos Gutkoski, Ph.D., research fields: science and food technology. E-mail: gutkoski@upf.br.
The Normative Instruction, which has been in effect since July 2012, has determined that, according to the new criterion for bread wheat classification, gluten strength (W) must rise from at least 180 10-4 J to 220 10-4 J and/or dough stability must be longer than 10 min [1]. Both gluten strength and stability are important parameters to assess the technological quality of wheat and are associated with two fundamental determinants: protein content, which is strongly influenced by the environment, and protein composition, which is determined by both genetic and environmental factors [2-4]. Over the last years, the strategy used to increase the protein content in wheat grains and obtain high quality bread-making products has been more focused on the N-fertilization management, which is
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specific to each genotype and environment, than on the development of new wheat cultivars [5]. However, the steady increase in productivity due to genetic advancements has led to a decreased protein grain starch ratio [6], causing lower bread-making quality. Besides that, another factor that has contributed to the reduced protein content in wheat is related to the induction of S deficiency in the grains. This is caused by the application of high N doses, thus originating the accumulation of non-protein compounds that are rich in N in the form of amides [7]. Higher N fertilization rates have shown a favorable effect on grain quality because they increase the protein concentration [4, 8, 9]. Nevertheless, different results have been reported regarding the effect of splitting the same dose of N in one or several applications along the wheat development cycle. Garrido-Lestache et al. [10] and Fuertes-Mendizbal et al. [5] have reported improvement of the quality parameters by splitting the dose of N applied, although the effects of N splitting have not been so evident [4]. For Ayoub et al. [11], the split application of N fertilization affected the protein content, but not their quality. On the other hand, Zhu and Khan [12] and Godfrey et al. [8] considered that a higher N content in wheat grains may have a negative effect on the protein quality, thus resulting in high dough extensibility due to the increased gliadin:glutenin ratio. Yet, according to these authors, such change in the viscoelastic properties of dough is not cultivar-related. Therefore, the decision about the N management strategy in wheat crop is a big challenge that has different dimensions and characteristics, and it should be defined in accordance with both genotype and environmental conditions. In such a context, this study aims at assessing the effects of rates, time and splitting of N fertilization on the technological quality of wheat cultivated in the Brazilian Southern region.

2.1 Site and Experimental Design The field experiment was carried out along the harvest period in 2010 at Agropecuria Sementes e Cabanha Buti Ltda., in the city of Coxilha, RS, Brazil, situated at latitude 280512.3 and longitude 522323. The soil in the experimental area, 0-0.15 m deep, classified as typical dystrophic red latosoil [13], presented the following chemical properties before the experiment: pH (water) = 5.7; O.M. = 2.7 g kg-1; Al3+ = 0.0 cmol L-1; Ca2+ = 5.0 cmol L-1; Mg2+ = 2.2 cmol L-1; P = 12 mg L-1; K = 68 mg L-1 and S = 13 mg L-1. On June 20th, 2010, Onix, Quartzo and Mirante cultivars, considered as appropriate for bread-making [14], were mechanically sown 5 cm deep in plots with 12.0 m2 of total area and 10.8 m2 of planted area. Distance between rows was 0.17 m, density of 300 seeds m2, in tillage system, on leftovers from soybean crops. Grains were harvested in the second half of November, 2010. Basal fertilization of plots consisted of 75 kg ha-1 of potassium chloride (KCl) applied before sowing and 200 kg ha-1 of diammonium phosphate (DAP) applied at sowing, which provided 36 kg ha-1 of N and 92 kg ha-1 of phosphorus pentoxide (P2O5) (Yara Brasil Fertilizantes, Brazil). Basal fertilization was conducted in the control management, called management zero (M0), as well as in the other managements, called management 1 (M1), 2 (M2) and 3 (M3). Topdressing fertilization was split, combined with basal fertilization, it reached the total rate of 100 kg ha-1 of N for M1 and 120 kg ha-1 of N for M2 and M3. Nitrogen was applied to the managements as follows: M0 = 36 kg ha-1 of N at sowing; M1 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering; M2 = 36 kg ha-1 of N at sowing + 84 kg ha-1 of N at tillering; M3 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering + 20 kg ha-1 of N at flowering. In topdressing fertilizations, N was applied as urea (Yara Brasil Ferilizantes, Brazil). Disease and pest
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control involved three applications of 0.7 kg ha-1 of Trifloxystrobin + Tebuconazole (Nativo, Bayer, Germany). The laboratory experiment was carried out in a completely randomized design, with a 3 4 factorial distribution (cultivars fertilization management), totalizing 12 managements. The analyses were conducted in quadruplicate. In the planted area of each plot, grains were mechanically harvested. The harvest was followed by procedures of pre-cleaning and drying in Bandeirante stationary dryer, model 20 second, Brazil, with forced ventilation, until obtaining 13% of water. Wheat samples were packed in cotton bags and taken to maturation in drying chamber at 20 C and 55% 5% of relative humidity for two months. Grain cleaning was performed in intecnial impurity separator, model Sintel, Brazil. Milling was performed in a UDY Corporation cyclone-type mill, model Sample Mill, USA, with a 0.05 millimeter-opening sieve. 2.2 Analytical Procedures The analyses to assess the technological quality were carried out at the Cereals Laboratory of the Food Research Center (CEPA) of University of Passo Fundo, Brazil. Grain yield in the planted area of each plot was determined through mechanical harvest and weighing of wheat grains, and the results were expressed in kg ha-1. Grain N content was determined according to AACC method nr. 46-12 [15]. Grain protein content was estimated through N concentration multiplied by conversion factor 5.7, and results were given in percent on a dry basis. The sequential extraction of different fractions of protein from whole wheat flour was performed in accordance to Wieser and Seilemeir [16], with changes in sample amount, solutions and material used in the nitrogen quantification (supernatant and non-precipitated). One gram of whole flour underwent three consecutive extractions with 5 mL of saline solution (0.4 mM NaCl/0.067 mM HKNaPO4, pH 6) to extract albumins and globulins, and three extractions with 5 mL of
aqueous solution of ethanol at 60% (v/v) to extract gliadins. The extractions were performed with the use of magnetic agitator for 15 min at room temperature. The suspensions were centrifuged for 20 min at 15,000 g at room temperature. Supernatants resulting from the three consecutive extractions of each protein fraction were collected. The protein content was determined through AACC method nr. 46-12 [15]. The protein content determined after the first stage of extractions corresponded to albumins and globulins. After the second stage of extractions with ethanol, supernatants were used to quantify gliadins. Glutenin content was estimated through the difference between the total protein content of the samples of whole wheat minus the sum of contents obtained at the two extraction stages. The results were given in percent of the total protein content of the sample. Grain S content was determined through sample combustion in LECO equipment, model TruSpec S, USA, according to ASTMD method nr. 4239 [17], and the results were given in percent on a dry basis. In order to perform alveography, promylography, bread specific volume and bread firmness analyses, wheat samples were conditioned at 15% of water. After 24 h, the samples were milled in a Chopin experimental mill, model CD1, France, according to AACC method nr. 26-10 [15]. The flour viscoelastic characteristics were determined in a Chopin alveograph, model NG, France, according to AACC method nr. 54-30 (2000), by weighing 250 g of flour and a volume of 129.4 mL of water, corrected on a basis of 14% water. Parameters considered were gluten strength (W), which corresponds to the mechanical work required to expand the bubble to its rupture, expressed in 10-4 J; dough tenacity (P), which measures the maximum overpressure on dough expansion (mm); and dough extensibility (L), which measures curve length (mm). The characteristics of the wheat flour blend were determined in a promylograph T6 apparatus, model Max Egger, Australia, according to AACC method nr. 54-21 [15].
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Effects of Rate, Time and Splitting of Nitrogen Fertilization on the Technological Quality of Wheat Table 1 Average performance of grain yield and S content in three wheat cultivars subjected to three different N fertilization managementsa. Effectb M0 M1 M2 M3 nix Quartzo
a
Dough stability was defined as the time difference between the point where the top of the curve intercepts the mean line of 500 units and the curve point that leaves the line expressed in minutes. Bread specific volume was determined in Vondel equipment, model MVP 1300, Brazil, through displacement of canola seeds, and specific volume was estimated through the ratio of loaf volume and mass measured in a semi-analytical balance. The determination of specific volume was performed one hour after the loaves were baked, and the results were given in cm3 g-1. Bread firmness was found through texture analyzer model TA.XTplus, England, according to AACC method nr. 74-09 [15]. A 36-millimiter cylindrical probe compressed the sample to 40% of its original size at a speed of 1.7 mm s-1, and the firmness parameter (g) was obtained. Twenty-five-millimeter-thick bread slices were used in each analysis. 2.3 Statistical Analysis Data processing and statistical analysis were conducted using Sisvar statistical software, Version 5.3, Build 75 [18]. Data significance was tested through an analysis of variance, with error probability of 0.01 and 0.05; in the significant models, means were compared through Tukey test, at a significance level of 0.95 (P > 0.05).
Grain yield (kg ha-1 at 13% water) Management 3870 b 4749 a 4825 a 4869 a Cultivar 4383 b 4885 a
Grain S content (% on a dry basis) 0.180 b 0.188 a 0.190 a 0.194 a 0.197 a 0.181 c
Mirante 4467 b 0.186 b Different letters indicate significant difference between the column according to Tukey test at a level of P 0.05; b M0 = 36 kg ha-1 of N at sowing; M1 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering; M2 = 36 kg ha-1 of N at sowing + 84 kg ha-1 of N at tillering; M3 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering + 20 kg ha-1 of N at flowering.

3.1 Traits Related to Wheat Grains Table 1 shows mean grain yield and S content of three wheat cultivars with different N fertilization managements. Grain yield increased 22.71% on average between M0 and M1. A dose of 64 kg ha-1 of N was added to the latter as topdressing fertilization at tillering. This result is in accordance with Li [19], who found that grain yield is defined at tillering in Triticum species. The addition of 20 kg ha-1 of N to M2 in comparison to M1, as well as splitting at flowering (M3), considering
the same N rate, did not change grain yield of the cultivars significantly (P > 0.05) (Table 1). The results obtained in this study are in agreement with those found by Lpes-Bellido et al. [20] and Garrido-Lestache et al. [4, 10], who did not observe any significant increase in grain yield by applying more than 100 kg ha-1 of N fertilizer, although, according to the authors, the protein concentration continued to increase. For Wuest and Cassman [21] N fertilization at vegetative growth raises grain yield by influencing the number of grains per spike as well as the number of spikes per plant; yet, when it is performed at flowering, it predominantly benefits the grain protein concentration. Average grain yield of Quartzo cultivar was 4,885 kg ha-1, which was significantly higher than yields of Onix and Mirante cultivars (4,383 kg ha-1 and 4,467 kg ha-1, respectively, with no significant difference between them). Such results were expected for those cultivars and were higher than the means obtained in that agricultural year in the South of Brazil, where the State of Paran evidenced the highest yield (2,891 kg ha-1), followed by Santa Catarina (2,755 kg ha-1) and Rio Grande do Sul (2,490 kg ha-1) [22].
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Grain S content increased significantly (P 0.05) with the addition of N to M1 in comparison to M0 (Table 1). Between M1 and the other managements, although S content showed the same behavior, the variation was not significant. This result was probably found because of the wider variation in the total protein content existing between M0 and M1. According to Zhao et al. [23], more than 90% of S in wheat is found in organic form, mainly associated with the composition of grain proteins. Zhao et al. [23] claimed that grains can be regarded as S-deficient when their S levels are lower than 0.120% and N:S ratio is above 17. In this study, mean S concentration and N:S ratio between managements were 0.188% and 12.01, respectively. Findings concerning increased N application and higher grain S content were reported by Dupont et al. [24], who obtained mean values of 0.200% of S and 14.5 of N:S ratio in wheat grains. However, contrarily to the results reported by Zhao et al. [23] and Flaete et al. [7], who claimed that the induction of S deficiency in the plants was due to unbalanced N:S ratio caused by high N dose, in this study, as well as in Ercoli et al. [25], the use of high N doses, such as 100 kg ha-1 and 120 kg ha-1, did not cause S deficiency in wheat grains. Such a result allows us to guarantee that, along the wheat cultivation cycle, S availability to quantitative and qualitative synthesis of proteins was provided. Table 2 shows the contents of total protein, gliadins and glutenins of the three wheat cultivars fertilized with different N managements. The fraction of metabolic proteins (albumins + globulins) remained constant, regardless the N fertilization management, however, there was a significant variation between different cultivars, with mean contents of 2.26%, 2.27% and 2.3% for Onix, Quartzo and Mirante, respectively. Similar results mentioning the low ratio between metabolic protein content and increased and/or split N fertilizer application in wheat were reported [2, 3, 5, 9, 26].
Onix cultivar showed a significant increase (P 0.05) in total protein content when N dose was raised from 36 kg ha-1 to 100 kg ha-1 (M0 and M1, respectively). When 20 kg ha-1 of N were added at tillering, there was no significant increase in this parameter, however, when the same N dose was applied at flowering, a new significant increase in total protein content was observed. For Quartzo and Mirante cultivars, the protein content increased significantly both with increased N dose and splitting of the same N dose (M2 and M3, respectively) (Table 2). Similar results reporting the effects of increasing N rates on higher protein content were reported by Garrido-Lestache et al. [4, 10], Bakht et al. [27] and Abedi [9]. A positive effect of splitting N at flowering on the protein content was found by Wuest and Cassman [21] and Woolfolk et al. [28].
Table 2 Total protein, gliadins and glutenins contents obtained from grains of three wheat cultivars subjected to three different N fertilization managementsa. Cultivar nix Quartzo Mirante Total protein (% on a dry basis) M0 A 12.50 c B 12.24 d C 11.81 d M1 A 13.40 b B 12.78 c B 12.67 c M2 A 13.50 ab B 13.12 b B 13.20 b M3 A 13.69 a B 13.39 a AB 13.50 a Gliadins (%) M0 AB 4.76 c A 4.79 d B 4.68 d M1 A 5.24 b B 5.08 c AB 5.18 c M2 AB 5.31 b B 5.26 b A 5.45 b M3 B 5.41 a B 5.36 a A 5.52 a Glutenins (%) M0 A 5.48 c B 5.17 c C 4.84 d M1 A 5.84 b B 5.43 b C 5.19 c M2 A 5.90 ab B 5.58 ab B 5.43 b M3 A 6.02 a B 5.74 a B 5.68 a a Different small letters in the same column and capital letters in the same row represent, respectively, significant differences between managements and cultivars according to Tukey significance test at a level of P < 0.05; b M0 = 36 kg ha-1 of N at sowing; M1 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering; M2 = 36 kg ha-1 of N at sowing + 84 kg ha-1 of N at tillering; M3 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering + 20 kg ha-1 of N at flowering. Managementb
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Effects of Rate, Time and Splitting of Nitrogen Fertilization on the Technological Quality of Wheat Table 3 Gluten strength (W), tenacity (P), extensibility (L) and dough stability of three wheat cultivars with different N fertilization managementsa. W P L Stability (10-4 J) (mm) (mm) (min) Management M0 201 c 69 b 74 b 18.05 b M1 224 b 72 ab 78 ab 18.65 ab M2 234 b 75 ab 81 ab 18.69 ab M3 250 a 77 a 85 a 19.28 a Cultivar nix 250 a 77 a 81 a 20.36 a Quartzo 217 b 67 b 87 a 16.85 c Mirante 215 b 75 a 70 b 18.79 b a Different letters indicate significant difference between the column according to Tukey test at a level of P < 0.05; b M0 = 36 kg ha-1 of N at sowing; M1 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering; M2 = 36 kg ha-1 of N at sowing + 84 kg ha-1 of N at tillering; M3 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering + 20 kg ha-1 of N at flowering. Effectb
Such results allow us to claim that in order to obtain high efficiency with the use of N, which is translated into a better benefit-cost relationship to producers, the definition of the fertilization period is as important as the doses applied. Besides, considering the fertilization strategies used in this study, similar response patterns among cultivars regarding the total protein content emphasize the importance of defining managements according to genotypes that belong to the same quality group to reach the best performance of the end products. Contrarily to albumins and globulins, gliadins and glutenins increased significantly (P 0.05) in response to fertilization managements (Table 2). This evidences that such fractions were responsible for increasing total proteins in wheat grains. These results are in accordance with those obtained by Johanssons et al. [29] and Fuertes-Mendizbal et al. [5] who mentioned that, once the requirement of metabolic protein needed for grain formation is fulfilled, additional N supplied to the plants is accumulated as reserve proteins. Consequently, according to these authors, changes in the bread-making quality of flour result mainly from the contribution of those proteins. The gliadin content showed variable behavior in the three cultivars according to the fertilization management applied, while the glutenin content was significantly higher in all the managements for Onix cultivar. 3.2 Rheological Analysis of Flour, Bread Volume and Firmness Table 3 presents the mean values of gluten strength (W), tenacity (P), extensibility (L) and dough stability (E) of three wheat cultivars fertilized with different N managements. The alveographic parameter gluten strength (W) varied significantly by increasing the N dose from 36 kg ha-1 to 100 kg ha-1 and splitting 120 kg ha-1 of N at three different wheat development stages, instead of two (M2 and M3, respectively). Studies pointing out
higher W with increased N rate were reported by Borghi et al. [30], Garrido-Lestache et al. [4, 10], and Fuertes-Mendizbal et al. [5]. Increased W resulting from splitting the same N dose (M2 and M3) is in agreement with results obtained by Borghi et al. [30] and Fuertes-Mendizbal et al. [5]. While raising N rate caused a mean increase of 16.41% in W, splitting N fertilization in three applications instead of two raised W in 6.83%. The latter percentage confirms the importance of N fertilization at flowering, not only to increase the protein content, but also to improve their quality, which in turn leads to the production of flours with stronger gluten. From the mean results of W, which is one of the criteria considered for wheat classification in Brazil, it was found that flours resulting from M0 did not reach the minimum value of W (220 10-4 J) required for wheat to be considered as suitable for bread-making [1]. From M1, W values gradually increased, allowing the cultivars to be included in that category. Such a result reinforces the theory advocated by Borghi et al. [30] that the same wheat cultivar produced either in different sites or in the same site, but with different fertilization practices, may show more variation in
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protein concentration and rheological properties of dough than different cultivars grown in the same site. Tenacity (P) and dough extensibility (L) of M0, M1 and M2 did not vary significantly with increased N rate and splitting. However, compared to M0 and M3, P and L showed a significant increase of 11.59% and 14.86%, respectively. Similar results showing increase in these parameters with higher dose of N and splitting were reported by Borghi et al. [30], Garrido-Lestache et al. [4] and Fuertes-Mendizbal et al. [5]. In accordance with our results, Garrido-Lestache et al. [10] and Fuertes-Mendizbal et al. [5] concluded that L was more sensitive than P to N fertilization. The authors claimed that the elastic behavior of dough (P) or its resistance to deformation, despite showing variation in relation to N managements, is closely related to the cultivar. Flour stability did not vary significantly (P 0.05) with increased N rate, but a significant increase in this parameter can be noticed when compared to M0 and M3 (Table 3). In different managements and cultivars, time of dough stability was above the minimum period of 10 min required for wheat to be classified as bread, according to the Normative Instruction nr. 38 from November 30th, 2010 [1]. Following this criterion, all the three wheat cultivars could be classified as improver wheat, of which the minimum stability time is 14 min. According to Mirlbes [31], stability is an indicator of tolerance of flours to blending, which tends to be more stable when flours are stronger. This was confirmed through the relation found between higher gluten strength and dough stability in M3 and Onix cultivar. The positive effects of N management on alveographic parameters of quality assessed in this study can be related to protein fractions. As the content of gliadins and glutenins increased, W, L and P changed (Table 3). According to Gianibelli et al. [32], the influence of gliadins on both extensibility and viscosity due to their monomeric nature is determined by the allelic composition and the amount
of such fractions. Glutenins, which are polymeric proteins, have an effect on elasticity and extensibility, and consequently, on dough strength, not only because of their composition and content, but also because of their level of polymerization. W and dough stability of Onix cultivar were significantly higher than the others, while Quartzo cultivar had the lowest P and dough stability. Table 4 shows bread volume and firmness values of three wheat cultivars fertilized with different N managements. Bread specific volume showed significant variation (P 0.05) with higher N rate and splitting (Table 4). As it was observed in this study, Thomason et al. [33] found a positive relationship between increased protein concentration and bread volume. Corroborating our results related to splitting the same dose of N in three applications instead of two, Rubio et al. [34] reported that the split application of 150 kg of urea ha-1 (50-50-50) to fertilize five bread wheat cultivars resulted in higher loaf volume in comparison to the fertilization with 150 kg of urea ha-1 applied at sowing.
Table 4 Bread specific volume and bread firmness of three wheat cultivars with different N fertilization managementsa. Cultivar nix Quartzo Mirante Bread specific volume (cm3/g) M0 A 4.90 d B 4.82 c C 4.55 d M1 A 5.23 c B 5.11 b C 4.91 c M2 A 5.39 b B 5.23 b B 5.25 b M3 A 5.54 a B 5.44 a C 5.38 a Bread firmness (g) M0 B 251.68 a C 206.44 a A 274.47 a M1 A 198.51 b A 205.52 a A 218.76 b M2 A 199.82 b A 207.38 a A 213.07 b M3 A 203.88 b B 183.21 b A 221.65 b a Different small letters in the same column and capital letters in the same row represent, respectively, significant differences between managements and cultivars according to Tukey significance test at a level of P < 0.05; b M0 = 36 kg ha-1 of N at sowing; M1 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering; M2 = 36 kg ha-1 of N at sowing + 84 kg ha-1 of N at tillering; M3 = 36 kg ha-1 of N at sowing + 64 kg ha-1 of N at tillering + 20 kg ha-1 of N at flowering. Managementb
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Mirante cultivar, except in M2, showed significantly lower bread specific volume, followed by Quartzo and Onix (Table 4). This result, associated with higher bread firmness of Mirante, was probably derived from the higher ratio of tenacity to extensibility shown by this cultivar. According to Gianibelli et al. [32] obtaining loaves with higher volume and silky texture requires a precise balance between the dough viscoelastic properties, since excessive elasticity may limit bread expansion, while insufficient elasticity may not retain the carbon dioxide released by yeasts, thus resulting in lower quality bread. Results of bread firmness, with the exception of Quartzo cultivar, which had a differentiated behavior, allowed to show that there was a significant reduction of this parameter with increased N rate between M0 and the other fertilization managements (Table 4). The research initiated by Timms et al. [35] mentioning an equivalent improvement in bread texture and volume with the determination of growing levels of protein in the flour. Reduced bread firmness with the same N dose split at two or three stages of wheat development (M2 and M3), respectively, was only seen in Quartzo cultivar.
the parameters that work as predictors of wheat quality and use in bread-making. A mean increase in the quality parameters of gluten strength (24.37 g 100 g-1), extensibility (14.86 g 100 g-1) and dough tenacity (11.59 g 100 g-1) among cultivars was noticed after application of 120 kg N ha-1, split at sowing, tillering and flowering of wheat. Bread specific volume increased, while bread firmness decreased with higher dose and splitting of N. The strategy of splitting N fertilization at sowing, tillering and flowering improves the grain technological quality and contributes to the inclusion of the cultivars into the bread category, according to the new wheat trading norms in Brazil.
Acknowledgments
Authors are thankful to the National Council for Scientific and Technological Development (CNPq) for the productivity grant, and to the Rio Grande do Sul Science and Technology Office for the financial resources.
References
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4. Conclusions
Grain yield of wheat cultivars used in this study does not show significant variation with N doses higher than 100 kg N ha-1 and after tillering. While the content of total and reserve proteins is significantly increased with a higher rate and splitting of N, the content of metabolic proteins remains constant. Wheat fertilization with a high rate of N (120 kg -1 ha ) does not cause induction to S deficiency in wheat grains. This guarantees the availability of this component to quantitative and qualitative synthesis of proteins. Quartzo cultivar shows the highest grain yield, while Onix outweighs the other cultivars concerning
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(2001) 71-76. [20] L. Lpez-Bellido, R.J. Lpez-Bellido, J.E Castillo, F.J. Lpez-Bellido, Effects of tillage, crop rotation and nitrogen fertilization on wheat under rainfed Mediterranean conditions, Agron. J. 92 (2000) 1054-1063. [21] S.B. Wuest, K.G. Cassman, Fertilizer-nitrogen use efficiency of irrigated wheat. 1. Uptake efficiency of preplant versus late-season application, Agron. J. 84 (1992) 682-688. [22] CONAB, National supply company, in brazilian harvest follow-up: Grains, sixth survey, 2010/2011 harvest, Brasilia: Conab [Online], 2011, p. 40, http://www.conab.gov.br/. [23] F.J. Zhao, M.J. Hawkesford, S.P. Mcgrath, Sulphur assimilation and effects on yield and quality of wheat, J. Cereal Sci. 30 (1) (1999) 1-17. [24] F.M. DuPont, W.J. Hurkman, W.H. Vensel, R. Chan, R. Lopez, C.K. Tanaka, et al., Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development, J. Cereal Sci. 44 (2006) 101-112. [25] L. Ercoli, L. Lulli, I. Arduini, M, Mariotti, A. Masoni, Durum wheat grain yield and quality as affected by S rate under Mediterranean conditions, Eur. J. Agron. 35 (2011) 63-70. [26] L. Pedersen, J.R. Jorgensen, Variation in rheological properties of gluten from three biscuit wheat cultivars in relation to nitrogen fertilization, J. Cereal Sci. 46 (2007) 132-138. [27] J. Bakht, M. Shafi, M.T. Jan, Z. Shah, Influence of crop residue management, cropping system and N fertilizer on soil N and C dynamics and sustainable wheat (Triticum aestivum L.) production, Soil and Tillage Res. 104 (2009) 233-240. [28] C.W. Woolfolk, W.R. Raun, G.V. Johnson, W.E Thompson, R.W Mullen, K.J. Wyn, et al., Influence of late-season foliar nitrogen applications on yield and grain nitrogen in wheat, Agron. J. 94 (2002) 429-434. [29] E. Johansson, M.L. Prieto-Linde, G. Svensson, Influence of nitrogen application rate and timing on grain protein composition and gluten strength in Swedish wheat, J. Plant Nutr. Soil Sci. 167 (2004) 345-350. [30] B. Borghi, G. Giordani, M. Corbellini, P. Vaccino, M. Guermandi, G. Toderi, Influence of crop rotation and fertilizer treatments on wheat bread-making quality, Eur. J. Agron. 4 (1995) 37-45. [31] C. Miralbs, Quality control in the milling industry using near-infrared transmittance spectroscopy, Food Chem. 88 (2004) 621-628. [32] M.C Gianibelli, O.R. Larroque, F. Macritchie, C.W. Wriley, Biochemical, genetic and molecular
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Effects of Rate, Time and Splitting of Nitrogen Fertilization on the Technological Quality of Wheat M.C.G. Nevrez, H.G. Rios, et al., Effect of split applications of urea on protein size distribution, physical dough properties, and baking performance of five experimental bread wheat lines, Agric. Sci. 2 (3) (2011) 1-11. [35] M.F. Timms, R.C. Bottomley, J.R.S. Ellis, J.D. Schofield, The baking quality and protein characteristics of a winter wheat grown at different levels of nitrogen fertilization, J. Sci. Food and Agric. 32 (7) (1981) 684-698.
characterization of wheat endosperm proteins, American Association of Cereal Chemists, Saint Paul [Online], 2001, p. 20, http://www.aaccnet.org/cerealchemistry/ freearticle/gianibelli.pdf. [33] W.E. Thomason, S.B. Phillips, T.H. Pridgen, J.C. Kenner, C.A. Griffey, B.R Beahm, et al., Managing nitrogen and sulfur fertilization for improved bread wheat quality in humid environments, Cereal Chem. 84 (5) (2007) 450-462. [34] A.R.I. Rubio, K.C. Quiroz, F.V. Lara, B.S. Espinoza,
DAVID
PUBLISHING
Response of Chlorophyll a, SPAD Values and Chlorophyll Fluorescence Parameters in Leaves of Apricot Affected Some Abiotic Factors
Adrijana Filipovi1, Milan Poljak2 and Dragan kobi3
1. Faculty of Agriculture and Food Technology, University of Mostar, Biskupa ule bb 88000, Mostar 2. Faculty of Agriculture, University of Mostar, Svetoimunska cesta 25, 10000, Zagreb 3. Faculty of Mathematics and Science Education, University of Mostar, Matice Hrvatske b.b. 88000, Mostar Received: October 29, 2012 / Published: January 20, 2013. Abstract: Experimental trail conducted in Jasenica, Bosnia and Herzegovina have showed apricot (Prunus persica L) leaf injures especially on the edges in the middle of the season. Leaf edges show chlorosis starting from the tip due to lack of Chlorophyll. Affected leaves start falling and renew leaves masse as respond to stress conditions which also delay fruits maturation. Technological development in detecting the stress of high plants trough the leaf fluorescence and Chlorophyll concentration have led to the introduction of instruments which utilize fluorescence as a basis for determining stress level. The paper was aimed to show whether the productivity values calculated from Chlorophyll fluorescence Fv/Fm were comparable to those produced by spectrophotometric method in detecting Chlorophyll a concentration and SPAD values detected by Chlorophyll meter (SPAD-502). Measurements of nitrogen content were also provided in order to obtain accuracy of SPAD meter since Chlorophyll molecules contain nitrogen in their structure. To test the relationship between these three technique regression relationship was obtained. Regression coefficient between Fv/Fm values and Chlorophyll a concentration mg g-1 was high. Also, strong regression coefficient was observed between Chlorophyll a and SPAD values indicating a good accuracy of this device which was also confirmed by good regression between nitrogen content and SPAD values. Fluorometer measurements on injured leaves samples of apricot have also reviled the Fv/Fm values below 0.83 which is in according with numerous authors who considered a line for indicating stress factors. Key words: Plant stress, apricot, Fv/Fm values, Chlorophyll fluorometer, Chlorophyll a concentration, SPAD values.
1. Introduction
The most common methods for measuring plant stress involve Chlorophyll fluorometer and Chlorophyll content meter because they are fast, reliable for measuring specific types of plant stress and relatively inexpensive. Chlorophyll fluorescence is one of the well-known indicators of photosynthetic stress status in various environments [1-3] have used Fv/Fm ratio as an abiotic stress index. Recently, it has been shown that a parameter derived from Chlorophyll fluorescence, the ratio of variable/maximum fluorescence, Fv/Fm, is a
Corresponding author: Adrijana Filipovi, assistant professor, research fields: plant physiology, plant nutrition. E-mail: adrijana.majic@sve-mo.ba.
quantitative measure for the photochemical efficiency of photo system II (maximum quantum yield of PSII) [4]. The accumulation of excessive excitation energy can cause photo inhibition or photo oxidation in the photosynthetic apparatus and the reduced values of Fv/Fm indicate that a proportion of PSII reaction centres were damaged. In this study, Fv/Fm distribution pattern was analyzed in the leaves of apricot affected some abiotic factors. Stress conditions provide the chlorosis and crowing of the leaves from the top and edge of the leaf resulting in total leaves draying and falling in the middle of the vegetation. In order to obtain basic knowledge for identifying the stress factor on apricot leaves OS-30p fluorometer (Fv/Fm),
20
Response of Chlorophyll a, SPAD Values and Chlorophyll Fluorescence Parameters in Leaves of Apricot Affected some Abiotic Factors
spectrometric measurement of Chlorophyll a, total Chlorophyll content (SPAD values) measured by Chlorophyll meter and total leaves nitrogen content were used. The Chlorophyll meter (or SPAD meter) is a simple, portable diagnostic tool for measuring the greenness or the relative Chlorophyll concentration of leaves [5]. Compared with the traditional destructive methods, this equipment might provide a substantial saving in time, space and resources. However, to determine the Chlorophyll concentration in samples calibration curve between meter readings and the Chlorophyll concentration in the tissue of samples must be made. Recent research indicates a close link between leaf Chlorophyll concentration and leaf N content which make sense because most of leaf N is contained in Chlorophyll molecules [6]. Chlorophyll concentration or leaf greenness is affected by a number of factors, one being N status of the plant. Since the Chlorophyll meter has the potential to detect N deficiencies, it could be used as a tool for improving N management [6]. Nitrogen supply has large effect on leaf growth because it increases the leaf area of plants and accordingly, it influences on photosynthesis. Samples represent leaves of apricot growing in sub-rural, mostly, industrial area of Mostar and were collected from 15 trees affected by stress factors during the May/June 2011. The objectives of this study were to assess Chlorophyll fluorescence (Fv/Fm), Chlorophyll a content, SPAD values and total N content as responses to environmental changes that affect the leaves of apricot in the South area of Mostar. This study was carried out to determine if there was a correlation and regression relationships between measured parameters Fv/Fm, Chlorophyll a content, SPAD values and nitrogen content on leaves of apricot trees.
Chl fluorescence is the emitted light that is not used for the photosynthetic process. Therefore, the fluorescence change is a useful index to reflect the photosynthetic efficiency [7]. When exposed to a weak light after a few minutes residence in a dark place, a plant leaf emits an amount of Fo, dark or initial fluorescence. In this case, the light absorbed by Chlorophyll is not available for photosynthesis and is emitted as fluorescence. The Fm value, maximum fluorescence, is the fluorescence emission when a dark-adapted leaf is exposed to saturated light. In general, the Fo value increases as plant stress increases while the Fm value decreases. The ratio of Fo/Fm is commonly used to remove the dependence on the amount of Chlorophyll, thickness and age of the leaf since F0 and Fm values vary with these factors [8]. Maximum variable fluorescence Fvvariable fluorescence is the difference between Fm and Fo. The ratio Fv/Fm indicates a maximum potential of plant photosynthetic ability. The advantage of complex analysis such as OS 30p is in the parameters obtained, most of the PII, that can detect stress in plants before the appearance of visible symptoms on leaves [9] (Fm Fo)/Fm = Fv/Fm (1) Measurements of Chlorophyll fluorescence started at May/June after the first leaves fallen and newly was developed using a fluorometer OS 30p. 2.2 Estimation of Chlorophyll a Content (Chl a) A known amount of fresh leaf tissue 30 mg was homogenized in 1.5 mL of 100% acetone. Supernatant was withdrawn after centrifugation (5000 rmp) and absorbance was recorded at 662, 645 and 470 nm on UV-VIS spectrophotometer Lambda 25 [10]. Measurements were run in three replicates. During the experiment samples of renew leaves masse were collected for photosynthetic pigment analysis. The amount of Chlorophyll was calculated according to Lichtenthaler [11] by next formula:

2.1 Chlorophyll Fluorescence (Chl fluorescence) Measurements
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11.24 A662 - 2.04 A645 I 1000 m AX = spectrophotometric absorbance nm V = volume (mL) l = length of optical path = 1 cm m = fresh weight = 30 mg C hla =
(2)
between these two parameters. Fv/Fm values have varied from 0.75 to 0.83 which correspond to content of Chl a, from 0.16 mg/g FW to 0.41 mg/g FW. According to Bjrkmann and Demmig-Adams [15] the estimated value of Fv/Fm under optimal growing condition in higher plants is
2.3 Chlorophyll Meter Reading (SPAD Values) The mean of 30 readings from the portable Chlorophyll meter (SPAD-502, Minolta, Japan) was obtained for six leaf samples from individual tree. After readings, each leaf was cut in fine strips and used for mentioned analyses. The meter makes instantaneous and non destructive readings on a plant based on the quantification of light intensity (peak wavelength: approximately 650 nm: red LED) absorbed by the tissue sample. A second peak (peak wavelength: approximately 940 nm: infrared LED) is emitted simultaneous with red LED to compensate the thickness leaf [12]. 2.4 Nitrogen Content Assessment The total nitrogen (TN) was determined by the micro-Kjeldahl method [13] after submitting the plant material to oxidation by sulphuric digestion (H2SO4). 2.5 Statistic Analysis Microsoft office software 2007 Excel option for providing a regression model and descriptive statistic for measured parameters were used.
Fig. 1 Calibration curves for Fv/Fm values recorded by fluorometer OS-30p versus leaf Chlorophyll (Chl a) content detected by spectrometer in leaves collected from 15 individual apricot trees in May/June 2011.

The Chl fluorescence originates from de-excitation of the first excited single-state of Chl molecules. Chl b molecules do not emit fluorescence because excitation energy from Chl b is very fast and efficiently transferred to Chl a [14] and that is the reason way Chl fluorescence was compared with content of Chl a. Fig. 1 shows the relationship between Fv/Fm values and Chl a content correspond to the regression coefficient R2 = 0.55, indicating a good relationship
0.832 0.004 but it depends on some different parameters of plant species. Healthy plant leaves generally show Fv/Fm value of 0.83 and a ratio below 0.83 means that plants are under stress [16]. As shown in Fig. 2, the relationship between the SPAD values and the extracted Chl a content was linear with the high regression coefficient R2 = 0.741. A relationship between the SPAD readings and the leaf Chl a content has been established for several crop species [17-19] and the regression model was different among species in the reports. Relationship linearly increased among 25 to 39 SPAD unit and 0.15 to 0.43 mg g-1 Chl a (FW). According to the study of Thambavani and Kumar [20] on few ornamental tree species leaves measurement of Chlorophyll a content was higher in control site compared with polluted site. The amount of Chlorophyll a varied from 1.18 mg g-1 to 1.78 mg g-1 (FW) in control site and from 0.61 mg g-1 to 1.33 mg g-1 (FW) in the polluted site. Data from Cresswell and Weir [21] indicated the percentage leaf N associated with woody plant health ranges between 1.7% and 2.5% with values less than
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1.7% generally associated with low foliar N content. Regarding to obtained N values results varies from 2.1% to 2.9% of total leaf nitrogen showing sufficient nutrient level. Fig. 3 corresponds linear regression model with a good coefficient R2 = 0.75. Relationship becomes a stable between 25 to 39 SPAD units and
Fig. 2 Calibration curves for SPAD meter values versus leaf Chlorophyll (Chl a) content detected by spectrometer in leaves collected from 15 individual apricot trees in May/June 2011.
Fig. 3 Calibration curves for SPAD meter values versus leaf N (%) content detected by determined by the micro-Kjeldahl method from leaves collected from 15 individual apricot trees in May/June 2011.
2.1% to 2.9% N percentages in apricot leaves. SPAD values vary between vegetation season and fertilization treatment, cultivars and vegetation period and according to the Maji et al. [22] study SPAD meter have been accurate in predicting N level deficiency at potato cultivars. Measurements on leaves of fruit trees show much lower values of SPAD units [23] then on vegetables plants [22].
As all values from collected leaves on these 15 trees shows Fv/Fm values below 0.83 and from the Figures above 2, 3 is obvious that main nutrient is not in insufficient level, it was assumed on some abiotic factor which damaging the leaves of apricot fruit on the mentioned area. The leaves injuries were connected with some climatic changes according to the data from Hydrometeorology station in Mostar for 2011 and may be the fact that area where samples were collected is near industrial area where plants could be exposed to air pollutant. The mean annual temperature for Mostar in 2011 was 16.2 C and it was 1.6 C higher than mean annual temperature. The total year amount of rainfall was 872.5l m-2. The year 2011 was extremely warm and third driest in Mostar ever since first official measurement [24]. Variable weather conditions occurred in May/June. Air temperatures in the first half of May were below 0 C and in the second half of month were positive with deviations up to 3.8 C as was registered in Mostar. Total precipitations in June were below long-term average sum for the same period and ranged from 30.6l m-2. High temperatures at the beginning of the third decades of June and drought have led to the drying of leaves on most of the cultivated plants as it was recorded in statistical newsletter for meteorology in 2011. According to a measured parameter Chlorophyll a content, Fv/Fm values, N content, SPAD values descriptive statistic (mean, standard error, median, variation coefficient, standard deviation and min. and max. values) and the data are presented in the Table 1. Plants can maintain and preserve all their vital functions under large variations of the environmental conditions. Among them, incident irradiance, ambient temperature, humidity, supply of water, CO2 and nutrients are of main importance. Nevertheless, if the changes exceed the limit of tolerance serious damages in the structure and function of individual plant cells and organs may occur. According to the general stress theory, the consecutively induced status of the
Response of Chlorophyll a, SPAD Values and Chlorophyll Fluorescence Parameters in Leaves of Apricot Affected some Abiotic Factors Table 1 Descriptive statistic for measured parameters (Chl a, Fv/Fm, N and SPAD values). Chl a values 0.31 0.02 0.33 25.13 0.08 0.16 0.41 Fv/Fm values 0.79 0.01 0.79 3.03 0.02 0.75 0.83 N values 2.65 0.07 2.72 10.25 0.27 2.1 2.9 SPAD values 33.58 1.30 34.40 15.04 5.05 25 39.9
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Measured parameters Mean Standard error Median Variation coefficient (cv) Standard deviation Minimum Maximum
plant, termed stress, generates specific responses on a cellular level (involving general signal responses, adaptation syndromes, and defense responses) [25]. Stress leads either to an acquirement of new homeostasis or under strong stress dysfunction leads to necrosis of the affected organ according to the situation on field.
Acknowledgments
This paper is provided from the data in project Plant reaction to abiotic stress financed by Federal Ministry of education and science B&H.
References
[1] U. Schreiber, W. Bilger, H. Hormann, C. Neubauer, Chlorophyll fluorescence as a diagnostic tool: Basics and some aspects of practical relevance, in: A.S. Raghavendra, (Ed.), Photosynthesis: A Comprehensive Treatise, Cambridge: Cambridge University Press, 1998, pp. 320-336. H.K. Lichtenthaler, Vegetation stress: An introduction to the stress concept in plants, J. Plant Physiol. 148 (1996) 4-14. F. Babani, H.K. Lichtenthaler, P. Richter, Changes of Chlorophyll fluorescence signatures during greening of etiolated barley seedlings as measured with the CCD-OMA fluorometer, J. Plant Physiol. 148 (1998) 471-477. K. Maxwell, G.N. Johnson, Chlorophyll fluorescenceA practical guide, Journal of Experimental Botany 51 (2000) 659-668. K. Kariya, A. Matsuzaki, H. Machida, Distribution of Chlorophyll content in leaf blade of rice plant, Nihon Sakumotsu Gakkai Kiji 51 (1982) 134-135. T.A. Peterson, T.M. Blackmer, D.D. Francis, J.S. Scheppers, Using a Chlorophyll Meter to Improve N Management, A Webguide in Soil Resource Management: D-13, Fertility, Cooperative Extension, Institute of Agriculture and Natural Resources, University of Nebraska, Lincoln, NE, 1993. L.N.M. Duysens, H.E. Sweers, Mechanism of the two photochemical reactions in algae as studied by means of fluorescence, in studies on microalgae and photosynthetic bacteria, Jpn. Soc. Plant Physiology, University of Tokyo Press, Tokyo, Japan, 1963, pp. 353-372.
4. Conclusions
The distribution patterns of Fv/Fm within the plants varied as the all trees were not affected in an equivalent way. Different stress factors produced different patterns of change of Fv/Fm in individual plants. These results suggest that the Fv/Fm distribution pattern in an individual plant may change according to the stress factor imposed. Analyzing the Fv/Fm distribution pattern may be an effective way of identifying stress factors since it was found that N nutrient was sufficient in all collected samples which implies on some other abiotic factor in our case. Not only the OS-30p fluorometer could be used as useful tool for detecting disturbance in photosynthesis of leaf apparatus but in comparing with Chlorophyll measurement (Chl a), we can provide reliable results. Nutrient stress detection by SPAD meter have shown also accurate results which were confirmed by good correlation relationship with N content detected by laboratory method, even the SPAD values had shown lower units on apricot trees then on same vegetables plants. All this measurement could provide data for plant disturbance in physiological function but reliable value ranges for each species and environmental conditions should be research.
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Response of Chlorophyll a, SPAD Values and Chlorophyll Fluorescence Parameters in Leaves of Apricot Affected some Abiotic Factors O.V. Kooten, J.F.H. Snel, The use of Chlorophyll fluorescence nomenclature in plant stress physiology, Photosynth. Res. 25 (1990) 147-150. D. Christen, S. Schnmann, M. Jermini, R.J. Strasser, Dfago, G. Characterization, Early detection of grapevine (Vitis vinifera) stress response to esca disease by in situ Chlorophyll fluorescence and comparison with drought stress. Environmental and Experimental Botany (2007) 60 504-514. D. Arnon, Plant Physiology 24 (1949) 1- 15. H.K. Lichtenthaler, Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes, Methods in Enzymology 148 (1987) 350-382. Minolta Camera Co. Ltd., Chlorophyll meter SPAD-502, Instruction Manual, Radiometric Instruments, Divisions, Osaka, Minolta, 1989, p. 22. Malavolta, E., Vitti, G.C., de Oliveira, J.G. Avaliacao do estado nutricional das plantas: Princpios e Aplicacoes. POTAFOS, Piracicaba, 1989, pp. 201. G. Papageorgiou, Bioenergetics of photosynthesis, in: Govindjee (Ed.), Academic Press, New York, London, 1975, p. 319. O. Bjrkman, B. Demmig-Adams, Photon yield of O2 evolution and Chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins, Planta 170 (1987) 489-504. H.K. Lichtenthaler, Application of Chlorophyll fluorescence in photosynthesis research, stress physiology, hydrobiology, and remote sensing, Kluwer Academic Publishers, Boston, USA, 1988, pp. 384. A. Yamamoto, T. Nakamura, J.J. Adu-Gyamfi, M. Saiqusa, Relationship between Chlorophyll content in leaves of sorghum and pigeon pea determined by extraction method and by Chlorophyll meter (SPAD-502), J. Plant Nutr. 25 (2002) 2295-2301. A.T. Netto, E. Campostrini, J.G.A. de Oliveira, R.E. Bressan-Smith, Photosynthetic pigments, nitrogen, Chlorophyll a fluorescence and SPAD-502 readings in coffee leaves, Scientia Horticulturae 104 (2005) 199-209. F.B. Fritschi, J.D. Ray, Soybean leaf nitrogen, Chlorophyll content, and Chlorophyll a/b ratio, Photosynthetica, 45 (2007) 92-98. S.D. Thambavani, S.R. Kumar, The monthly changes of chloroplast pigments content in selected plant species exposed to cement dust pollution, Journal of Research in Biology 8 (2011) 660-666. G.C. Cresswell, R.G. Weir, Plant Nutrient DisordersOrnamental Plants and Shrubs, Inkata Press, Melbourne, Australia, 1997, pp. 132221. A. Maji, M. Poljak, Z. Knezovi, A. Sabljo, E. Sefo, Chlorophyll Meter as diagnostic tool for nitrogen fertilization management in potato crop (Solanum tuberosum L.), 16th Nitrogen Workshop Turin, Italy, 2009, pp. 253-254. C.B. Netoab, C. Carrancaa, J. Clementec, A. de Varennesc, Assessing the nitrogen nutritional status of young non-bearing Rocha pear trees grown in a Mediterranean region by using a Chlorophyll meter, Journal of Plant Nutrition 34 (2011) 5. Hydro meteorological newsletter of Federation B&H: Climatologically analysis of year, 2011. H. Selye, Nature 138 (1936) 32.
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DAVID PUBLISHING
Optimization of High EPA Structured Phospholipids Synthesis from -3 Fatty Acid Enriched Oil and Soy Lecithin
Teti Estiasih1, Kgs Ahmadi2, Erliana Ginting3 and Arya Ulil Albab1
1. Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University Jl. Veteran, Malang 65141, Indonesia 2. Department of Agroindustrial Technology, Faculty of Agriculture, Tribhuwana Tunggadewi University Jl. Telaga Warna, Tlogomas, Malang 65144, Indonesia 3. Indonesian Research Institute for Legumes and Tuber Crops (ILETRI), Kendalpayak, Malang, Indonesia Received: October 15, 2012 / Published: January 20, 2013. Abstract: The molecular structure of phospholipids can be changed enzymatically to obtain different tailor-made phospholipids. Incorporation of -3 fatty acids into phospholipids structure increased their oxidative stability, suggesting more health beneficial phospholipids. This study aimed to optimize eicosapentaenoic acid (EPA) incorporation into phospholipids structure by acidolysis reaction using free lipase (EC 3.1.1.3) from Rhizomucor miehei. Deoiled soy lecithin from anjasmoro variety was used as phospholipids source, while -3 fatty acid enriched oil was used as acyl source. Oil enriched with -3 fatty acids was obtained from low temperature solvent crystallization of lemuru (Sardinella longiceps) by-product. Response surface methodology (RSM) was used in this study to determine the relationship between the three factors (enzyme concentration, reaction time and substrate ratio) and their effects on EPA incorporation into soy lecithin structure. The results showed that the relation between EPA content with three factors (reaction time, enzyme concentration and substrate ratio) was quadratic. The significant factors were substrate ratio and reaction time. Optimum conditions at a ratio of 3.77:1 between -3 fatty acids enriched oil and soy lecithin, 30% lipase concentration, and 24.08 h reaction time, gave 22.81% of EPA content of structured phospholipids. Key words: Structured phospholipids, enzymatic acidolysis, EPA, lipase, deoiled soy lecithin, -3 fatty acids enriched oil, lemuru.
1. Introduction
Soy lecithin or phospholipids (PL) is widely used as an emulsifier in food industries. Soy lecithin comprises of 18% phosphatidylcholine (PC), 14% phosphatidylethanolamine (PE), 9% phosphatidylinositol (PI), 2% phosphatidic acid (PA), other minor phospholipids, 11% glycolipid, and 27% neutral lipid [1]. Some studies reported that the major fatty acid in soy lecithin was linoleic acid [2, 3]. Modification is usually performed to obtain desired
Corresponding author: Teti Estiasih, PhD, research fields: lipid chemistry and technology. E-mail teties@yahoo.co.id and teties@ub.ac.id.
functional properties of lecithin. The molecular structure of phospholipids can be changed enzymatically or chemically to obtain different tailor-made technological and/or physiological properties compared to those of natural substrate. Enzymatic modification being of interest as enzymes can be used to modify phospholipids in different ways [4]. Structured phospholipids with certain fatty acid profile can be obtained by enzymatic synthesis [5]. This synthesis process gives benefits as the reaction conditions are not extreme and the enzyme is highly specific [6]. Incorporation of -3 fatty acids into phospholipids structure increases their oxidative
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stability [7], suggesting more health beneficial phospholipids. The health beneficial effects of -3 PUFA are currently well known and have been almost exclusively attributed to cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) [8, 9]. The beneficial effects of EPA include reductions in heart diseases severity and various inflammatory disorders [10, 11]. EPA has been used in the prevention and treatment of atherosclerosis, arthritis, thrombosis, inflammation, diabetes, and cancer [12]. EPA also exhibits various physiological functions on being incorporated into membrane phospholipids [13, 14]. Some studies showed that enzymatic incorporation of -3 fatty acids into phopsholipids structure used lipase or phospholipase. A mobile lipase from Rhizomucor meihei (Lipozyme TM) with 1,3 regiospecific had been used by Haraldsson and Thorarensen [15] in PC containing EPA and DHA synthesis. Recent research suggested egg yolk was used as a source of PC and acyl source was -3 fatty acids concentrates. However, information on structured phospholipids synthesis using specific EPA incorporation is still lacking. Almost all of the acidolysis studies in structured phospholipids However, synthesis on used certain type of phospholipids, such as PC as a substrate [6, 15-20]. studies structured phospholipids synthesis using a mixture of phospholipids such as soy lecithin are still limited. Therefore, there is a call for research in different properties of phospholipid forms in soy lecithin. In general, enzyme used in structured phospholipids synthesis is specific 1,3-lipase and PLA2, which can modify fatty acid in sn-1 and sn-2 positions. Incorporation of such fatty acid into phospholipids structure can be controlled during reaction [21]. Some factors that may affect acidolysis reaction in -3 fatty acids incorporation into phospholipids structure are enzyme concentration, reaction time, and ratio of acyl
to phospholipids [15]. While enzymes used for structured phospholipids synthesis are lipase that may derive from Rizhomucor miehei [15, 17] or Thermomyces lanuginose [5, 21], and phospholipase A1 [12]. This study aimed to optimize EPA incorporation into soy lecithin from anjasmoro soybean variety. Anjasmoro is one of improved varieties cultivated in Indonesia that has high productivity (2.3 t/ha) and tolerant to puddle. As a acyl source, we used -3 fatty acid enriched oil that was prepared from a by-product of Lemuru (Sardinella longiceps) canning processing using low temperature solvent crystallization. This study focused on the generation of soy lecithin that is highly enriched with EPA from inexpensive EPA source. Response surface methodology (RSM) was used in this study to determine the relationship between the three factors (enzyme concentration, reaction time and substrate ratio) and their effect on EPA incorporation into soy lecithin structure during lipase-catalyzed acidolysis.

2.1 Materials Crude soy lecithins were obtained from water degumming of soybean oil extracted from anjasmoro variety. Lemuru (Sardinella longiceps) oil as a by-product of fish meal processing was purchased from a local fish meal plant. -3 fatty acids enriched oil was obtained using low solvent crystallization of hydrolyzed lemuru oil. Other materials were lipase derived from Rhizomucor meihei, fatty acid methyl ester mixture, phospholipids standard, and BF3-methanol 14% (Sigma Co.), other chemical reagents, and TLC plate (silica gel G60 F254) (Merck). 2.2 Soy Lecithin Preparation from Anjasmoro Soybean Variety Lecithin extraction was performed according to Eshratabadi [22] with a minor modification. Lecithin
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was extracted from soybean oil using water degumming. About 20 g 0.1 mg of soybean oil was placed in a beaker glass and 10% (w/v reaction mixture) distilled water and 3 mL of hexane was added. The mixture was stirred and heated at 60 C for 30 min. Oil and water phase were separated by centrifugation at 300 rpm for 30 min. Gum or crude lecithin was formed in the bottom layer (subnatant) and then dried in a vacuum oven at 25 C and 1 atm for 1 h. This dried crude lecithin was then purified using the method employed by Nasir et al. [23] to remove neutral oil. Acetone was added into the crude lecithin with the ratio of 6:1 (v/w). The mixture was stirred for 1 h. The solvent was decanted to separate the lecithin. This treatment was repeated until the solvent was colorless. Lecithin was flushed with nitrogen to discard residual solvent. 2.3 Acidolysis Reaction of Oil Enriched with -3 Fatty Acids and Deoiled Soy Lecithin
Table 1 No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
This activity was referred to Haraldsson and Thorarensen [15] with a slight modification. Oil enriched with -3 fatty acids and soy lecithin (phospholipids) were put into a number of reaction tubes at different ratios as shown in Table 1. Free enzyme of Lipozyme (Rhizomucor miehei) at different concentrations (Table 1) were subsequently added. The mixture was shaked slowly at 300 rpm in a shaking water bath with temperature of 40 C for different reaction times (Table 1). Afterwards, the mixture was centrifuged at 1,500 rpm for 15 min to precipitate structured phospholipids. Then the precipitate was washed with 1 mL hexane to discard residual free fatty acid. The optimized response in this study was EPA content in structured phospholipids. Data were analyzed using Design Expert 7.0 Trial Version. Structured phospholipids from optimum conditions were analyzed for Hydrophilic Lipophilic Balance (HLB) (saponification number and acid value) according to the method of AOCS [24], phospholipids
Second ordo central composite design with three factors to optimize EPA content of structured phospholipids. Actual variable Substrate ratio* 2.5:1 2.5:1 2.5:1 2.5:1 4.5:1 4.5:1 4.5:1 4.5:1 3.5:1 3.5:1 3.5:1 3.5:1 3.5:1 3.5:1 1.818:1 5.182:1 3.5:1 3.5:1 3.5:1 3.5:1 Enzyme concentration** 10 10 30 30 10 10 30 30 20 20 20 20 20 20 20 20 3.18 36.82 20 20 Reaction time (h) 15.84 18.46 19.78 16.77 21.24 15.51 19.38 21.57 19.70 23.38 20.39 24.26 23.84 19.34 14.16 16.07 18.88 22.02 12.28 13.75 Coded variable X1 X2 -1 -1 -1 -1 -1 +1 -1 +1 +1 -1 +1 -1 +1 +1 +1 +1 0 0 0 0 0 0 0 0 0 0 0 0 -1.682 0 +1.682 0 0 -1.682 0 +1.682 0 0 0 0 X3 -1 +1 -1 +1 -1 +1 -1 +1 0 0 0 0 0 0 0 0 0 0 -1.682 +1.682 Response of EPA content (%) 15.84 18.46 19.78 16.77 21.24 15.51 19.38 21.57 19.70 23.38 20.39 24.26 23.84 19.34 14.16 16.07 18.88 22.02 12.28 13.75
*oil enriched with -3 fatty acids to deoiled soy lecithin, **% w/w substrate.
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profile, and fatty acid profile. 2.4 Analysis of Phospholipids Phospholipids analysis was performed using TLC according to Nzai and Proctors method [25] with slight modification. About 1 mg of sample or standard was dissolved in chloroform:methanol (95:5 v/v) and 10 L of sample or standard solution was spotted into TLC plate. The TLC plate was activated by heating for 10 min at 100 C prior to analysis. The plates were developed for 40 min in a mobile phase solution containing chloroform, methanol, and distilled water at a ratio of 75:25:3 (v/v/v). Visualization was performed by charring using H2SO4:distilled water (1:1), and quantification by TLC scanner at 500 nm wavelength (Dual Wave Length Chromato Scanner CS-930 and Data Recorder DR-2 Shimadzu). 2.5 Fatty Acid Analysis Fatty acid profile of -3 fatty acids enriched oil, deoiled soy lecithin, and structured phospholipids from optimum conditions of acidolysis reaction were analyzed using gas chromatography (Shimadzu GC 8A). The column used for separation was capillary CBP20 0.25 m bonded silica column with dimension of 50 mm in length, i.d. 0.22 mm and o.d. 0.33 mm.
Table 2 Fatty acid Lauric acid (C12:0) Myristic acid (C14:0) Palmitic acid (C16:0) Palmitooleic acid (C16:1-7) Stearic acid (C18:0) Oleic acid (C18:1-9) Linoleic acid (C18:2-6) Linolenic acid (C18:3-3) Arachidonic acid (C20:4-3) Docosanoic acid (C22:0) Docosaenoic acid (C22:1) Eicosapentaenoic acid/EPA (C20:5-3) Docosahexaenoic acid/DHA (C22:6-3) EPA + DHA nd = not detected.
Nitrogen was used as a gas carrier with a pressure of 200 kg/m2, while for supporting and burning gas, air and hydrogen were used with a pressure of 0.15 kg/cm2 and 0.6 kg/cm2, respectively. Injector, column, and detector temperature was 230 C, 250 C and 230 C, respectively. Samples and standard were injected at the volume of 2 L. Methylation of phospholipids was performed according to Park and Goins method [26]. Analysis of fatty acid composition of phospholipids was conducted after phospholipids separation using TLC. Each spot was scrapped off and extracted according to Christie [27]. Derivatization prior to GC analysis was performed using Park and Goins method [26].
3. Results and Discussions

The predominant fatty acids in -3 fatty acids enriched oil were EPA and DHA (Table 2). In original lemuru oil, the EPA and DHA content were 15.87% and 13.58%, respectively. Meanwhile, EPA and DHA content in enriched oil were 1.79 and 1.72 times higher than the original oil. Lemuru oil is a good source of EPA rather than DHA. According to Howe et al. [28], fish oil generally contains higher EPA than DHA, except for tuna oil. Hence, in this study, we used lemuru oil as the source of EPA in the
Fatty acids profile of -3 fatty acids enrihed oil, deoiled soy lecithin, and high EPA structured phospholipids. De-oiled soy lecithin 0.60 1.29 26.97 1.05 4.80 11.86 48.64 5.26 1.37 3.59 1.40 nd nd nd -3 fatty acids enrihed oil nd nd 3.33 19.57 5.00 15.68 2.79 1.90 nd nd nd 28.36 23.38 51.74 High EPA structured phospholipids 0.53 0.39 1.79 0.80 nd 1.23 54.52 3.94 nd nd 7.82 24.36 4.13 28.49
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preparation of high EPA structured phospholipids from soy lecithin. Linoleic acid was a predominant fatty acid obtained in soybean lecithin derived from Anjasmoro variety (Table 2). Normally, soybean oil has high linoleic acid content that may comprise 55.83% of the total fatty acids [29]. Soybean lecithin consisted of 32.63% PI, 29.18% PC, 27.15% PE and 11.01% PA with the order PI > PC > PE > PA (Table 3). Wu and Wang [1] reported that soy lecithin had 18% PC, 14% PE, 9% PI, 2% PA, 2% minor PL, 11% glycolipid, 5% sugar complex and 37% neutral lipid. Commercial lecithin has different phospholipids composition with a predominant phospholipids is PC or PE [30]. Higher PC or PE concentration in commercial lechitin is due to fractionation treatment. Wang [3] reported that predominant fatty acid in PE or PC was linoleic acid, followed by palmitic acid. The results of optimization revealed that the suitable model for acidolysis reaction with three factors (enzyme concentration, reaction time, ratio of oil enriched with -3 fatty acids to soy lecithin) was quadratic. However, the model was not significant. Factors that affected the response in quadratic manner were the ratio of -3 fatty acids enriched oil to soy lecithin and reaction time, while the effect of enzyme concentration was not significant. The effect of substrate ratio and enzyme concentration was quadratic (Fig. 1). At high enzyme concentration, enzyme was sufficient for hydrolysis of fatty acid from original phospholipids, and subsequently esterification of -3 fatty acid enriched oil into phospholipids structure. At low enzyme
Table 3 Phospholipids profile of deoiled soy lecithin and structured phospholipids. Deoiled soy lecithin Phosphatidylinositol 32.63 Phosphatidylcholine 29.18 Phosphatidylethanolamine 27.15 Phosphatidic acid 11.01 Phospholipids High EPA structured phospholipids 22.96 25.85 22.32 28.87
concentration, it was supposed that enzyme would hydrolyze fatty acid from phospholipids structure, however, the synthesis was limited. This led to low EPA content of structured phospholipids. Similarly, increasing substrate ratio also enhanced EPA content, but then it decreased. According to Vikbjerg et al. [5], fatty acid composition of structured phospholipids depended on the substrate ratio (mole of acyl donor/mole of phospholipids) and incorporation of acyl increased a long with the availability of acyl or mole substrate ratio. This study showed that increasing acyl donor decreased incorporation of EPA into phospholipids structure. Previous study reported that through different ratios of PC to palmitic acid as substrate (1:2 to 1:10) and 20% enzyme used, the maximum incorporation obtained at the ratio of 1:5. Increasing substrate ratio from 1:5 to 1:10, however, did not increase the incorporation level [6]. This study showed that high amounts of acyl donor did not always increase the level of incorporation. This was due to the preference of enzyme to hydrolyze and esterify acyl from and into phospholipids structure. At high acyl concentration, reaction tended to synthesize structured phospholipids. However, an increase of acyl availability could limit the hydrolysis due to inhibition effect of acyl [31]. The effect of substrate ratio and reaction time on the EPA incorporation into structured phospholipids was quadratic (Fig. 2). Maximum incorporation obtained at certain reaction time. Longer reaction time did not increase EPA incorporation into phospholipids structure. Reddy et al. [6] observed that incorporation of palmitic acid and stearic acid into PC was maximum at reaction time of 24 h. Extended reaction time did not increase the level of incorporation. That finding was similar to thoseresults obtained in this study. After maximum reaction time, longer reaction time was supposed to lead the hydrolysis, including hydrolysis of incorporated EPA from structured phospholipids. Enzymatic reaction was hydrodynamic
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a b Fig. 1 Response surface (a) and contour (b) of enzyme concentration and substrate ratio in acidolysis reaction of high EPA structured phospholipids synthesis.
process and lipase was able to hydrolyze and synthesize depending on particular factors. One of them was water availability that stimulated reaction into hydrolysis. The availability of water in this study was limited to 10% (w/w substrate) that aimed to promote esterification reaction. The effect of reaction time and enzyme concentration on EPA incorporation into structured
phospholipids was also quadratic (Fig. 3). High incorporation of EPA tended to occur at high enzyme concentration. At low enzyme concentration, increasing reaction time enhanced EPA incorporation. However, longer reaction time decreased incorporated EPA of structured phospholipids. It was supposed that at a low enzyme concentration, hydrolysis of fatty acid
a b Fig. 2 Response surface (a) and contour (b) of reaction time and substrate ratio in acidolysis reaction of high EPA structured phospholipids synthesis.
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a b Fig. 3 Response surface (a) and contour (b) of reaction time and enzyme concentration in acidolysis reaction of high EPA structured phospholipids synthesis.
from original phospholipids occurred at the beginning of reaction, followed by esterification of EPA. At long reaction time, incorporated EPA can be hydrolyzed from the structured phospholipids. Optimum incorporation of EPA was supposed to be higher than enzyme concentration of 30% and reaction time about 24 h (Fig. 3). Among the three factors, ratio of substrate and reaction time gave significant effect on EPA content and the relation was quadratic (Fig. 2). The quadratic equation to predict the response was Y = -36.76 9.059X1 + 2.05X2 + 3.78X3 0.033X1X2 + 0.19X1X3 0.031X2X3 + 0.70X12 0.029X22 0.056X32 with X1 = ratio of oil enriched with -3 fatty acids to soy lecithin, X2 = enzyme concentration, X3 = reaction time, and Y = EPA content (%) of structured phospholipids. Optimum conditions were obtained at a ratio of oil enriched with -3 fatty acids to soy lecithin 3.77:1, enzyme concentration 30%, and reaction time 24.08 h. At these optimum conditions, the response was predicted to be 22.81% of EPA. Verification showed that the response of EPA content of structured phospholipids was 24.23%. There was an interesting phenomenon in fatty acid composition of structured phospholipids (Table 2). Incorporation of EPA decreased palmitic acid content sharply, while linoleic acid increased. This reflected that palmitic acid was replaced by EPA in phospholipids structure. Acidolysis reaction using
lipase was reversible [32]. At the beginning, lipase hydrolyzed fatty acids from phospholipids structure. Afterwards, EPA from -3 fatty acids enriched oil was esterified. Therefore, the increase of linoleic acid content was due to hydrolysis of other fatty acids rather than linoleic acid from phospholipids structure. HLB value of structured phospholipids was 8.24, while original soy lecithin had HLB of 5.0. This suggested that high EPA structured phospholipids was suitable for oil in water emulsion. Fatty acid composition of structured phospholipids would influence the HLB value [33]. Increasing desaturation of fatty acid attached to phospholipids structure by incorporation of EPA, increased the polarity, as well as the HLB value. Additionally, fatty acid composition of structured phospholipids may affect its functional properties [34].
4. Conclusions
Factors affecting high EPA structured phospholipids synthesis were ratio of substrates and reaction time. Relation of three factors (ratio of substrates, reaction time, and enzyme concentration) was quadratic. EPA was supposed to replace palmitic acid in phospholipid structure. Optimum condition was achieved at ratio of oil enriched with -3 fatty acids to soy lecithin 3.77:1, enzyme concentration 30%, and reaction time 24.08 h. At these optimum conditions, the response was predicted to be 22.81%
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Optimization of High EPA Structured Phospholipids Synthesis from -3 Fatty Acid Enriched Oil and Soy Lecithin [12] S.P.J.N. Senanayake, F. Shahidi, Structured lipids containing long-chain Omega-3 polyunsaturated fatty acids, in: F. Shahidi (Ed.), Seafood in Health and Nutrition, Transformation in Fisheries and Aquaculture Global Perspectives, Science Tech Publishing, St. Johns, NF, Canada, 2000, pp. 29-44. [13] P. Mancuso, J. Whelan, S.J. De Michele, C.C. Snider, J.A. Guszcza, K.J. Claycombe, et al., Effects of eicosapentaenoic and -linolenic acids on lung permeability and alveolar macrophage eicosanoid synthesis in endotoxic rats, Crit. Care Med. 25 (1997) 523-532. [14] J.E. Gadek, S.J. De Michele, M.D. Karlstad, E.R. Pacht, M Donahoe, T.E. Albertson, et al., Effect of enteral feeding with eicosapentaenoic acid, -linolenic acid, and antioxidants in patients with acute respiratory distress syndrome, Crit. Care Med. 27 (1999) 1409-1420. [15] G.G. Haraldsson, A. Thorarensen, Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase, J. Am. Oil Chem. Soc. 76 (1999) 1143-1149. [16] A. Mustranta, T. Suorti, K. Poutanen, Transesterification of phospholipids in different reaction conditions, J. Am. Oil Chem. Soc. 71 (1994) 1415-1419. [17] D. Adlercreutz, E. Wehtje, An enzymatic method for the synthesis of mixed-acid phosphatidylcholine, J. Am. Oil Chem. Soc. 81 (2004) 553-557. [18] M. Hosokawa, K. Takahashi, N. Miyazaki, K. Okamura, M. Hatano, Application of water mimics on preparation of eicosapentaenoic and docosahexaenoic acid containing glycerolipids, J. Am. Oil Chem. Soc. 72 (1995) 421-425. [19] A.M. Aura, P. Forsell, A. Mustranta, K. Poutanen, Transesterification of soy lecithin by lipase and phospholipase, J. Am. Oil Chem. Soc. 72 (1995) 1375-1379. [20] J.T. Lin, S. Wani, X. He, T. Nguyen, T.A. McKeon, Incorporation of laurate and hydroxylaurate into phosphatidylcholines and acylglycerols in castor microsomes, J. Am. Oil Chem. Soc. 82 (2005) 495-499. [21] A.F. Vikbjerg, H. Mu, X. Xu, Monitoring of monooctanoyl phosphatidylcholine synthesis by enzymatic acidolysis between soybean phosphatidylcholine and caprylic acid by thin-layer chromatography with a flame ionization detector, Agric. Food Chem. 53 (2005) 3937-3942. [22] P. Eshratabadi, Effect of different parameters on removal and quality of soybean lecithin, Res. J. Bio. Sci. 3 (2008) 874-879. [23] M.I. Nasir, M.A. Bernards, P.A. Charpentier, Acetylation of soybean lecithin and identification of components for solubility in supercritical carbon dioxide, J. Agric. Food Chem. 55 (2007) 1961-1969. [24] AOCS, Official Methods and Recommended Practices of the American Oil Chemistry Society, 5th ed., AOCS, Champaign, Illinois, 2001.
of EPA, meanwhile, verification showed 24.23%. Further work is needed to know degree of EPA incorporation into phospholipids classes as well as physiological effects of high EPA structured phospholipids ingestion.
Acknowledgments
The authors thank to Directorate General of Higher Education, Ministry of Education and Culture, Republic of Indonesia for the funding of this work through Strategic National Research Grant with Grant Contract No. 404/SP2H/PL/Dit.Litabmas/IV/2011.
References
[1] Y. Wu, T. Wang, Fractionation of crude soybean lecithin with aqueous ethanol, J. Am. Oil Chem. Soc. 81 (2004) 697-704. [2] S. Das, D.K. Bhattacharyya, Preparation and surface-active properties of hydroxy and epoxy fatty acid-containing soy phospholipids, J. Am. Oil Chem. Soc. 83 (2006) 1015-1020. [3] G. Wang, T. Wang, Oxidative stability of egg and soy lecithin as affected by transition metal ions and pH in emulsion, J. Agric. Food Chem. 56 (2008) 11424-11431. [4] A.F. Vikbjerg, J.Y. Rusig, G. Jonsson, H. Mu, X. Xu, Comparative evaluation of the emulsifying properties of phosphatidylcholine after enzymatic acyl modification, J. Agric. Food Chem. 54 (2006) 3310-3316. [5] A.F. Vikbjerg, L. Peng, H. Mu, X. Xu, Continuous production of structured phospholipids on a packed bed reactor with lipase from Thermomyces lanuginose, J. Am. Oil Chem. Soc. 82 (2005) 237-242. [6] J.R.C. Reddy, T. Vijeeta, M.S.L. Karuna, B.V.S.K. Ra, R.B.N. Prasad, Lipase-catalyzed preparation of palmitic and stearic acid-rich phosphatidylcholine, J. Am. Oil Chem. Soc. 82 (2005) 727-730. [7] A.M. Lyberg, E. Fasoli, P. Adlercreutz, Monitoring the oxidation of docosahexaenoic acid in lipids, J. Am. Oil Chem. Soc. 40 (2005) 969-979. [8] M.E. Stansby, Nutritional properties of fish oil for human consumptionearly development, in: M. Stansby (Ed.), Fish Oils in Nutrition, Van Nostrand Reinhold, New York, 1990, pp. 268-288. [9] J.A. Nettleton, Omega-3 Fatty Acids and Health, Chapman and Hall, New York, 1995. [10] J. Jumpsen, M.T. Clandinin, Brain Development: Relationship to Dietary Lipid and Lipid Metabolism, AOCS Press, Champaign, 1995. [11] B.F. Haumann, Nutritional aspects of n-3 fatty acids, Inform 8 (1997) 428-447.
Optimization of High EPA Structured Phospholipids Synthesis from -3 Fatty Acid Enriched Oil and Soy Lecithin [25] J.M. Nzai, A. Proctor, Phospholipids determination in vegetable oil by thin layer chromatography and imaging densitometry, Food Chem. 63 (1998) 571-576. [26] P.W. Park, R.E. Goins, In situ preparation of fatty acids methyl ester for analysis of fatty acids composition in foods, J. Food Sci. 59 (1994) 1262-1266. [27] W.W. Christie, Lipid Analysis, 2nd ed., Pergamon Press, USA, 1982. [28] P.R.C. Howe, J.A. Downing, B.F.S. Grenyer, E.M. Grigonis-Deane, W.L. Bryden, Tuna fishmeal as a source of dha for n-3 pufa enrichment of pork, chicken, and eggs, Lipids 37 (2002) 1067-1076. [29] E.A.A. Sanibal, J. Mancini-Filho, Frying oil and fat quality measured by chemical, physical, and test kit analyses, J. Am. Oil Chem. Soc. 81 (2004) 847-852. [30] W. Nieuwenhuyzen, M.C. Toms, Update on vegetable lecithin and phospholipid technologies, Eur. J. Lipid Sci. Technol. 110 (2008) 472-474.
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[31] H.F. Castro, P.C. Oliveira, E.B. Pereira, Influence of substrate partition coefficient on the performance of lipase catalyzed synthesis of citronellyl acetate by alcoholysis, Braz. J. Chem. Eng. 17 (2000) 1-4. [32] K.D. Mukherjee, Lipid biotechnology, in: C.C. Akoh and D.B. Min (Eds.), Food Lipids: Chemistry, Nutrition, and Biotechnology, Second Edition, Revised and Expanded, Marcel Dekker Inc., New York, 2002, p. 783. [33] H.G. Bueschelberger, Lecithins, in: R.J. Whitehurst (Ed.), Emulsifiers in Food Technology, Blackwell Publishing Ltd., Oxford, 2004, pp. 6-10. [34] T. Estiasih, K. Ahmadi, F.C. Nisa, Synthesis of Natural Emulsifier from a Byproduct of Fish Canning Processing and Phospholipids from Waste of Palm Oil Processing, Fundamental Incentive Research Report, Research Center, University of Brawijaya, Malang, Indonesia, 2009.
DAVID
PUBLISHING
Effect of Radiant Energy Vacuum on Physical and Microbial Properties of Beef Jerky
John Scott Church1, Carley Marie MacIntyre2, Wade Robert Archambault3, Paul Edward Moote2, Jason Laco Cochran4, Timothy Douglas Durance5 and Jonathan Douglas Van Hamme2
1. Department of Natural Resource Sciences, Thompson Rivers University, Kamloops, British Columbia V2C0C8, Canada 2. Department of Biological Sciences, Thompson Rivers University, Kamloops, British Columbia V2C0C8, Canada 3. Department of Physical Sciences, Thompson Rivers University, Kamloops, British Columbia V2C0C8, Canada 4. Department of Culinary Arts, Thompson Rivers University, Kamloops, British Columbia V2C0C8, Canada 5. Department of Food Science, University of British Columbia, Vancouver, British Columbia V6T1Z4, Canada Received: October 3, 2012 / Published: January 20, 2013. Abstract: To test the feasibility of radiant energy vacuum (REV) dehydration, a technology that couples microwave cooking with vacuum pressure, on beef jerky preparation, the physical and microbial properties of the final jerky product was compared to conventional preparation methods. Physical characteristics assessed using puncture and shear tests of samples prepared using REV dehydration compared to the traditional method were not statistically different (P < 0.05). Moisture content and water activity levels were also very similar between the two products. To test microbiological quality, samples were homogenized in a stomacher and a variety of 3 M Petrifilms were used to evaluate the microbial load. Raw beef harboured low numbers of microbes, but the post-marination pasteurization/smoking step used in both treatments eliminated all culturable microorganisms tested for. To further investigate the ability of REV dehydration to kill microbes, samples were spiked with Listeria innocua after the pasteurization/smoking step but prior to REV dehydration. Samples were taken at different time intervals for microbial enumeration, and a decimal reduction time of 1 min was calculated, with 99.99% of 1.98 107 CFU g-1 Listeria being killed in five min. Improved drying times were observed for jerky samples prepared using the REV method offering potential energy savings during jerky preparation. Key words: Microwave, food safety, dehydration, preservation, Listeria innocua, beef jerky.
1. Introduction
In the last decade, the market for meat snacks, of which over 80% is beef based, has grown by 14% [1]. Jerky, which is among the best known of the meat snacks, is very popular with consumers such as hikers, bikers and travelers for reasons such as its compact nature, shelf stability and high protein content. The name jerky is derived from the Spanish word Charque, meaning dried meat. Jerky is typically made by slicing meat into strips and salting and drying to improve shelf life. The increasing market share for shelf-stable meat products increases the need for
Corresponding author: John Scott Church, researcher, research field: meat science. E-mail: jchurch@tru.ca.
alternate, efficient processing methods. Jerky is in high demand, the typical jerky consumer is a 35 to 54 year old male, and data indicate that if jerky were softer and easier to chew, it would be more appealing to woman and children [1]. Texture is affected by drying jerky at high temperatures for extended periods of time, and as product quality mainly relies on textural properties, it is vital to investigate the effects of different process parameters such as method of drying [2]. Previous attempts to prepare softer jerky by incorporating dehydrated vegetables with meat failed in sensory evaluations due to the unappealing appearance [2]. In addition, the American Association of Meat Processors [3] has reported that traditional jerky processing either at
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home or in commercial smokehouses takes between 6-10 h at 60 C. Reductions in the typical processing time could lead to substantial reductions in cost, while increasing production efficiency [4]. During jerky preparation, drying reduces water content in order to prevent undesirable changes in both the sensory quality and nutritive value of the final product through the control of microbial growth. The high water content of raw meat makes it extremely perishable, making it a good target for preservation by dehydration. In general, drying reduces the water content of perishable products such as meat, which ultimately lowers the water activity to the point where micro-organisms are no longer able to access sufficient water necessary for their growth. However, the choice of drying process also impacts the color, texture and odor of the final meat product because, along with the transient heat and mass transfer associated with water removal, structural transformations to the final product occur such as shrinkage, puffing or crystallization [5, 6]. During conventional drying, the heat required to evaporate moisture is transmitted inward through the moist material from the surface. Dielectric heat generated by microwave radiation volumetrically heats material internally through electromagnetic waves, requiring approximately 75% less energy to dry materials compared to conventional methods [7]. An interaction occurs in a microwave field which forces the polar molecules, such as water, and ionic species to constantly attempt to realign themselves by reversing an electric field around the food product. This molecular movement is extremely fast, and the molecular friction produced by dipole rotation and by the migration of ionic species under the influence of the oscillating electromagnetic field, generates heat inside the food by energy dissipation, which dramatically increases the drying rate [8]. In addition, microwave heating is drawing attention from researchers in developing novel pasteurization and sterilization processes for packaged foods. It has been
demonstrated that microwaves can be used for short time surface sterilization of sliced beef in gravy prepackaged in polymeric trays, enhancing food safety [9]. While microwave drying is more rapid, uniform and energy efficient than conventional hot air drying, it can sometimes improve product quality [10]. It is highly unlikely, however, that bulk water removal by microwave heating alone will be successful, or show an economic advantage [11]. During the falling rate period, because of the evaporative cooking effect and low thermal conductivity, high product temperatures will be difficult to obtain, and anticipated surface hardening and thermal gradients will provide further resistance to moisture transfer, as well as reduce jerky texture [11]. Radiant energy vacuum (REV) has been developed as a novel way of dehydrating food products. By combining microwave energy within a vacuum, a product can be dehydrated without losing its characteristic shape. REV dehydration of tomatoes has been shown to be much faster than conventional hot-air dehydration, particularly towards the end of the drying process [12]. Microwave vacuum-dehydration also caused puffing of the tomato tissue such that the dry product was half as dense as the 100% air-dried tomato. As jerky quality mainly relies on its textural properties to maximize its market value, it is vital to investigate the effect of different parameters involved in its processing such as water activity and drying method. The goal of this research was to investigate the potential for this new REV dehydration technology to be used in the commercial production of beef jerky with a focus on improving the sensory and microbial properties of the final beef jerky product.

2.1 Beef Jerky Sample Preparation Five inside rounds obtained from a local abattoir were used for jerky preparation. The rounds were cut into strips, 3 cm 12 cm 0.5 cm, and individually marinated (31 g curing salt, 6 g white sugar, 2 g black
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pepper, 0.5 g cayenne pepper, 1.5 g ginger, 1.5 g granulated garlic, 2.5 mL lemon juice, 10 mL teriyaki sauce, 2 g maple extract, and 40 mL water for every 1 kg of meat) for two days in a 4 C cooler. 2.2 Drying The marinated meat strips were placed in a single layer on wire mesh racks and placed in a Maurer-atmos smokehouse. This was because the REV was not found to produce an equivalent tasting product when it is used to pasteurize the meat samples, which were first subjected to a pasteurization/smoke step prior to dehydration by each of the two processes. The pasteurization/smoke step included heating the meat at 90% humidity until the surface temperature reached 71 C, followed by 20 min of further drying at 80 C and 15 min of smoking at 80 C to add flavor prior to the final dehydration step. The traditional samples were further dehydrated to 37%, 34% and 31% mean moisture content by drying in the smokehouse for 100 min, 130 min and 160 min; and triplicate samples at each moisture content were taken to compare to the REV process. Jerky samples were produced in triplicate by REV using a lab-scale batch 1.8 kW, 2,450 MHz vacuum microwave (VM) (nutra REVTM lab Model 1.8, EnWave Corp., Vancouver, BC, Canada). Beef jerky (105 g to 230 g initial weight) was placed in a 27 cm diameter perforated polyethylene drum inside the VM chamber. The drum was rotated on its horizontal axis at 6 rpm throughout the drying process. Absolute pressure was maintained at 4.0 kPa throughout the process and microwave power was maintained at 1.2 kW, while dry air (1.0 L min-1) was flushed through the chamber to accelerate the removal of water vapor. An on-board water detector in the unit was utilized to produce jerky samples. Run times were recorded once target water loss was reached for each REV sample. 2.3 Microbial Measures To determine the best conditions for stomaching
To determine the best conditions for stomaching jerky for microbiological analysis, jerky was stomached under a variety of conditions. Conditions altered included the duration of mastication (15 s, 30 s, 60 s or 90 s) and the presence of sand. In each case, samples sat in buffer for fifteen minutes prior to being stomached. The diluent from each bag was serially diluted with 0.1% (w v-1) peptone water. Fifty microlitres of each dilution was spread on nutrient agar plates and the plates incubated for 24 h at 37 C. In order to understand the impact of the vacuum microwave treatment on the microbiological safety of beef jerky, Aerobic count, Yeast and Mould, Enterobacteriaceae, E. coli/Coliform, Environmental Listeria, and Staph Express Petrifilms (3 M) were used. The colonies on the plates were counted and the mean of triplicates determined. In order to further test the antimicrobial ability of REV dehydration, a second set of samples were spiked with 1.98 107 1.07 107 CFU g-1 Listeria innocua (ATCC 33090) following the pasteurization/smoke step previously described, and dehydrated in the VM. 2.4 Water Activity and Moisture Content Water activity, a critical parameter used to assure product safety, was measured using a Rotronic HydroLab3 Meter, Hauppauge, NY. Dried samples were crushed into small pieces and placed in the measurement cup and readings were noted. The moisture content of the samples was determined by drying 2 g to 5 g of pre-weighed sample in a 105 C oven. 2.5 Physical Measurements Both puncture and shear tests were performed using a Lloyd Instruments texture analyzer with a 50 kN load cell (C.S.C. Force Measurement Inc. Agawam MA), with a 4 mm diameter stainless steel probe and shear blade. Jerky strips measuring 4.5-5.5 mm thick, cut into 20 20 mm slices, were laid over a 5 mm hole and the probe was set for 5 mm s-1. The probe
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was programmed to punch completely through the jerky, and the force required for puncture was the value used. Jerky samples were cut in 20 mm 40 mm slices with a measured thickness between 4.5 mm to 5.5 mm. For each treatment, three samples were tested and the average value from each treatment was used in statistical analysis.
3. Results
To evaluate the effect of REV on the physical characteristics of beef jerky, jerky was prepared using a traditional smokehouse method, and using a combination of smokehouse preparation combined with REV treatment. No statistically significant differences between products were observed in either the puncture or the shear tests between the two treatments. However, pores were visually observed in the jerky that was subjected to REV drying, but pores were visually absent in traditional preparations (Fig. 1). A general decline in water activity was observed between both the traditional and the REV jerky treatments as the moisture content decreased (Fig. 2). When comparing the two preparation techniques, only samples at 37% moisture were statistically different in water activity, with REV-treated jerky having slightly lower Aw. For microbiological analysis, preliminary experiments showed that stomaching samples for 90 s without sand was sufficient to achieve good recovery of culturable microorganisms for analysis (not shown). In all cases, the post-marination smoking step used in both treatments effectively eliminated all culturable
microorganisms. As such, smoked samples were spiked with L. innocua in order to evaluate the pasteurization abilities of the REV technology. In spiked samples, there was a steady decline in microbial viability over time (Fig. 3). Culturable counts of L. innocua dropped from 2.0 107 (SD 1.07 107, n = 3) to 1.4 103 (SD1.71 107, n = 3) over the course of 5 min, a decimal reduction time of 1 min. While no statistically significant differences were observed between treatments for either meat quality or microbial measures, it was observed that samples processed by REV were dehydrated much faster than what was observed using traditional preparation methods in smokehouses (Table 1). For example, when trying to achieve the lowest moisture content of approximately 31%, the REV process took only 4 min, compared to 160 min using the traditional smokehouse method.
4. Discussion
Although jerky is in high demand in the marketplace, minimal research effort has been conducted to date in optimizing its process conditions. Recently, electromagnetic technologies in food processing such as radio frequency and microwave heating have gainedincreased industrial interest and have potential to replace traditional well-established preservation processes [13]. In addition, meat snack textural characteristics are critical for gaining consumer acceptance. Even though, microwave ovens have been universally adopted for household use, microwave energy to date has not been exploited to its fullest
(a)
37
Fig. 1
(b) Traditional beef jerky (a), compared to the REV produced jerky (b). Table 1 Summary of weight loss and run times for individual samples processed by REV. Jerky Wt (g) 105.5 127.5 124.1 167.4 207.1 194.8 204.0 201.7 214.2 218.9 200.3 228.9 181.6 181.6 177.0 Target Wt Loss (g) 26.63 36.51 31.32 48.09 66.18 68.00 56.18 62.04 72.33 53.51 55.84 70.99 49.29 55.31 59.26 Actual Wt loss (g) 27.9 34.8 30.4 46.9 68.1 69.0 56.5 61.5 72.8 50.3 51.9 70.8 46.7 56.1 58.5 Total time (s) 125 122 123 208 270 224 215 296 286 185 130 200 200 225 221
Fig. 2 Comparison of water activity of jerky samples prepared using traditional preparation and by REV. Error bars indicate standard deviation.
Fig. 3 Microbial numbers steadily decreased during application of radiant energy vacuum (REV), error bars indicate standard deviation.
potential in industrial food processing applications. Major problems related to non-uniform heating during microwave processing have often resulted in poor quality, microbial safety concerns and overheating in the final end product that have inhibited widespread commercial adoption [14]. It has been well established
that beef jerky texture is negatively affected by drying jerky at high temperatures for extended periods of time [10]. Microwave energy in the use of jerky production has the potential of reducing the drying time, while enhancing the food safety of the final product. The REV drying method was identified as an alternate way of processing beef jerky. There has been limited research to date on the drying behavior of beef jerky, and the only method used commercially in practice to prepare jerky is traditional smokehouse processing. REV technology dried the jerky product much faster than what is observed using traditional drying methods. This difference might translate into substantial energy savings in terms of processing time and merits further investigation. Energy and cost/benefit analysis of utilization of REV in jerky
38
processing on an industrial scale needs to be done as there is potential to commercialize REV as an alternative drying method in jerky processing. The importance of dielectric properties and material interactions of beef jerky during microwave heating under vacuum needs to be further studied for complete understanding of the heating behavior that leads to both the increased efficiency of water removal as well as the porosity observed in the final jerky product. The textural characteristics of beef jerky are one of the most important parameters that defines the commercial value and consumer acceptability of the product. REV dehydration as carried out in this study did not produce a tenderer product according to our instrumental texture analysis than traditionally prepared jerky. It is important to point out that it did not produce a less tender product either, and in fact both samples were very similar. No surface hardening was observed that could potentially occur during traditional microwave heating. Samples produced by REV were not significantly different in product quality from traditionally prepared samples as evidenced by either the puncture or the shear tests. Further sensory analysis is warranted to assess the impact of the increased porosity observed in jerky samples produced by REV, as instrumental texture analysis does not always agree with sensory analysis. A density decrease of more than 50% was previously reported for REV dried tomatoes as compared to air dried tomatoes, both at Aw of 0.43 [12], due to pores created in the tissue by steam bubbles during REV drying. The increased porosity observed in the REV produced jerky samples might interact with saliva during mastication, providing a different eating experience for the consumer. And finally, many of the instances of food poisoning reported in the media are associated with meat products. The major limitation for using microwave heating for pasteurization and sterilization of food products in the past is the existence of non-uniform temperature distribution [13].
Non-uniform temperature distribution that results in cold spots during conventional microwave heating is a serious concern in meat products because pathogen survival is an obvious health hazard. A serious problem in variable temperature distribution after microwave heating has been observed in studies with poultry [14, 15], ground pork patties [16] and beef-pork loaves [17] that would support the survival of pathogenic microorganisms rather than eliminate the pathogens present. The REV process appeared very capable of reducing, the microbial load on samples intentionally spiked with high levels of Listeria. We speculate that the tumbling action of the polyethylene drum inside the VM chamber may have contributed to a more uniform temperature distribution not typically seen during conventional microwave heating. Also, water is typically boiling during REV drying, albeit at moderate temperature due to the vacuum. Therefore, product temperature in the drying chamber rapidly equilibrates to the boiling point of water in that product and vacuum and tends to be uniform through the load.
5. Conclusions
REV technology demonstrated superior efficiency in dehydrating jerky samples compared to traditional smokehouse processing which might translate into substantial energy savings. Product quality was maintained with respect to jerky tenderness, which may or may not be due to increased porosity inherent in the process. And finally, combining microwave energy within a vacuum, and placing the product in a tumbler resulted in very effective pathogen reductions in jerky samples spiked with Listeria. This major improvement in pathogen reduction may be due to improved temperature distribution during microwave heating under vacuum and/or tumbling that may result in maximum utilization of microwave heating in industrial processes that to date have been impeded by poor food safety performance.
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Acknowledgments
The authors gratefully acknowledge both EnWave Corporation and the Natural Sciences and Research Council of Canada for their financial support of this research.
[8]
[9]
[10]
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[1] A. Bose, M. Boland, Dried beef industry profile [Online], http://www.agmrc.org/commodities_products/livestock/b eef/dried_beef_profile.cfm/. E.E. Ray, L.H. McKee, B.J. Gardner, D.W. Smith, Properties of an extruded jerky-type meat snack containing potato flour, J. Muscle Foods 7 (1996) 199-212. American Association of Meat Processors, The Jerky Journal, Chapter 1. Introduction, American Association of Meat Processors, Pennsylvania, United States, 2004. I.W. Thiagarajan, Combined microwave-convection drying and textural characteristics of beef jerky, M.Sc. Thesis, College of Graduate Studies and Research, Department of Agricultural and Bioresource Engineering, University of Saskatchewan, Saskatchewan, Canada, 2008. M. Calicioglu, J.N. Sofos, J. Samelis, P.A. Kendall, G.C. Smith, Deystruction of acid and non-adapted Listeria monocytogenes during drying and storage of beef jerky, Int. J. Food Microbiol. 19 (2006) 45-59. G. Monin, P. Marinova, A. Talmant, J.F. Martin, M. Cornet, D. Lanore, et al., Chemical and structural changes in dry-cured hams (Bayonne Hams) during processing and effects of dehairing technique, Meat Sci. 47 (2000) 29-47. N.M. Quenzer, E.E. Burns, Effect of microwave steam and water blanching on freeze dried spinach, J. Food Sci. 46 (1981) 410.
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DAVID
PUBLISHING
The Study of Supply and Demand of Organic Products in the European Union and Serbia
Ljubomir Pupovac, Tomislav Sudarevi and Suzana Salai
Faculty of Economics, University of Novi Sad, Segedinski Subotica 24000, Serbia Received: September 24, 2012 / Published: January 20, 2013. Abstract: One of the phenomena that marked 20th century is rapid technology progress. This process did not avoid food production and processing. Thanks to rapid development of technology, there has been a development of conventional food production, as well as appearance of genetically modified food. As a consequence of this trend, as well as the concern among some part of human population for their health, we had the return to natural food production in the form of the development of organic food production. One of the characteristics of organic food market in the European Union is the constant growth of demand for this product group, and insufficient quantities of organic products available in this market. These information led to the conclusion that organic food represents an opportunity for less developed countries, i.e., that countries like Serbia can manufacture and sale these products on the market with the highest purchasing power in the worldthe European Union market. In this paper, the characteristics of demand for organic foods in the European Union were presented, then it was explained in detail why are these products export chance of Serbia, as well as reasons why is the export of organic food from Serbia to the EU very low at the time being. At the end, some recommendations that could help Serbia to increase export of organic product to EU countries were proposed. Key words: Organic products, supply and demand, European Union, Serbia.
1. Introduction
The technological revolution that started in the twentieth and resumed in the twenty-first century did not pass the food products market. Bearing in mind the constant population growth on one side and limited amount of the arable land on the other, mankind found a new challengehow to provide food for this ever increasing world population. In order to solve this problem, new technologies in food production have been developed and their goal was to achieve increased yield per hectare [1]. That was the reason why people started using large number of chemical substances throughout the twentieth century, such as pesticides and fertilizers in order to improve efficiency of food production. The research went one step further and made the production of genetically modified food possible. This type of production
Corresponding author: Ljubomir Pupovac, MS.c., research fields: direct and indirect customer value and organic food marketing. E-mail: ljubomirp@ef.uns.ac.rs.
includes changing of the genetic make up of an organism in order to increase its useful properties such as weight, size or resistance to various diseases. This trend has twofold consequences. The first consequence is the fact that the yield of agricultural crops over the past hundred years or so has increased several times. For example, according to the Cimmyt-International Maize and Wheat Improvement Center [2], crop production increased three times on the same amount of land over the period 1951 to 1991. In that way, possible food shortages around the world have been avoided. The second consequence is related to health challenges in the production of conventional and genetically modified food. Even though, because of the methodological requirements, it is extremely hard to prove that both conventional and genetically modified food can be harmful to human health. Today a growing number of consumers believe that the food produced in this way can have adverse consequences.
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There is a new methodology of food production as a response to the problems we stated, it is called organic farming. There are several definitions of organic food. According to Allen and Albala [1], organic food represents food produced using methods which do not include modern synthetic pesticides, chemical fertilizers, and which do not include genetically modified food, the food produced without the use of radiation, industrial solvents or chemical food additives. According to International Federation of Organic Agriculture Movements [3], organic farming is a food production system that sustains the health of soils, ecosystems and people. It relies on ecological processes, biodiversity and cycles adapted to local conditions, rather than the use of inputs with adverse effects. Organic agriculture combines tradition, innovation and science to benefit the shared environment and promote fair relationships and a good quality of life for all involved. We can conclude from these two observations that this type of production is based on the natural way of producing food without addition of any artificial substances and by respecting the principles of health, ecology and equity concerns. Starting with the stated problems perceived by consumers when it comes to consumption of conventionally produced agricultural products, demand for organic agricultural products is on the rise in high-income countries, especially in most EU member states. On the other hand, there is not enough unpolluted soil suitable for this type of production, so we can conclude that the possibilities of producing organic agricultural products are limited. The only possible solution to meet consumers needs on given markets, at the moment (and in the near future), would be the import of organic products. Organic food market in the European Union is one of the rare markets where demand consistently exceeds supply which makes it extremely attractive to bidders from different countries. Hence, the optimism that one of the
opportunities for the development of Serbian economy lies in the right investment in both production and export of organic food. The purpose of this study is to explore the prospects of the export of organic products from Serbia to the markets of developed countries, primarily the European Union.
2. Current State of Demand for Organic Food in the EU

When someone should describe an ideal market in which he/she wishes to perform, that person would almost certainly state the two such market characteristics: the fact that demand exceeds supply and that market is poised for continued stable and predictable growth [4]. Organic food market is one of the few in which the demand for products exceeds supply and in which we have a constant stable growth over the years. According to the EU report about the organic sector [5], organic food market for the EU-15 countries (members which joined the Union before 2004) in 2006/2007 amounted to EUR14.4 billion. Up to 80% of that amount (around EUR11.5 billion) goes to four largest consumersGermany, United Kingdom, France and Italy. When it comes to relative amounts, the consumption was the highest in Austria (around 5% of population buys organic products), followed by Germany, Denmark and Luxembourg (3.7%/3.8%) [5]. What we can notice from the report is the significantly lower demand for organic products in the EU-12 countries (countries which joined the EU in 2004, together with Bulgaria and Romania). In these countries, demand for these types of products is less than 0.2%, while only in Czech Republic (0.5%), it is higher than the total demand for food products [5]. Another interesting trend is the significant growth of organic food in the EU over the last ten years. According to the same report there has been a growth of demand for organic food of 18.1% in France for the period 2005 to 2009, 14.0% in Germany for the period
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2000-2008, 8.7% in Italy for the period 2001-2009, and 11.9% in United Kingdom for the period 2000-2008 [5]. It is interesting that the economic crises caused a drop in demand for organic food only in the UK, while it recorded a growth in Germany, France and Italy. The report also reveals the fact that the demand for organic food exceeds supply. As a result of this trend, we can come to the conclusion that the majority of member states will be forced to import organic food in order to solve this problem. It is expected that a trade between members and non-members of EU in the field of organic food will record a significant increase in the future, thus solving the problem of lacking these types of products. In addition to this report, we can find the research in organic food conducted by Richter and Padel [6]. The results are presented in the Table 1. Results are related to the European countries in 2005. Research by Richter and Padel confirms analysis of the European Union that the organic food market is relatively large with tendency of growth. Market of the four largest countries in the European Union for 2005 exceeds EUR10 billion. The biggest market in Europe is Germany with EUR3.9 billion, followed by Italy, France and United Kingdom with over EUR2 billion each [6]. What is interesting is that the United Kingdom achieved the highest rate of market growth for the period 2004-2005, with over 30% which might explain the decline in demand for organic foods during the crisis [6]. Switzerland has the highest consumption of organic food per capita by far, it amounts to EUR103 which is more than twice the one of Denmark and Austria, the countries with the highest consumption of organic food per capita within the European Union [6]. This data shows that Switzerland, although the market outside the European Union, should not be neglected.
Table 1
Demand for organic food in Europe for 2005 [6]. Market Organic growth market in percentages billions of compared to Euros 2004 11% 30% 10% 12% 1.4 -1% 30% Consumpti on per capita in euros () 47 42 39 37 57 56 25 7 103 10.2 1.14 Market share of organic food compared to conventional food around 3% around 3% around 4.5% -
Country
Germany 3.9 Italy 2.4 Great Britain2.33 France 2.2 Denmark 0.307 Austria 0.450 Holland 0.419 Spain 0.300 Switzerland 0.763 Norway 0.041 Czech 0.012 Republic
from Serbia, followed by the current status of organic food production in Serbia, and finally the factors that negatively affect the current balance of exports from Serbia to European Union. 3.1 Organic Food as an Export Opportunity for Serbia There are several important reasons why organic food represents great opportunity for the development of Serbian economy. They are reflected in cheap labor, large amount of arable land, proximity to the markets of the European Union, and high demand for organic products. Cheap laborthe way of production and processing of organic food are the reason why this type of production is considered to be labor-intensive industry, that is to say, in order to obtain the same amount of output for the production of organic food, it is necessary to employ more labor than conventional food production. This is considered to be one of the primary reasons why organic food is more expensive than conventional food. Since the average salary in Serbia is several times lower than the average salary in certain European countries (according to the Bureau of Statistics average net wage salary in Serbia currently amounts to over RSD38,000 (around EUR380) [7], we can say that the production of
3. Organic Food in Serbia

In the remainder of this paper, we stated the main advantages of production and export of organic food
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organic food is far more profitable in Serbia than in EU countries. The unemployment rates are among the highest in Europe (around 22%), so we can conclude that the production of organic food could be great opportunity to launch Serbian economy and reduce unemployment. A large amount of arable landone of the main characteristics of Serbia is large areas of arable and fertile land [8]. The soil fertility makes it possible to produce large amounts of food without the addition of synthetic inputs such as fertilizers and pesticides. On the other hand, the soil diversity makes it possible to grow various types of crops. This is very important because it is necessary to round up the production process in order for organic production to take place; it is not enough to produce only one crop (Fig. 1). So for example, after grains have been processed they can be used for direct sale at the market (flour, cornflakes, etc.) or they can be used as an ingredient in animal feed. The benefits of livestock production in an organic way may be multiply beneficial: after meat have been processed it can be marketed, it is possible to produce milk and milk products, eggs as well, which can be placed on the final consumers market. Manure can also be used for fertilizing fields in order to get higher yields from organic fruits, vegetables and grains. Organic fruits and vegetables can be sold directly on the market, but can be processed as well (juice, jam, salad in a jar, etc.), and then can be placed on the market. Organic food makes sense only if all listed manufacturing segments are covered, which can best be seen on the example of Austria, which achieves the best results in the production of organic food in Europe.
Livestock production Manure
In addition, the land in Serbia is still largely preserved for organic production [7] because of poor use of pesticides and fertilizers due to farmers low purchasing power, which is not common in most EU countries. Proximity to EU marketsAnother advantage for Serbia could be the fact that it is close to many EU markets, primarily Austria, Italy, Germany and even France, so the transportation costs would not be high and there would be more time to sell the goods, especially if we take into consideration the short-term duration of most food products. The duty-free agreement between Serbia and European Union allows export with no additional export taxes [9]. High demand for organic productsas it was stated in the previous part of the study, demand for this group of products goes beyond the production possibility, and is poised for continued growth, so we can assume that this trend will continue. We can therefore conclude that the organic food market is a very attractive market. After defining the factors that are contributing to the development of organic food in Serbia, we are going to describe the current state of this agricultural sector in Serbia. 3.2 The Limiting Factors Given the chances that organic food production carries, but a small area of land in Serbia on which this type of production is being used right now as well, it is necessary to define the specific reasons for modest production level of organic food in Serbia. The authors made a list of the limiting factors affecting the level of production of organic food in
Fruits/Vege tables
Cereals
Food
Product Consumer Product
Processing Fig. 1 Possible alternatives to organic food production.
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Serbia by reviewing the literature and by interviewing the experts in the field of organic production. High capital costsone of the characteristics of the organic production is the fact that it is necessary to have higher initial investment compared to conventional production. This investment can later be compensated by higher prices for the organic products. In Serbia, the problem lies in the fact that capital is very expensive, often several times more expensive compared to the countries of the European Union, which makes the manufacturing process more expensive as well. Also, banks in Serbia require a high degree of collateral, often in the form of mortgages and/or guarantors, so only few people in Serbia decide to enter into any risky venture because miscalculation may lead to loss of home. Many estates in Serbia are very small and it often happens that the land of the farmers is located in several locations, which further complicates the production process. If one considers that organic farming to be labor-intensive, fragmentation of arable land in several locations that each household has, can be a serious problem. On the other hand, many farmers produce the same crops over the years, and even generations back, in more or less the same way, possibly with some technological advances, and that is why they are not willing to experiment with new varieties and new production methods. Lack of trainingalthough organic farming is nothing new for the experts, this type of production represents the great unknown. What needs to be done to increase the volume of organic production is a general education of the producers on both the benefits and characteristics of organic farming. It is realistic to expect that the rapprochement between scientific research institutions and manufacturers will lead to increased production of organic products in Serbia. One way of solving this problem might be the employment of a large number of unemployed agronomists who would receive salary from the state
which would result in the farmers education. Of course, education about organic food must be a priority in the education of Serbian farmers. Lack of varietyaccording to some authors [10], another big problem is a lack of suitable varieties for organic production. This type of production requires the return of some forgotten varieties which posses far better characteristics for this type of production, in relation to available, modern varieties. The trouble is that it is very difficult to get those varieties in Serbia due to low demand for these sorts among the existing manufacturers. The development of this market is sure to find a motive for the production and import of some varieties that are missing. Low demand for organic products in Serbiademand for organic products in Serbia is low and we can say that this market is still in its infancy. It is extremely hard to find organic food and the price is far higher than the price of conventional food, especially if take into consideration prices in European Union. Logical development of any company is to become a stable company in the domestic market and then continue to grow its global market performance. Modest demand for organic food in Serbia can be one of the reasons why there has not been a large enough company that could successfully perform in the EU market. The solution to this problem could be the association of producers and sellers of organic food in Serbia which would educate the citizens of Serbia and generate the primary demand for organic products. Something like this would enable them to increase sales, and to build a cooperation as well, and in that way welcome the joint appearance on third markets. In regards to the specific data, it is very difficult to find the exact data from secondary sources on the demand for organic products in the Serbia. In this study, as a source of data was used a study status and prospects of consumption of organic products [11]. The advantage of this study is that it was done in 2010,
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so the data can be considered relevant, while the main disadvantage is the small number of respondents. The study was conducted exclusively in Novi Sad which can be another drawback, however, taking into account that it is the urban population that is primarily interested in organic food in Serbia, this research can be a solid indicator of demand for the domestic organic market. The survey [11] showed that 89% of respondents are familiar with the concept of organic food. On the other hand, 14% of respondents regularly buy organic products, while 38% of respondents sometimes or rarely purchase organic foods. 48% of respondents do not buy organic products at all. As the main motive for buying organic food [11] are cited health reasons (50% of respondents), then the quality of food (32% of respondents), environmental care is the third most important reason for buying these types of products (15% of respondents), and 3% of respondents did not state the reason for purchasing organic food. Lack of security in the marketing of organic agricultural productsthe leading producers of organic agricultural products are, in most of the cases, the earlier highly successful farmers of conventional agricultural products. It only indicates that the success in this type of agriculture requires systematic, hard work and willingness to learn. Despite all, the biggest problem for transition to this type of production is the possibility of exporting products at prices that are reasonable given the increased costs and reduced yields. Therefore, as a necessary condition appears the requirement that organic farmers provide the appropriate conditions of purchase by the cooperative association, or under contract with the proven organizers of production for export to international markets.
4. Conclusions
Conclusion of the complete study could fit in one sentencetaking into account the characteristics of the organic food market in the European Union and the characteristics of Serbia regarding the
productionpossibility when it comes to this type of products, export of organic food from Serbia to the European Union would have to be much higher. The question arises, what to do next? What is necessary to do in order to improve the situation? There are two groups of subjects that can facilitate the process in the positive direction: producers and the state. The first thing that manufacturers should do is to form the association in order to approach the EU market together, but also to generate a common deterrent demand for organic products in Serbia. As far as joint appearance is concerned, the chronic problem of Serbian farmers to meet the demands of distributors in the EU markets is already known (not in terms of quality but in terms of quantity instead). It often happens that foreign traders find a quality product in Serbia, but later realize that manufacturers not nearly meet their needs. This problem could be solved by manufacturers association, but also by focusing only on certain varieties that would have the best pass and/or which would wear the highest return on invested resources. Association would make it possible to generate greater demand for the organic products in Serbia. The state can help the manufacturers with both financial and non-financial measures. As regards the financial measures, providing strong subsidies for production of certain crops or animals under organic production can positively influence farmers to adopt this form of food production. In cooperation with commercial banks, it is possible to offer more favorable loans where the state would be taking on given cost (interest) or accepting at least a part of the risk if the loans are not returned. These activities would lead to a spillover of risks from the manufacturer to the state which might have beneficial effects on the increase of the organic production. Another direction would certainly be the education of agricultural holdings on the use and characteristics of organic food production. This could be done by linking scientific research institutions and the farms
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The Study of Supply and Demand of Organic Products in the European Union and Serbia nds/wft9596/htm/wft9596part2.htm. [3] International Federation of Organic Agriculture Movements [Online], http://www.ifoam.org/growing_organic/definitions/doa/in dex.html. [4] P. Kotler, Principles of marketing [Online], Prentice Hall, upper Saddle River, New Jersey, USA, 2011, Organic Eprints, http://orgprints.org/13170/. [5] European Commission [Online], http://ec.europa.eu/agriculture/analysis/markets/organic_2 010_en.pdf. [6] T. Sudarevi, Marketing of agricultural products as a determinant of agricultural development in Serbia (translated from Serbian), Unpublished Ph.D., Dissertation, Faculty of Economics Subotica, University of Novi Sad, 1999. [7] Statistical Office of the Republic of Serbia [Online], http://webrzs.stat.gov.rs/WebSite/Public/PageView.aspx? pKey=2. [8] Radio Television of Vojvodina [Online], http://www.rtv.rs/sr_lat/ekonomija/srbija-godisnje-gubi-2 5.000-hektara-obradivog-zemljista_279998.html. [9] T. Sudarevi, S. Salai. L. Pupovac, Marketing aspects of the supply of organic agricultural products in Serbia and the region, Anali Ekonomskog Fakulteta u Subotici, 47 (26) (2011) 33-48. (in Serbian) [10] J. Curi, S. Cerani, The value chain of organic food in Serbia, in: Proceedings of the XXV Consulting of Agronomists, Veterinarians and Technologists, 2010, pp. 3-4. [11] Center for Organic Production Selena [Online], http://organiccentar.rs/14.pdf.
together. On the other hand, creating certain institutions that would help sales of organic food would certainly help both existing and potential producers of these types of products. Also, import of certain varieties that are suitable for organic production and which can not be found on domestic market would be much easier if certain ministries participated in the enterprise. The state made the first step in hiring agronomists and in providing subsidies to the producers of organic food, but the question is whether this is enough in order to significantly increase production and export of this type of products.
Acknowledgments
The authors gratefully acknowledge financial support from the Ministry of Education and Science of the Republic of Serbia (Project III 46005).
References
[1] G.J. Allen, K. Albala, The business of food: Encyclopedia of the Food and Drink Industries, ABC-CLIO, USA, 2007, p. 278. [2] CIMMYT-International maize and wheat improvement center [Online], http://apps.cimmyt.org/Research/economics/map/facts_tre
DAVID
PUBLISHING
Sensory and Organoleptic Characteristic, Zinc and Iron Content of Fortified Chips from Cassava Flour
Siti Helmyati1, Nindya Putri Pamungkas1, Lily Arsanti Lestari1 and Narendra Yoga Hendarta2
1. School of Health Nutrition, Faculty of Medicine, Universitas Gadjah Mada, Jalan Farmako, Sekip Utara, Yogyakarta 55281, Indonesia 2. Health Analyst, Polytechnic of Health, Ministry of Health, Ngadinegaran MJ III/62, Yogyakarta, Indonesia Received: October 8, 2012 / Published: January 20, 2013. Abstract: Iron fortification can cause several biophysicochemical modifications. Those depend on many factors, such as iron fortificant and the food carrier. There were four groups of chips: 1) non-fortified wheat flour chips (K1); 2) non-fortified cassava flour chips (K2); 3) fortified cassava flour chips, each with ZnSO4 30 ppm and NaFe EDTA (K3) 30 ppm and 4) fortified cassava flour chips, each with ZnSO4 50 ppm and NaFe EDTA (K4) 50 ppm. The chips were evaluated for sensory characteristic (color, taste, flavor, and texture), organoleptic characteristics tested by preference test, as well as zinc and iron contents. Zinc and iron contents were analyzed by Atomic Absorption Spectophotometric method. The results showed that both fortificants did not affect the sensory characteristic of cassava flour chips. The preference test showed that color, taste, and flavor of K1 chips as a control, were mostly liked, but there was no significant difference preference of texture. Moreover, preference test using K2 as control showed that color of K3 was mostly liked, but there was no significant difference preference of taste, flavor and texture. Fortification can increase the contents of zinc and iron in cassava flour chips. The panelist can accept the fortified cassava chips as well as wheat flour chip, as a consequence, both can be a potential way to combat the iron deficiency anemia. Key words: Fortification, ZnSO4, NaFe EDTA, sensory characteristics, organoleptic, cassava.
1. Introduction
Zinc and iron deficiency are one of the main nutrition problem in Indonesia, especially in the relation to their essential roles in human body [1, 2]. The deficiency of both minerals is related, because both are present in same type of food products. The absorption of both is also inhibited by same inhibitors such as phytate, polyphenols, calcium, and phosphate. Zinc and iron deficiency can affect not only to the children, but also to adults [3, 4]. Food fortification can be taken into account as an effective way to overcome micromineral deficiency. Compared to supplementation program, food fortification is a half less effective [5], but it is less costly. Besides, food fortification can be used for
Corresponding author: Siti Helmyati, lecturer, research fields: fortification, micronutrients and community nutrition. E-mail: siti_helmyati@yahoo.com.
longer time and larger population [6, 7]. In Indonesia, food fortification program has been widely done, especially in wheat flour. The guideline for wheat flour fortification program has been stated in Indonesian Health Ministry Statement No. 1452/Menkes/SK/X/2003. In the other hand, because of not wheat-producing country, Indonesia depends on the other country for providing wheat flour as basic ingredients of biscuits [8]. As a consequence, it is needed to substitute wheat flour with local food. Cassava is considered suitable to substitute the wheat flours role regarded to its availability, consumption level, and potency as flour [9]. Basically, fortifying food with iron is challenging, because some iron fortificants tend to produce undesirable sensory changes [7]. Unlike iron fortification, sensory changes due to zinc fortification do not appear as a major concern [10]. To reduce
48
unacceptable sensory changes on food fortified by iron fortification, NaFe EDTA is suitable to be used, especially for flours [11]. NaFe EDTA also indicates 2-4 times better absorption than ferrous sulphate (FeSO4) in high phytate food including cassava [12], not oxidizing fat or precipitate peptides, and is stable at least for 12 months [13]. One of the important aspects on food fortification is the safety and the acceptance of the product by consumers. The objective of this research is to evaluate the sensory attributes, organoleptic characteristics, to determine zinc and iron content of cassava flour chips fortified by ZnSO4 and NaFe EDTA, and thereby, help to develop a palatable, safe, and nutritionally important food to overcome iron and zinc deficiency.
Table 1
The ingredients of the chips.
Amount Chips K1 500 g K2, K3, K4 Wheat flour 800 g K1 K2, K3, K4 300 g Corn starch All chips 200 g K1, K2 ZnSO4 K3 30 mg K4 50 mg NaFe EDTA K1, K2 K3 30 mg K4 50 mg K1: nonfortified wheat flour chips; K2: nonfortified cassava flour chips; K3: cassava flour chips fortified by 30 ppm of each ZnSO4 and NaFe EDTA; K4: cassava flour chips fortified by 50 ppm of each ZnSO4 and NaFe EDTA.
Ingredient Cassava flour

Cassava flour was bought in supermarket in Yogyakarta, Indonesia. We used two kinds of fortificants, namely NaFeEDTA as iron fortificant and ZnSO4 as zinc fortificant. The preparation of NaFe EDTA followed the method of Layrisse and Martinez-Torres [14], while ZnSO4 was bought from Merck (Darmstat, Germany). 2.1 Preparation of Chips There had been a preliminary experiment to determine the best composition of flour that would be used to make chips with the best sensory characteristic. The results of flour compositions are listed in Table 1. Chips were made from dough obtained from ingredients listed in Table 1, added by the other additional ingredients such as garlic, salt, butter, parsley leaves, water, and coriander in the same amount for four kinds of chips. The chips were made according to the method reported in Sutomo [15]. Below is the detail procedure for preparation of chips: Each ingredient was weighed as necessary (Table 1); Flours were mixed with fortificants for 5-7 min; The mixture of flour and fortificants was added
with grounded garlic and coriander, chopped parsley, and salt Water was poured into the flour to make dough; Dough was flattened to about 1 mm thickness by doughroller; The flattened dough was cut to small square shape; The square shape dough then was fried in hot cooking oil until it turned to yellow-brownish chips. 2.2 Fortification The fortificants were added to the flour in the early step of dough preparation. The dose levels of fortificants were based on minimum level of zinc and iron premix of flour fortification suggested by Indonesian government. The doses used were 0 ppm (non fortification), 30 ppm of each fortificant (30 mg of each fortificants/1,000 g flour) and 50 ppm of each fortificant (50 mg of each fortificants/1,000 g flour). 2.3 Sensory and Organoleptic Test Sensory attributes consisting of color, taste, flavor, and texture were analyzed subjectively by researcher. Organoleptic characteristic (preference) was evaluated by 25 semi-skilled panelists using hedonic scale method. The method used six preference scale: 6 =
49
like very much; 5 = like moderately; 4 = like slightly; 3 = dislike slightly; 2= dislike moderately; 1 = dislike very much. Twenty five panelists were students of Scholl of Health Nutrition, Faculty of Medicine, Universitas Gadjah Mada, Indonesia who ever did the similar test before. Four samples were evaluated in the same time, and panelists rinsed their mouth using water between samples. 2.4 Determination of Zinc and Iron Contents Zinc and iron contents were measured using Atomic Absorption Spectrophotometry. 2.5 Data Analysis Sensory test data were explained descriptively. Chips preference data were analyzed statistically by Kendalls W test continued by Wilcoxon test, if there had any significant difference among samples. Zinc and iron content data were analyzed by ANOVA continued by post hoc test if there had any significant difference among samples.
3. It can be inferred that there was significant difference of panelist preference among samples in color, flavor, and taste attribute. Comparisons of mean value in each attribute shows that K1 chips mean value was the highest among the samples. This means that color, flavor, and taste of K1 were mostly liked by the panelists. While, the preference to texture attribute tend similar in all samples (P = 0.061). Table 3 also showed the second statistical analysis. Flavor, taste, and texture of three samples did not differ significantly (P > 0.05). This means that dose level of fortificants did not affect the panelist preference on those three sensory attributes. Meanwhile, there were significant differences of panelist preference on chips color, where K3 chips color was mostly liked. It was shown from its highest mean score in preference test compared with the other chips. The panelists also scored the chips from its overall characteristic. K1 chip was mostly preferred on its overall characteristic. 3.3 Zinc and Iron Content Zinc and iron content was declined between K1 and K2, but there was increment of zinc and iron contents from K2 to K4. More fortificants added would increase zinc and iron contents in cassava flour chips (Table 4). As much 50 ppm iron fortificants added, it will increase the iron content in cassava flour chips to 70.027 0.802 ppm, while the cassava flour contains 40.655 0.802 ppm.
3. Results
3.1 Sensory Characteristic of Chips There was no effect of increasing doses of fortificants on the sensory attributes of the chips. These could be inferred from same characteristics of K2, K3, K4 in Table 2. However, there was difference between wheat flour chips (K1) and cassava flour chips (K2, K3, K4). Even, the difference could be noticed in the dough making process. Unlike wheat flour dough, the cassava flour dough was not elastic and quickly be dry. 3.2 Preference Difference of Chips Two statistical analyses were done in this study. The first analysis used four samples (K1, K2, K3 and K4) with K1 as control. The second analysis used 3 samples (K2, K3 and K4) with K2 as control. The results of first statistical analysis were listed in Table
4. Discussion
4.1 Sensory Characteristic of Chips Fortificants of NaFe EDTA and ZnSO4 added to flour did not affect the sensory characteristic of chips. This report was in agreement with that reported by Hurrel [11] stating that NaFe EDTA was suitable fortificant for flour to minimize undesirable sensory changes. Meanwhile, there were a few studies about the effect of zinc fortification on sensory changes of
50
Table 2
Sensory characteristics analyze result.
Samples of chips K1 K2 K3 K4 Color Bright yellow Slightly dark brown Slightly dark brown Slightly dark brown Tasty, musty taste Tasty, musty taste Tasty, musty taste Flavor Tasty (characteristic of cassava flour) (characteristic of cassava flour) (characteristic of cassava flour) Texture Crispy and crunchy Slightly crispy (crumbly), oily Slightly crispy (crumbly), oily Slightly crispy (crumbly), oily Tasty, there was after-taste Tasty, there was after-taste Tasty, there was after-taste Taste Slightly plain sensation sensation sensation K1: nonfortified wheat flour chips; K2: nonfortified cassava flour chips; K3: cassava flour chips fortified by 30 ppm of each ZnSO4 and NaFe EDTA; K4: cassava flour chips fortified by 50 ppm of each ZnSO4 and NaFe EDTA. Characteristics Table 3 Results of panelists preference statistical analysis using K1 as control. Mean Variables Color Flavor Taste Texture K1 5.36 4.68a 4.32a 4.68
a
K2 2.72 3.40b 3.08b 3.96

b1
K3 3.48 3.88b,c 3.12b,c 4.32

c2
K4 3.16b,c,d,1,2 3.88b,c,d 3.28b,c,d 3.96
P of Kendalls W test* 0.000 0.000 0.014 0.061
P of Kendalls W test# 0.030 0.125 0.759 0.125
*results of panelists preference statistical analysis using K1 as control; # results of panelists preference statistical analysis using K2 as control; a,b,c,d,1,2 mean scores in rows with the same superscript notation letters are not significantly different (P > 0.05); K1: nonfortified wheat flour chips; K2: nonfortified cassava flour chips; K3: cassava flour chips fortified by 30 ppm of each ZnSO4 and NaFe EDTA; K4: cassava flour chips fortified by 50 ppm of each ZnSO4 and NaFe EDTA. Table 4 Samples K1 K2 K3 K4 Iron and zinc content of the chips. Fe content (ppm) 45.479 0.801 40.655 0.802 55.234 0.526 70.027 0.802 Zinc content (ppm) 13.791 0.016 10.057 0.199 17.973 0.120 22.146 0.482
K1: nonfortified wheat flour chips; K2: nonfortified cassava flour chips; K3: cassava flour chips fortified by 30 ppm of each ZnSO4 and NaFe EDTA; K4: cassava flour chips fortified by 50 ppm of each ZnSO4 and NaFe EDTA; a,b,c,d mean scores in rows with the same superscript letters are not significantly different (P > 0.05).
fortified food [7]. However, since all of zinc fortificants are white or colorless [7], besides, sensory
changes due to zinc fortification do not become major problem [16]. As mentioned before, the differences were observed between chips made from wheat flour (K1) and other chips made from cassava flour (K2, K3, K4). The wheat flour dough was more elastic because wheat flour contains more gluten than cassava flour does. Gluten is a kind of protein which will form a visco elastic linkage if it mixes with water. Cassava flour substitution will decrease protein contents [17, 18] and it will decrease the dough ability in making that viscoelastic linkage. Cassava flour chip had a darker color than wheat flour chips did (Fig. 1). Color of chips can be influenced by the color of flour itself. Wheat flour is whiter than cassava flour. Cassava flour used in this study has dark yellow color. The color of cassava flour is affected by cassava variety itself and polyphenolase enzyme activity [19]. There would be a bluish-brown color appearing in cassava which is resulted formoxidation by polyphenolase enzyme. Winarno [20] also reported that color of cassava flour is influenced by Maillard reaction. Maillard reaction is browning process resulted from reducing sugar and primary amina group interaction. This reaction is more lasted in traditional process of cassava flour making which uses sunlight to dry the cassava. The darker color appearing in cassava flour chips is not only caused by the natural color of the composing flour but also caused by caramelization occurred during the frying. Caramelization is a heating process of sugar
51
Fig. 1 Non-fortified Chips. K1 = unfortified wheat flour chips; K2 = unfortified cassava flour chips.
outer environment. Second process is absorption of oil into the food replacing the water loss during the evaporation. More water contained in the food, more oil will be absorbed into the food [25]. Cassava flour has higher water content than wheat flour [24, 26]. This caused why chips produced from cassava flour have oily texture. 4.2 Panelists Preference of Chips Two statistical analyses were done in this study. First analysis used 4 samples (K1, K2, K3 and K4) with K1 as control. This was to evaluate the panelists preference between chips made from wheat flour and cassava flour. Second analysis used three samples (K2, K3 and K4) with K2 as control. This was to evaluate the effect of fortificants dose level on panelists preference of the chips. Panelists are more preferred to wheat flour chips (K1) than cassava flour chips (K2, K3 and K4) in color, taste, and flavor properties. It is estimated because wheat flour chips have brighter color and do not have acid taste and flavor such as cassava flour chips do. However, panelists preference to texture property was same in all kinds of chips. Assessment of foods texture is started when the foods are started to be cracked, chewed, and swallowed. Panelists tended to like crispy chips similar with wheat flour chips. Crumbly and slightly crispy texture of cassava flour chips is little bit improved by corn starch existence. Corn starch is usually added in the cookies dough to create crispy and crunchy texture [15]. Another analysis showed that preference score of taste, flavor and texture properties comparing three samples of chips fortified by different level of fortificants dose (K2, K3 and K4) did not differ significantly. It means that increasing level of fortificants did not affect panelists preference on that three sensory properties. Meanwhile, the texture of chips made from cassava flour fortified by 30 ppm of each NaFe EDTA and ZnSO4 was mostly liked. The similar colour of fortified cassava flour chip in
Fig. 2 Fortified chips. K3 = cassava flour chips fortified by 30 ppm of each ZnSO4 and NaFe EDTA; K4 = cassava flour chips fortified by 50 ppm of each ZnSO4 and NaFeEDTA; For overal preference chips properties, of 18 panelist prefer the K1, 2 panelist K2, 4 for K3 and 1 for K4.
in high temperature resulting brownish compound named melanoidin [21]. Caramelization in cassava flour is easier and faster to happen because cassava flour contains simple sugar resulted from fermentation during cassava drying process. The carbohydrate contents of cassava is 86%-88% while wheat flour is 70%-80% [22]. Texture property is influenced by two kinds of starch contained in the flour. Those starches are amylose and amylopectin. High amylose containing food will have hard texture with high density. Meanwhile, high amylopectin containing food will have crumbly texture with low density [23]. Cassava flour contains more amylopectin than amylose [24] so that chips produced have crumbly and slightly crispy texture (determined subjectively by researcher). Another difference property was that cassava flour chips had oily texture. This is related to water content in flour. During the frying process, there are two processes happened respectively. First process is evaporation of water from the inner part of food into
52
different dosage (Fig. 2) depends on the fortificant. Compared to other iron fortificant, NaFe EDTA is one of iron fortificant which produces least organoleptic characteristic changes including color changes [27]. Then, related to zinc fortification, previous study [28] resulted that there was no undesirable organoleptics changes noticed in bread fortified by more than 100 mg/kg of zinc. In overall performance, wheat flour chips were mostly liked. It was because the chips had bright color, crispy and crunchy texture, and did not have acid and musty taste. 4.3 Zinc and Iron Content The highest zinc and iron contents were found in the cassava flour chips fortified by of each NaFe EDTA 50 ppm and ZnSO4 50 ppm. Besides the fortificants themselves, the ingredients which partly contributed to both mineral content in the chip was wheat flour. Wheat flour used in this research was one of Indonesian manufactured wheat flour that had been fortified by zinc and iron. This was why the zinc and iron content in wheat flour chips was higher than non-fortified cassava flour chips content (Table 4). Compared to unfortified cassava flour chip, there should be increment of iron content as much as 3.978 ppm and 6.630 ppm in cassava flour chips fortified by each 30 ppm and 50 ppm of NaFe EDTA respectively. However, based on data in Table 4, the increment of Fe content is 14.579 ppm in cassava flour chips fortified by 30 ppm of NaFe EDTA and 29.372 ppm in cassava flour chips fortified by 50 ppm of NaFe EDTA. This higher incremental is caused by the existence of free Fe, which are not bound with EDTA molecule and counted by AAS. NaFe EDTA fortificant used in this study was made by the researcher with FeSO4 as iron source. Less properly rinsing with aquabidest and NaOH done in the last stage of it making process caused the presence of free iron molecules of FeSO4 which were not bound with EDTA molecules. Unlike iron content, the increment of zinc content
in fortified cassava flour chips was lesser than expected. According to Rosado [29], the percentage of zinc content ion ZnSO4 was 32%. So, there should be increment of zinc content as much as 9.6 ppm and 16 ppm in cassava flour chips fortified by each 30 ppm and 50 ppm of ZnSO4 respectively. However, based on Table 4, there was only 7.916 ppm and 12.089 ppm of zinc content increment. This lesser content was caused by losing zinc molecules during cooking process. Data of zinc and iron content of the cassava flour chip can be used to estimate how much portion of chips should be eaten by the target population to prevent zinc and iron deficiency. The permitted intake of EDTA was 2.5 mg EDTA/kg body weight/day [30], whereas recommended iron intake from NaFe EDTA was 0.2 mg/kg body weight/day [31]. School children are one of population group who are commonly affected by zinc and iron deficiency. If its generally assumed that school childrens average weight are 30 kg, so they only could consume maximum NaFe EDTA as much as 6 mg/day. In 1 kg cassava flour chips fortified by 50 ppm of each NaFe EDTA and ZnSO4 there was about 22 mg zinc and 70 mg iron. That one kilograms chips could be divided into 67 portions of chips (@ 15 g. This portion was based on average portion of snacks sold in Indonesian markets). Thus, in 1 portion, chips contains about 0.33 mg zinc and 1.04 mg iron. Based on National Workshop on Food & Nutrition 1998, daily requirement of zinc and iron for school children was 10 mg. So, by the calculation above, maximum consumption of 10 cassava chips portions/day hopefully could help to prevent the children from zinc and iron deficiency. However, this fortified cassava flour chips is planned as additional food/snack so it is needed another source food of zinc and iron to meet the requirements. The bioavaibility of Fe from NaFe EDTA is about 51% and Zn from ZnSO4 is about 33% in whole wheat flour in rats [32]. So using that
53
assumption, the consumption of 10 portions of chip will only meet about 50% of requirements. Other zinc and iron food source such as egg, meat, fish, beans and green vegetables should be eaten daily to help meeting the requirements. Although the fortified cassava flour chip has a worse sensory characteristic compared to wheat flour chips, the fortified cassava flour chip is potential way to prevent nutritional problem such as iron and zinc deficiency. However, there should be improvements in some points to produce better chips. One of problem in the making process of cassava flour chips is that the dough was quickly dry and the dry dough will be difficult to be processed henceforth. It needed a specified machine used to process the dough quickly so it can be fried soon. Flavoring powder such as barbeque flavor, cheese flavor, chili taste, etc. can be added to the chips to reduce the undesirable taste in the chips.
[6]
[7]
[8]
[9] [10]
[11] [12]
[13]
5. Conclusions
There was no significant effect of both ZnSO4 and NaFe EDTA on sensory characteristics of cassava flour chips. There were different levels of preference of each cassava flour groups. There was significant difference of zinc and iron contents in each cassava flour groups. Consequently, it can be a potential way to improve the value of cassava as basic ingredients of chips. Then the effectiveness of these fortified chips to improve the hemoglobin and zinc status of people needs further research.
[14]
[15]
[16]
[17]
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Sensory and Organoleptic Characteristic, Zinc and Iron Content of Fortified Chips from Cassava Flour the role of iron EDTA, Archivos Latino Americano de Nutrition 49 (2) (1999) 23-33. D. Romaa, K.H. Brown, J.X. Guinard, Sensory trials to assess the acceptability of zinc fortificants added to iron-fortified wheat products, Journal of Food Science 67 (2002) 461-465. J.L. Rosado, Zinc and copper: Proposed fotification levels and recommended zinc compounds, J. Nutr. 133 (2003) 2985-2989. T.H. Bothwell, A.P MacPhail, The potential role of NaFe EDTA as an iron fortificant, International Journal of Vitamine and Nutrition Res. Nov. 74 (6) (2004) 421-434. R. Hurrell, I. Egli, Optimizing the bioavailability of iron compounds for food fortification, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Zurich, Switzerland, 2006. S.Z. Akhtar, Z. Rehman, F.M. Anjum, Z. Ali, A. Nisar, Bioavaibility of iron and zinc fortified whole wheat flour in rats, Pakistan J. Zool 42 (6) (2010) 771-779.
2nd ed., Gramedia, Jakarta, 2002. (in Indonesian) [22] L.K. Mahan, S. Escott-Stump, Krauses food, nutrition & diet therapy, Saunders, Philadelphia, 2004. [23] S. Koswara, Technology of starch modification [Online], 2009, http://ebookpangan.com. (in Indonesian) [24] Z. Nahdlah, The characteristics of cassava flour (Manihotesculenta, Crantz) and modified cassava flour (Modified Cassava Flour/MOCAF) and their application in food product, Bachelor Thesis, Universitas Gadjah Mada Yogyakarta, 2010. (in Indonesian) [25] B. Jamaluddin, P. Raharjo, Rochmadi, Mathemathical model of heat and mass movement on fruit frying process in anaerob condition, Proceedings on National Seminar of Agricultural Technique, Yogyakarta, 2008, pp. 18-19. [26] M.K. Mahmud, N.S. Hermana, R.R. Zulianto, I. Apriyantono, Ngadiarti, Hartati, Budi, Bernadus, Tinexcelly, Indonesian food composition table, Elex Media Komputindo, Jakarta, 2009. (in Indonesian) [27] T.H. Bothwell, Iron fortification with special reference to
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Journal of Food Science and Engineering

Volume 3, Number 1, January 2013 (Serial Number 18)

David Publishing Company www.davidpublishing.com

T. Karupaiah (Malaysia) Zbeyde ner (Turkey)

David Publishing Company www.davidpublishing.com

Journal of Food Science and Engineering

Journal of Food Science and Engineering 3 (2013) 1-8

2. Materials and Methods

3. Results and Discussion

The initial and final pH values and sugar contents

Initial pH and sugar Sugar (%)

Fig. 3 Changes in pectin composition of peach during pressurizing and heating.

High-pressure-induced jam, Heat-induced jam.

Fig. 5 L-, a- and b-values of peach jam.

Journal of Food Science and Engineering 3 (2013) 9-18

2. Materials and Methods

3. Results and Discussion

Journal of Food Science and Engineering 3 (2013) 19-24

2. Materials and Methods

3. Results and Discussion

Journal of Food Science and Engineering 3 (2013) 25-32

2. Materials and Methods

3. Results and Discussions

Journal of Food Science and Engineering 3 (2013) 33-39

2. Materials and Methods

Journal of Food Science and Engineering 3 (2013) 40-46

2. Current State of Demand for Organic Food in the EU

3. Organic Food in Serbia

Product Consumer Product

Processing Fig. 1 Possible alternatives to organic food production.

Journal of Food Science and Engineering 3 (2013) 47-54

The ingredients of the chips.

Ingredient Cassava flour

2. Materials and Methods

Sensory characteristics analyze result.

K2 2.72 3.40b 3.08b 3.96

K3 3.48 3.88b,c 3.12b,c 4.32

K4 3.16b,c,d,1,2 3.88b,c,d 3.28b,c,d 3.96

P of Kendalls W test* 0.000 0.000 0.014 0.061

P of Kendalls W test# 0.030 0.125 0.759 0.125

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