Proceedings of the Fourth International Conferencemultilingual.bionetsyst.com/images/other/2013_Volume_2.pdf · Proceedings of the Fourth International Conference Publisher Bulgarian

ISSN 1313-7735

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES

Proceedings of the Fourth International Conference

Publisher

Bulgarian National Multidisciplinary Scientific Network of the

Professional Society for Research work

Volume 2

LOZENEC, BULGARIA 12– 16 JUNE, 2013

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES

Fourth Conference

Publisher

Bulgarian National Multidisciplinary Scientific Network of the Professional Society for Research Work

with the support of

Bulgarian National Society of Agricultural Engineers “Engineering and Research for Agriculture”

“Angel Kunchev” University of Rousse, Bulgaria National Institute of Research-Development for Machines and Installations

Designed to Agriculture and Food Industry, Bucharest, Romania University of Agricultural Sciences and Veterinary Medicine of Banat Timişoara,

Faculty of Zootechny and Biotechnology, Romania Society “ECOLOGICA”, Belgrade, Serbia

Volume 2

Social and human sciences. Economic sciences. Ecology and Environment protection, Land & Water Use

and Environment, Physics, Chemistry, Biology, Computer Systems and Technologies

LOZENEC, BULGARIA 12– 16 JUNE, 2013

Organizing Committee

Atanasov Atanas, Bulgaria - Editor in Chief Mihailov Nikolay, Bulgaria Vezirov Chavdar, Bulgaria Kehayov Dimitar, Bulgaria Bikic Sinisa, Serbia Vlăduţ Valentin, Romania Dochev Veselin, Bulgaria Bungescu Sorin, Romania Atanasova Polina, Bulgaria Kneževiš Dario, Croatia Biriş Sorin-Ştefan, Romania Sandric Maria, Romania

International Scientific Committee Vezirov Chavdar, Bulgaria Banaj Đuro, Croatia Paradjikovic Nada, Croatia Vulkov Lubin, Bulgaria Inoue Keiichi, Japan Vlăduţ Valentin, Romania Martinov Milan, Serbia Popescu Simion, Romania Bilalis Dimitrios, Greece Fröba Norbert, Germany Kehayov Dimitar, Bulgaria Perkoviš Anica, Croatia Zheliazkova Irina, Bulgaria Bungescu Sorin, Romania Kaya Yalcin, Turkey Biriş Sorin-Ştefan, Romania Jovanoviš Larisa, Serbia

ISSN 1313-7735

Printed in Bulgaria Printing House “Angel Kunchev” University of Rousse 8, Studentska Street, 7017 Rousse, Bulgaria

PREFACE

Dear Colleagues! The International Scientific Conference “Research People and Actual

Tasks on Multidisciplinary Sciences” is fourth International Conference organized in Bulgaria with basic purpose to create the framework for the presentation, debate and publication of the valuable scientific results obtained by both the young members.

United by the idea of Multidisciplinary Sciences, the researchers and faculty will report the results of their research. Thus, the scientists will contribute is to promote exchange of research results, scientific ideas and their practical implementation and development work in the various disciplines.

We hope this meeting will initiate new joint research projects, new friendships. We owe special thanks to all participants, and especially to the supporting organizations.

Chief Editor Assoc. Prof. Dr. Atanas Atanasov

CONTENTS VOLUME 2

Social and human sciences. Economic sciences. Ecology and Environment protection, Land & Water Use

and Environment, Physics, Chemistry, Biology, Computer Systems and Technologies

1. STABILIZATION OF L-ASCORBIC ACID WATER SOLUTIONS DURING ITS DETERMINATION BY HORSERADISH PEROXIDASE

Vladan Djuriš, Nebojša Deletiš, Slaviša Stojkoviš, Nenad Draškoviš, Suzana Stankoviš…………………………………………………………………………1

2. IRON CONTENT IN PSEUDOGLEYOF WESTERN SERBIA AS DETERMINED BY DIFFERENT EXTRACTION METHODS

M. Jelic, J. Milivojevic, G. Dugalic, O. Nikolic, S. Stamenkovic………………………6

3. THE ASSESSMENT OF ANTIOXIDANT CAPACITY OF MEDICINAL HERBS IN LOW PH CONDITIONS

D.S. Stef, I. Gergen, T.I. Trasca, A. Rivis, L. Stef, R. Heghedus-Mindru………..….11

4. THE EVALUATION OF FOOD QUALITY BY USING DIFFERENT METHODS

D.S. Stef, R. Heghedus-Mindru, L. Stef, G. Heghedus-Mindru, M. Cazacu, G.Bujanca, F. Berbentea………………………………………………………………..16

5. ANGIOTENSIN I CONVERTING ENZYME INHIBITORY ACTIVITY OF PUMPKIN OIL CAKE 12S GLOBULIN HYDROLYSATES Ž. Vaštag, Lj. Popoviš, S. Popoviš, D. Peričin, and V. Krimer-Maleševiš................21

6. THE PLANTLETS ROOTING OF CHILLI PEPPER OBTAINED IN VITRO CULTURE CONDITIONS A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar………………………………………………………………….…26

7. THE SUCROSE INFLUENCE ON CHILLI PEPPERS PLANTLETS GROWING IN THE IN VITRO CULTURE CONDITION A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar………………………………………………………………….…31

8. THE DETERMINATION OF THE OPTIMAL CHLORINE DOSE FOR THE DISINFECTION OF A MICROBIAL CONTAMINATED WASTEWATER V.V. Safta, Viviana-Carmen Ciucă...........................................................................35

9. STUDY ON MEAT QUALITY DEPENDING ON OXIDATIVE STRESS OF THE ANIMALS BEFORE SLAUGHTER Iuliana Manea, Laur Constantin Manea………………………………………………40

10. THE DYNAMIC OF PHENOLS ACCUMULATION AND THE INFLUENCE ON THE ANTIOXIDANT ACTIVITY OF VITIS VINIFERA L. CALLUS CULTURE EXTRACTS R. Mihai, F. Helepciuc, and M. Mitoi……………………………………………………44

11. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS

FROM NORTH REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) R. C. Heghedűş-Mîndru, G. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac……………………………………….…50

12. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM WEST REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac…………………………………………..56

13. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM CENTRE REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac………………………………………….62

14. MICROARCHITECTURE OF QUATERNIZED POLYSULFONE/POLY (VINYLIDENE FLUORIDE) BLEND FOR SCAFFOLDS IN TISSUE ENGINEERING L. I. Buruiana, E. Avram, A. Popa, and S. Ioan........................................................67

15. ANTIOXIDANT CAPACITY OF SALVIA OFFICINALIS EXTRACT REGARDING THE SUNFLOWER OIL USED IN FOOD THERMAL APPLICATIONS Nadina Buta, N. Popa, Nicoleta Bordea, Mariana Poiană and T.I. Traşcă..............72

16. IMPROVE PERFORMANCE OF THE POLYSULFONE MEMBRANES INDUCED BY PRESENCE OF POLYVINYL ALCOHOL A. Filimon1, E. Avram1, and S. Ioan1…………………………………………………...77

17. DEVELOPMENT AND ANALYSIS OF A TOPICAL PHARMACEUTICAL PRODUCT CONTAINING RIBES NIGRUM LEAVES EXTRACT L.E. Dutu, .T.D.Balaci, C.E.Gird, L.M. Popescu, I. Dutu……………………………..82

18. PRELIMINARY RESEARCH REGARDING A DERMATO-COSMETIC PRODUCT WITH VEGETAL EXTRACTS USEFUL FOR THE TREATMENT OF HYPERPIGMENTATION C. E. Gîrd, T. D. Balaci, L. E. Duţu, I. Nencu, E. A. Ciocan.....................................88

19. ISOTROPIC AND ANISOTROPIC PHASES IN CELLULOSE LIQUID CRYSTALLINE SOLUTIONS M. Dobos1, M. D. Onofrei1, and S. Ioan1..................................................................95

20. DIELECTRIC PROPERTIES OF NEW POLYSULFONES WITH CHELATING GROUPS R.M. Albu1, E. Avram1, V.E. Musteata1, and S. Ioan1………………………………100

21. EFFECT OF AMMONIUM VANADATE ON VIABILITY AND PROLIFERATION OF CULTURED ANIMAL CELLS Abdulkadir Abudalleh, Pavel Mitrenga, Tanya Zhivkova, Lora Dyakova, Stoyan Shishkov, Radostina Alexandrova……………………………………………..……105

22. THE FORMULATION, PREPARATION AND CHARACTERIZATION OF A

COMPRESSED MULTIPARTICULATE SYSTEM Mircea Hîrjău, Dumitru Lupuliasa, Mirela Mitu, Anca Nicoară, Victoria Hîrjău…..111

23. STUDIES CONCERNING THE INCORPORATION OF ORIGANUM L. SYRIACUM ESSENTIAL OIL IN LIQUID AND SOLID FORMULATIONS FOR ORAL CAVITY USE Zeinab Lakis, Teodora Balaci, Cerasela Gird, Ancuţa Fiţa, Andreea Stanescu, Denisa Mihele…………………………………………………………………………..118

24. THE MICROBIOLOGICAL MONITORING OF SOME BATCHES OF MILK ORIGINATED FROM PRIVATE HOUSEHOLDS IN THE WEST PLAIN OF ROMANIA Mişcă Corina Dana, Mişcă C. B., Rinovetz A., Bujancă G., David I., Rădoi P. B., Rinovetz Adina.......................................................................................................124

25. THE MONITORING OF ENZYME ACTIVITY OF GLUCOAMYLASE AND ENDOXYLANASE ON THE BREAD DOUGH David I., Misca Corina, Bujanca G., Rinovetz A., Berbentea F., Georgescu L., Danci M..................................................................................................................129

26. VARIATION OF HEAT TRANSFER AND TEMPERATURE OF TOMATO PASTE AT STERILIZATION AND COOLING PROCESSES G. Bujancă. C. Jianu, A. Rinovetz, F. Crista, I. David, Corina Dana Mişcă, D. Ştef, Antoanela Cozma, Mihaela Cazacu, Zorica Adina Riniovetz……………………..136

27. AMENDING FACTORS OF ANTHOCYANIN DYES IN FRUIT JUICES G. Bujancă, C. Jianu, A. Rinovetz, Corina Dana Mişcă, I. David, B. Rădoi, D. Ştef, Ariana Velciov, Mihaela Cazacu, Adina, Zorica Adina Rinovetz............................142

28. IMPROVEMENT OF PUMPKIN OIL CAKE PROTEIN GELATION PROPERTIES THROUGH ENZYMATIC POLYMERYZATION WITH TRANSGLUTAMINASE Lj. Popoviš, Ž. Vaštag, S. Popoviš, D. Peričin, V. Krimer-Maleševiš.....................148

29. VIABILITY ASSESSMENT OF Ruscus aculeatus L. GERMPLASM EXPOSED TO EXTREME LOW TEMPERATURES Banciu Cristian, Anca Aiftimie-Păunescu…………………………………………...153

30. STUDIES CONCERNING THE VARIABILITY OF SOME BLACK PINE (PINUS NIGRA) POPULATIONS FROM ROMANIA AND GREECE I.Sotiropoulos., E.Madoşă, Oana Danci, and M.Danci……………………………...158

31. AN ASSESSMENT ON THE TROPHIC STATE OF LAKE ŠELIJE, SERBIA S. Širiš, B. Miloševiš, J. Kneževiš and Z. Spasiš...................................................163

32. DISCOVER TRADITIONAL GASTRONOMY ORIGINATING FROM THE VRANCEA COUNTY C. C. Vişan and C. I. Băra......................................................................................168

33. ENVIRONMENTAL DAMAGES, WHICH MUST BE COMPENSATED DURING THE PROCESS COMPULSORY PURCHASE OF LAND IN UKRAINE

Alina Lizunova, Mariia Mykhalova…………………………………………………….173 34. SYSTEMATIC AND ECOLOGICAL ANALYSIS ON STINK BUGS

(PENTATOMIDAE, HEMIPTERA) IN THE ECOSYSTEMS OF DURRESI REGION, ALBANIA Eltjon Halimi1,Hesat Aliu2 , Anila Paparisto1, Dritan Topi1…………………………..177

35. INTERDISCIPLINARY STUDY ON INFLUENCE OF VARIOUS TYPES OF WATER ON HERBS AND AROMATIC PLANTS IN CULTURE Gabriela Vlăsceanu1, Ştefan Manea1, Georgeta Negru1, Marosy Zoltán2, Alexandru Cristian Ion3............................................................................................................184

36. NEW PERSPECTIVES FOR SPIRULINA PLATENSIS G. Iordachescu, G. Vlasceanu, St. Manea.............................................................190

37. DNA IDENTIFICATION OF HUMAN REMAINS EXHUMED IN KOSOVO AND METOHIA DURING THE PERIOD 2001-2011 Milanka Miletiš, Nebojša Deletiš, Zvezdan Milanoviš, Suzana Matejiš, Sonja Smiljiš……………………………………………………………………………..195

38. ANALYSIS OF THE MOTIVATING FACTORS AND THE SATISFACTION OF THE EMPLOYEES OF A COMPANY FROM TIMISOARA (ROMANIA) C. C. Ruset, G. Buzamat, G. Popescu, K.I. Lato, and I. Cretescu…………………200

39. THE ANALYSIS OF THE FINANCIAL POSITION IN AN AGRICULTURAL COMPANY FROM ROMANIA Genoveva Buzamat, Corina Ruset, Elena Pet, Iuliana Cretescu...........................205

40. THE PROBLEM OF SYNTACTIC STRUCTURES IN CONTEMPORARY LINGUISTICS L.Ramazanova.......................................................................................................210

41. ENGLISH FOR SPECIFIC PURPOSES IN CAREER OPPORTUNITIES Rodica Silvia Stan………………………………………………………………………215

42. Opportunities and prospects of improvement of differentiation of lands in Ukraine A.N. Lychogrud…………………………………………………………………………..220

43. Usage of renewable energy resources in Serbia Larisa Jovanoviš…………………………………………………………………………224

44. Application of the multi-criteria decision analysis for assessing the student’s programming task performance I. Zheliazkova and O. Kir…………………………………………………………….....229

45. Environmental applications of zeolite-based nanomaterials. hospital wastewater treatment investigation C.I.Covaliu, L.Toma, G. Paraschiv, S.Şt. Biriş……………………………………….237

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

1

STABILIZATION OF L-ASCORBIC ACID WATER SOLUTIONS

DURING ITS DETERMINATION BY HORSERADISH PEROXIDASE

Vladan Djuriš, Nebojša Deletiš, Slaviša Stojkoviš, Nenad Draškoviš, Suzana Stankoviš

Abstract: Metaphosphoric acid has been shown to be the best medium for homogenization of vitamin C, and it stabilized the vitamin even in the presence of oxygen. Metaphosphoric acid is often used for extraction of ascorbate from natural products, in order to precipitate proteins from sample and to inhibit endogenous ascorbidase. During the study, several different stabilizing substances have been tested and compared with the protection effect of metaphosphoric acid. Mixture of EDTA and acetic acid showed the greatest protective effect and expressed synergism in regard to simple components.

Key words: Ascorbic Acid, Horseradish, Peroxidase, Stability, Determination.

INTRODUCTION

Globalization of modern society sets new standards in every area of human activity, so a special attention is paid to production and processing of food and water, as precondition of living quality. Nutritionists and pharmacists additionally advocate correction of food nutritive value using supplementation and fortification by various vitamins and minerals, with the aim to protect and improve nutritive properties of food products. This concept necessarily includes systems for safety control in every stage of food production and distribution, together with inventing new methods and modifying existing ones for chemical analysis of versatile sample types.

The all vitamins are essential nutrients that ought to be present in diet, and their deficiency cause a row of structural and functional disorders. For that reason their presence in dietary supplements is almost inevitable.

Vitamin C is L enantiomer of ascorbic acid and represents a derivative of glucose soluble in water. Although it is necessary cofactor for several enzymes involved in various metabolic processes, the highest attention in last few decades is attracted by its antioxidant activity (Padayatty et al., 2003). Thus, vitamin C is one of the most important nonenzymatic antioxidants, capable to neutralize most of reactive oxygen species (by donating hydrogen), above all thanks to its low redox potential (0.18 and 0.08 V at pH 4.5 and 6.4, respectively) (Shekhovtsova et al., 2006).

L-ascorbic acid is highly reactive compound, sensitive to action of air and light. Stability of its water solutions depends on pH, temperature, pressure and presence of metal ions and organic matter. During oxidation, ascorbic acid is converted to dehydroascorbic acid (Welch et al., 1995).

For that reason, water solutions and extracts containing vitamin C ought to be stabilized immediately after sampling by adding suitable substances, in order to get more relevant data. L-ascorbic acid is stabler at lower pH values, so that solutions of acids (metaphosphoric, phosphoric, hydrochloric, etc.) are usually used as mediums. Metaphosphoric acid ((HPO3)3) has been shown to be the best medium for homogenization of vitamin C, and it stabilized the vitamin even in the presence of oxygen. (HPO3)3 is often used for extraction of ascorbate from natural products, in order to precipitate proteins from sample and to inhibit endogenous ascorbate oxidase (Davey et al., 2000).

Although there are plenty of methods for determination of vitamin C published in literature, as regarding food products as biological fluids, a constant effort is directed to find better and more accurate technics.

Within a wider study aimed to standardize method by which the total antioxidant capacity of certain sample would be expressed as equivalent of corresponding amount of


2

vitamin C, for determination of vitamin itself was used the enzymatic kinetic method with horseradish peroxidase (Arnao et al., 1996). It is highly sensitive method with detection limit below 0.1 μM. As the most of kinetic methods, this method is also limited by numerous interferences with complex chemical system composed of many reactants and having relatively low frequency of carrying out. Involving new components, such as stabilizers of vitamin C, gives an additional load to this system, especially because metaphosphoric acid, like other mineral acids, shows strong inhibitory effect to peroxidase enzyme, which affects final results of measurement (Đurić et al., 2012).

For that reason several alternative stabilizing substances have been tested, such as acetic, oxalic and ethylenediaminetetraacetic acid (EDTA), with the aim to obtain consistent results during enough long period of time, needed for carrying out the experiment.

MATERIAL AND METHODS

The trial was set so that different concentrations of L-ascorbic acid in the presence of various stabilizing agents were exposed to laboratory atmosphere in open glasses at room temperature. Concentration of ascorbic acid was determined by the standard volumetric method with 2,6-dichlorophenolindophenol (DCPIP) and by spectrophotometry, based on horseradish peroxidase activity. Coefficient of direction of the obtained curves was used as a measure of vitamin C oxidation speed.

During this study, the following solutions and equipment were used: Solutions of L-ascorbic acid (Galenika, Serbia) used in this trial were within range of

concentration between 0.1 and 10 mM. Buffer systems: acetate buffer pH 4 (made in the laboratory), citrate buffer pH 5

(Farmitalia Carlo Erba, Italy), citrate buffer pH 6 (Farmitalia Carlo Erba, Italy), phosphate buffer pH 7 (Fisher Scientific, UK) and borate buffer pH 8 (Farmitalia Carlo Erba, Italy).

The following stabilizing substances were used: metaphosphoric acid, ortophosphoric acid, hydrochloric acid, acetic acid, oxalic acid and EDTA.

Preparation of horseradish peroxidase of purity level 200 kU/g was made by Merck (Germany). Enzyme solutions were prepared immediately before trials. Relative activity of horseradish peroxidase was determined by spectrophotometry, measuring dependence of absorbance of o-tolidin oxidized form (Centrohem, Serbia), at wavelength of 630 nm, from the moment of H2O2 injection. For that purpose spectrophotometer Beckman DU-65 (UK) was used.

Solutions of hydrogen peroxide (Merck, Germany) were checked by standard permanganometric method.

As the dilutant for preparing basic solutions we used deionized water, with 0.5 μS/cm2 of conductivity, while for preparing series of ascorbic acid water solutions we used distilled water, with 15 μS/cm2 of conductivity.

RESULTS AND DISCUSSION

Stability of L-ascorbic acid water solutions is time-dependent and decreases by rise of dilution (figure 1a).

Speed of vitamin C degradation is a reaction of first order (Vieira et al., 2000; Van der Broeck et al., 1998) and, as can be seen, dependent on concentration. Logarithm of ascorbic acid concentration is linearly dependent on time (Imer et al., 2003). Solutions of higher concentration showed a lower degree of degradation, which pointed to certain degree of autoprotection of L-ascorbic acid molecules.


3

a) b)

0 24 48 72 96 120

1E-5

1E-4

1E-3

0.01

AscH2 0.1mM

AscH2 1mM

AscH2 10mM

lo

g C

AscH

2

Time (h)

0 24 48 72 96 120

1E-4

1E-3

pH 8

pH 7

pH 6pH 5

pH 4

log

C A

scH

2

Time (h)

Figure 1 a) Speed of L-ascorbic acid transformation to dehydroascorbic acid in water solutions 10; 1 i

0.1 mM of concentration during 96 hours. Coefficients of line direction were: -0.0126; -0.0144; and -0.0169, respectively. b) Dependence of L-ascorbic acid transformation speed to dehydroascorbic acid of medium pH, without use of stabilizing substances.

The speed of transformation AscH2 into DHA, in aerobic conditions, is faster at higher

pH values, in other words, L-ascorbic acid is stabler in acid environment, as presented on figure 1b.

Content of vitamin C in acetate buffer pH 4; citrate buffers pH 5 and pH 6; phosphate buffer pH 7 and borate buffer pH 8, after 96 hours amounted: 59%, 40.7%, 30.05%, 6.6% and 1.53% of the initial value, respectively.

Application of standard stabilizing substances significantly decelerated degradation of ascorbic acid in water solution (figure 2a).

a) b)

0 24 48 72 96 120

1E-3

8E-4

(HPO3)

3 2%

(HPO3)

3 1%

H3PO

4 3%

HCl 1%

HCl 2%log C

AscH

2

Time (h)

0 24 48 72 96 120

1E-3

5E-4

CH3COOH 0.1M + EDTA 0.5mM

CH3COOH 0.1M + EDTA 1mM

CH3COOH 0.05M

EDTA 0.5mM

H2C

2O

4 0.1M

CH3COOH 0.1M

EDTA 1mM

log C

AscH

2

Time (h)

Figure 2 a) Speed of ascorbic acid degradation when standard (inorganic) stabilizing substances were

used. b) Speed of ascorbic acid degradation when organic stabilizing substances were used.

As expected, the most efficient stabilizing agent was metaphosphoric acid and, when

it was present, concentration of vitamin C during 24 hours, stayed almost unchanged. After 96 hours, content of ascorbic acid determined by the standard DCPIP method, in 2% and 1% solutions of metaphosphoric acid, 3% solution of ortophosphoric acid and 2% and 1% solutions of hydrochloric acid, amounted 98.0%, 95.5%, 90.5%, 88.2% and 72.4% of the initial value, respectively.

Results of the same samples, obtained using kinetic spectrophotometric method, differed significantly in regard to the expected values, and showed unrealistically higher content of vitamin C (tab. 1).

Application of organic acids as stabilizing agents in the prescribed doses increased content of ascorbic acid after 96 hours by more than 10 times in regard to the reference


4

value without using stabilizers (figure 2b). As organic stabilizers, CH3COOH, H2C2O4 and EDTA, as well as their mixtures, were applied. Different concentrations of these substances showed different protective effect on content of ascorbic acid in solutions.

A satisfactory stabilizing effect, during first 48 hours, for determination by the standard DCPIP method and enzymatic method using horseradish peroxidase, was shown by mixture of CH3COOH and EDTA (tab. 1).

Table 1 Stabilizing effect of some substances on vitamin C in water solutions during

48 hours (presented as percent in regard to 1 mM of vitamin C).

Reaction type No

stabilizers (HPO3)3

2% HCl 2% H2C2O4 0.1M

CH3COOH 0.1M

EDTA 1 mM

CH3COOH 0.1M EDTA 1mM

DCPIP 0h 98.6 100 100.2 101.0 100.6 99.5 100.1

DCPIP 24h 61.4 100.1 91.2 94.1 94.5 85.6 99.3

DCPIP 48h 19.2 99.5 80.5 89.2 85.5 79.7 98.5

Peroxidase 0 h 103.1 137.4 129.3 107.0 100.8 99.7 101.4

Peroxidase 24 h 66.0 135.8 121.6 103.3 95.4 87.8 99.8

Peroxidase 48 h 27.3 135.0 120.2 96.6 88.1 82.3 97.8

Mixture of CH3COOH and EDTA showed a synergistic effect in regard to simple

components and did not change activity of horseradish peroxidase enzyme.

CONCLUSIONS AND FUTURE WORK

On the basis of the above mentioned, the following conclusions can be drawn out:

L-ascorbic acid, physiologically active form of vitamin C, can easily undergo to oxidation process of transformation to inactive dehydroascorbic acid, which can significantly affect content of this vitamin in regard to the declared values for various food products.

Lower concentrations of L-ascorbic acid undergo faster to oxidation than solutions of higher concentration, which suggests possible autoreduction mechanism.

Process of L-ascorbic acid oxidation accelerates significantly by increasing pH value of medium.

Among the applied classical stabilizers ((HPO3)3, H3PO4 and HCl), the highest protective effect was shown by metaphosphoric acid. Mixture of CH3COOH and EDTA (ratio 100:1) showed almost the same protective effect as metaphosphoric acid.

Mixture of CH3COOH and EDTA did not affect peroxidase activity at any observable level which could disturb measuring L-ascorbic acid using the mentioned enzymatic system, while inorganic stabilizers inhibit peroxidase activity, and so they are useless for application in this enzymatic method.

Price of (HPO3)3 is several times bigger than price of CH3COOH and EDTA. REFERENCES

[1]. Arnao, M.B., Cano, A., Hernandez-Ruiz, J., Garcia-Canovas, F. and M. Acosta. 1996. Inhibition by L-ascorbic acid and other antioxidants of the 2,2‘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation catalyzed by peroxidase: a new approach for determining total antioxidant status of foods. Anal. Biochem., 236, 255-261.


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[2]. Davey, M.W., Montagu, M.V., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I.J., Strain, J.J., Favell, D. and J. Fletcher. 2000. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agric., 80, 825–860. [3]. Djuriš, V.R., Deletiš, N.R., Stankov-Jovanoviš, V.P. and R.M. Simonoviš. 2012. Inhibitory effect of retinol acetate on horseradish peroxidase. Hemijska industrija, On Line-First (00), 95-95. [4]. Imer, F., Sonmezoglu, I.C. and M. Kozcaz. 2003. The role of buffers on thr kinetics of l-ascorbic acid oxidation catalyzed by copper (II). Italian Journal of Food Science, 15, 521-529. [5]. Padayatty, S.J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J., Chen, S., Corpe, C. and A. Dutta. 2003. Vitamin C as an antioxidant: evaluation of its role in disease prevention. Journal of the American College of Nutrition, 22(1), 18-35. [6]. Shekhovtsova, T.N., Muginova, S.V., Luchinina, J.A. and A.Z. Galimova. 2006. Enzymatic metods in food analysis: determination of ascorbic acid. Anal. Chim. Acta, 573-574, 125-132. [7]. Van der Broeck, I., Ludikhuyz,e L., Weemaes, C., Van Loey, A. and M. Hendricks. 1998. Kinetics of isobaric-isothermal degradation of L-ascorbic acid. Process optimisation and minimal processing of foods. 4, 12-17. [8]. Vieira, M.C., Teixeira, A.A. and C.L.M Silva. 2000. Mathematical modeling of the thermal degradation kinetics of vitamin C in cupuaçu (Theobroma grandiflorum) nectar. Journal of Food Engineering, 3(1), 1-7. [9]. Welch, R.W., Wang, Y., Crossman, A., Park, J.B., Kirk, K.L. and M. Levine. 1995. Accumulation of vitamin C (Ascorbate) and Its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem., 270(21), 12584-12592.

ABOUT THE AUTHORS

V. Djuriš, MSc, Teaching Assistant, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected]

N. Deletiš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected]

S. Stojkoviš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected]

N. Draškoviš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected]

S. Stankoviš, PhD, Senior Research Associate, Mining and Metallurgy Institute, 35 Zeleni Bulevar 19210 Bor, Serbia, E-mail: [email protected]

mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


6

IRON CONTENT IN PSEUDOGLEYOF WESTERN SERBIA AS

DETERMINED BY DIFFERENT EXTRACTION METHODS

M. Jelic, J. Milivojevic, G. Dugalic, O. Nikolic, S. Stamenkovic

Abstract: This study was conducted on pseudogley soil having different physico-chemical characteristics. Soil samples were collected from tilled fields and meadows at six different locations in Western Serbia. A extraction methods was applied with the extraction of total iron (HNO3-HclO4-HF) and available forms (0.1 M HCl- and 0.005 M DTPA-extractable). The physico-chemical characteristics of the test pseudogleys had a significant effect on the content and forms of iron in different locations in Western Serbia. Soil pH, CEC and the content of finer fractions (silt and clay) had the highest effect on the distribution of different forms of iron The extraction methods showed similar forms of iron in tilled field and meadow pseudogleys. However, the content of DTPA-extractable iron did not correspond to the total content, which led to the common incidence of iron deficiency in Western Serbian pseudogleys regardless of the high levels of total iron in the soil.

Key words: Content, Extraction method, Iron, Location, Pseudogley, Soil.

INTRODUCTION

Pseudogleys cover significant areas of Serbia, accounting for about 285,000 ha or 78.73% of the total land area in Western Serbia (Tanasijevic et al., 1966). Pseudogleys are rather poor in alkalis, being medium to strongly acid in reaction. They have a highly unfavorable structure, and a low content of organic matter. The acid reaction of pseudogley, its low humus content, and a low supply with available phosphorus and potassium are limiting factors for higher crop yields (Dugalic et al., 2005).

Iron is one of the most common elements in the Earth´s crust, ranking fourth in abundance after oxygen, silicon and aluminium. In the soil, iron forms a variety of minerals such as hematite, goethite, limonite, notronite, and pyrite. Iron is also present in the form of grain-coatings in oxidized material, and in many secondary minerals (Kostic et al., 2002). Iron hydroxides play a key role in the biogeochemical cycle, in the bioavailability of iron itself and in the bioavailability of other elements (Hesterberg, 1998).

The bioavailability, mobility and chemical reactivity of heavy metals in soils are often associated with their distribution among certain soil fractions and the dynamic equilibrium among them (Kreamer, 2004; Sharma et al., 2008). It is well known that main factors affecting the mobility of heavy metals in soil include: pH, content and quality of soil organic matter, content and quality of clay fraction, iron and manganese oxides. Apart from soil pH, which is a key parameter, the content and mainly the quality of soil organic matter can influence the availability of heavy metals in soil (Makovnikova, 2000).

Iron is an important micronutrient whose availability could be significantly affected by soil properties. Generally, the total iron increases with increasing cation exchange capacity (CEC) and the clay and silt content (Sharma et al., 2008). Complexation of iron by soil organic matter may result in increased plant availability, and microbial exudates can supply additional iron to plant routes (Mackowiak and Grossl, 2001). Likewise, exchangeable iron absorbed onto inorganic sites and DTPA extractable iron increase with increasing soil organic matter but decrease with higher soil pH and calcium carbonate content (Sharma et al., 2008; Jakovljevic et al., 2005).


The investigations were conducted on pseudogley soil taken from the Ah horizon at six different localities in Serbia: Kraljevo, Koceljevo, Lajkovac, Gorobilje, Mionica and


7

Vladimirci. The sub-samples were taken from field and meadow ecosystems at a depth of 0 to 20 cm, after which they were air-dried, crushed in a porcelain mortar and sieved through stainless steel screens. Particles 2 mm in size were used for soil characterization and Fe-fraction analyses

Soil pH was determined in a suspension with water and 1M KCl mixture, with the ratio of soil/solution 1:2.5 after a 0.5 hour equilibration period; the organic content was determined using the humus method by Kotzmann (1966), available P2O5 and K2O - using the Al method by Egner-Riehm (Egner et al., 1960). CEC - using 1M NH4OAc, pH 7, and particle size distribution - by a pipette method. The total iron content was determined by atomic adsorption spectrophotometry (AAS, Model Carl Zeis Jena AAS 1N) after soil digestion in acid (HF, HNO3 and HClO4) mixtures. A cold extraction method was used for the determination of the total and pseudo-total iron in which 0.5±0.001 g of sample was transferred into a centrifuge tube and then 10 ml of 0.5 M HCl was added. The solution was shaken, subjected to vortex for a short time and placed on a shaking-table for agitation for 1 h. After agitation, the solution was centrifuged at 3000 rpm (1.900xg) for 15 min, and them filtered through a 0.45-µm syringe filter to remove particulates (Ure, 1995).

The results obtained for different contents of iron (total iron, accessible iron and different chemical fractions) in pseudogley soil were subjected to statistical analysis using Student and Pearson correlation coefficients (Mead et al., 1996).


Table 1. gives main characteristics of the test pseudogley samples. The test soils had very poor physical and chemical characteristics. Particle size analysis showed that, apart from an average content of clay particles (< 2 μm) of 48.3% (tilled fields) and 45.4% (meadows), the soils had a high percentage of silt particles (2-50 μm), averaging 39.3% (tilled field) and 42.9% (meadow). The total percentage of silt and clay particles in the soil was very high, i.e. above 86%, whereas the sand fraction content (0.05-2.0 mm) was very low – about 12%. According to the above properties, these soils were classified as very silt loam soils.

Table 1. The physico-chemical characteristics of pseudogleys in Western Serbia

Soil characteristics Tilled field Meadow

Mean Range Standard deviation

Mean Range Standard deviation

pH(H2O) 5.4 4.9-6.1 0.4 5.5 5.1-3.2 0.4

pH(KCl) 4.3 3.9-5.6 0.4 4.4 4.0-4.9 0.4

Humus content,% 2.9 2.2-3.7 0.5 4.2 2.9-6.6 1.2

P2O5 mg 100 g-1 7.6 3.4-10.1 2.2 7.6 4.4-10.2 1.9

K2O 100 g-1 10.8 8.8-13.5 1.6 9.7 8.2-11.6 1.3

CEC m.e 100 g-1 19.0 14.1-24.8 4.0 18.2 13.3-25.7 4.3

Sand, % 12.4 5.5-21.8 5.7 11.7 4.8-20.6 5.8

Silt, % 39.3 30.2-48.2 6.9 42.9 31.0-55.2 8.3

Clay, % 48.3 44.0-53.9 3.0 45.4 37.9-50.2 3.7

Silt + Clay, % 86.6 78.6-92.4 4.9 88.3 80.3-95.4 5.8

Soil pH ranged from 3.9 to 5.6, averaging 4.3, which makes this soil extremely acid.

The cation adsorption capacity (CEC) was relatively high, ranging from 13.3 to 25.7 m.e 100 g-1 soil, with an average of about 19.0 m.e 100 g-1. The average humus content of tilled field was low (2.9%), whereas that of meadow soil was considerably higher (4.2%).


8

The content of readily available phosphorus in the test locations was low, ranging from 3.4-10.1 mg 100 g-1 in samples taken from tilled fields and 4.4-10.2 mg 100 g-1 in meadow soil samples. The pseudogleys in the test locations had a higher supply of K2O than of P2O5.

The average content of total iron (HNO3-HClO4-HF extraction) in the test pseudogley samples was 2.76% (tilled field) and 2.73% (meadow), the values being within the range of 1.88 to 3.61% (Table 2). The values were typical of the test soil type. The variations between samples were the result of differences between the test soil samples in both physical and chemical characteristics across locations. Soil metals having a higher clay level and higher CEC values are more strongly bound to soil components and they are not susceptible to leaching into deeper soil layers. Hence, the total iron content in these soils is higher than in soils having a lower content of clay fraction and lower CEC values (Adriano, 1986).

The available forms of 0.1 M HCl- and 0.005 M DTPA-extractable iron were extremely low as compared to their total amount in the test soil (Table 2). The content of 0.1 M HCl-extractable iron was twofold higher than that of 0.005 M DTPA- extractable iron. The average content of 0.1 M HCl-extractable iron was 177.6 mg kg-1 (tilled field) and 159.2 mg kg-1 (meadow), whereas that of available 0.005 M DTPA-extractable Fe was 85.3 mg kg-1 (tilled field) and 82.3 mg kg-1 (meadow).

The relative values of HNO3-extractable iron (pseudo-total content) show considerable variations in both groups of test pseudogleys. The average content of pseudo-total forms of iron was about 2.4% in both pseudogley varieties. The percentage of HNO3-extractable forms as compared to the total iron content (HF) in the test locations was 88.5% on average in tilled field pseudogley and 88.1% in meadow pseudogleys. The data suggest that 11 to 12% of the total iron in the soil is bound to the structure of primary and secondary minerals. The results are in agreement with those obtained by Han et al. (2007) who studied the content of 4 M HNO3 extractable iron in vertisol in the Mississippi River Delta. Table 2. Iron content in the tested pseudogley of West Serbia as determined using different extraction methods (X±sd and interval, mg kg-1)

Location HF HNO3 0.1 M HCl 0.005 DTPA

Field (n=6) 27600±5946 24300±4694 177.6±20.5 85.3±15.9

18800-35000 17500-31000 150.0-220.0 60.0-120.0

Meadow (n=6)

27300±5192 24000±4076 159.2±58.5 82.3.±14.9

20500-36100 18500-29800 70.0-240.0 60.0-110.0

t-test 0.615 0.650 1.227 0.581

There is no statistical significance at p ˂ 0.05 The percentage of water-soluble iron in relation to the total iron content is given in

Table 3. In the tilled field pseudogley, the average percentage of water-soluble 0.1 M HCl-extractable iron relative to the total iron was very low (0.63%), whereas that in the meadow pseudogley was lower (0.58%), regardless of similarity in their total and pseudo-total contents. The content of HCl-extractable iron in certain samples showed significant variations, ranging from 0.31 to 0.88, and was found to be independent of the total iron content. Therefore, in most locations, the relative content of 0.1 M HCl-extractable iron decreased with increasing concentration of total iron. However, similar differences were observed between certain samples in the distribution of 0.1 M HCl-extractable iron, resulting from differences in pH between the test samples. Particularly important were the samples with the lowest pH values which had the highest content of HCl-extractable Fe.


9

During 0.005 M DTPA extraction, the average percentage of iron was identical in both pseudogleys (0.31%). As compared to 0.1 M HCl-extractable Fe, the obtained values for DTPA-extractable Fe decreased by over 50% on average in both soils. The distribution of DTPA-extractable iron in both pseudogley soils exhibited marked heterogeneity. The observed distribution dynamics across locations was consistent with the distribution of 0.1 M HCl-extractable iron, but inconsistent with the total and pseudo-total iron contents of the test soil. The values of DTPA-extractable Fe in the soil samples confirm the common incidence of iron deficiency regardless of the high amount of total iron in the soils (Sharma et al., 2008).

Table 3. The relative content of iron in different extraction agents (in % of HF- total)

Location/Extraction method

HF HNO3 0.1 M HCl 0.005 M DTPA

Tilled field

Kraljevo 33000 93.9 0.63 0.26

Koceljevo 18800 93.6 0.87 0.36

Lajkovac 35000 80.3 0.44 0.28

Gorobilje 30200 82.1 0.55 0.30

Mionica 26500 91.3 0.67 0.26

Vladimirci 22000 90.0 0.88 0.39

X 27600 88.5 0.67 0.31

Meadow

Kraljevo 30600 92.4 0.76 0.21

Koceljevo 20600 89.3 0.87 0.47

Lajkovac 36000 82.7 0.61 0.28

Gorobilje 28400 85.4 0.49 0.26

Mionica 24500 90.1 0.45 0.37

Vladimirci 23800 88.6 0.31 0.27

X 27300 88.1 0.58 0.31

The results by Jeliš et al. (2011) show that the test vertisols in Serbia had a similar

distribution and content of total and pseudo-total forms of Fe, and an extremely low i.e. critical content of DTPA- extractable Fe.


The physico-chemical characteristics of the test pseudogleys had a significant effect on the content and forms of iron in different locations in Western Serbia. Soil pH, CEC and the content of finer fractions (silt and clay) had the highest effect on the distribution of different forms of iron.

The extraction methods showed similar forms of iron in tilled field and meadow pseudogleys. However, the content of DTPA-extractable iron did not correspond to the total content, which led to the common incidence of iron deficiency in Western Serbian pseudogleys regardless of the high levels of total iron in the soil.

ACKNOWLEDGEMENTS

The authors would like to thank the Serbian Ministry of Science and Environmental protection for supporting this work through project No. TR 31054.


10

REFERENCES

[1]. Adriano, D. C., 1986. Trace elements in the Terrestrial Environmen, Springer–Verlag, Inc, New York.

[2]. Dugalic, G., et al. 2005. Agrochemical characteristics of pseudogley in the Kraljevo basen. Proceedings. XL. Croatian Symposium on Agriculture, 15-18. February, Croatia, pp 415-416.

[3]. Egner, H., et al. 1960. Laboratory Manual of Soil and Water Chemistry. Berlin, 26, (In Germany).

[4]. Han, X. F., et al., 2007. Effects of land uses on solid-phase distribution of micronutrients in selected vertisols of the Mississippi River Delta. Geoderma, 142, 96-103.

[5]. Hesterberg, D., 1998. Biogeochemical cycles processes leading to changes in mobility of chemical in soils. Agric. Ecos. Emviron. 67, 121-133.

[6]. Kostic, M. N., et al., 2002. Geochemistry of iron in soils of Vojvodina, Sumadija and Northern Pomoravlje. Proceedings for Natural Sciences, Matica Srpska, Novi Sad, 103, 17-28.

[7]. Kreamer, M. S. 2004. Iron oxide dissolution and solubility in the presence of siderophores. Aquat. Sci. 66, 1, 130-138.

[8]. Jakovljevic, M., et al. 2005. Some negative chemical properties of acid soils. J. Serb. Chem. Soc., 70 (5) 765-774.

[9]. Jelic, Z. M., et al., 2011. Distribution and forms of iron in the vertisols of Serbia. J. Serb. Chem. Soc. 76, 781-794.

[10]. Mackowiak, C. L. and R. P. Grossl, 2001. Beneficial effect of humic acid on micronutrient availability to wheat. Soil Sci. Soc. Ame. J. 65, 1744-1750.

[11]. Makovnikova, J., 2000. Relationship between selected soil parameters and available content of cadmium, lead, copper and zinc. Rostl. Vyr., 46, 289-296.

[12]. Manual for Soil chemical Analysis. Yugoslav Society of Soil Science. Belgrade, 1966. [13]. Mead, R. R., et al. 1996. Statistical methods in agriculturae and experimental biology.

Chapman & Hall, London, p. 410. [14]. Sharma, B. D., et al. 2008. Forms of iron and their association with soil arid

properties in four soil tahonomic orders of arid and semi soils of Punjab, India. Communications in Soil Science and Plant Analysis 39, 2550-2567.

[15]. Tanasijevic, Đ., et al., 1966. Pedologic composition of the western and northwestern Serbia. Belgrade, Belgrade.

[16]. Ure, M. A., 1995., in Heavy metals in soils, 2nd ed., B. J. Allovay, Blackie Academic & Professional, Glasgow, UK, p.58.

ABOUT THE AUTHORS

M. Jelic, University of Pristina-Kosovska Mitrovica, Faculty of Agriculture, Kopaonicka bb, 38219 Lesak, Serbia, E-mail: [email protected]

J. Milivojevic, Small Grains Research Center Kragujevac, S. Kovacevic 31, 34000 Kragujevac, Serbia, E-mail: [email protected]

G. Dugalic, University of Kragujevac, Faculty of Agronomy Cacak, cara Dusana 34, 32000 Cacak, Serbia, E-mail: [email protected]

O. Nikolic, University of EDUCONS, Faculty of Ecological Agriculture,Svetog Save 60, 35210 Svilajnac, Serbia, E-mail: [email protected]

S. Stamenkovic, University of Pristina-Kosovska Mitrovica, Faculty of Agriculture, Kopaonicka bb, 38219 Lesak, Serbia, E-mail: [email protected]

mailto:[email protected]:[email protected]:[email protected]


11

THE ASSESSMENT OF ANTIOXIDANT CAPACITY OF MEDICINAL

HERBS IN LOW PH CONDITIONS

D.S. Stef, I. Gergen, T.I. Trasca, A. Rivis, L. Stef, R. Heghedus-Mindru

Abstract: The aim of this paper was to evaluate the antioxidant activity of medicinal herbs in extraction solution with lower pH, to simulate the acidity of the stomach. Eighteen medicinal plants were analyzed both for total phenols content and for total antioxidant capacity. The results were compared with experimental variants which used alcoholic extracts. The highest TACFRAP value was obtained for Rosa canina (362 μM Fe2

+ ·g

-1), followed by Crataegus monogyna (192 μmoli Trolox/g), Hippophae rhamnoides (169 μM Fe2

+ ·g

-1)

and Echinaceae herba (134 μM Fe2+ ·g

-1). The smallest value was noticed for Pinus (31 μM Fe2

+ ·g

-1). The

highest total phenols values were identified for Rosa Canina (730 μmoli Trolox/g), followed by Arnica montana (490 μmoli Trolox/g) and Rhamnus frangula (317 μmoli Trolox/g). The medium values were noticed for: Thy mi herba (227 μmoli Trolox/g), Echinaceae herba (250 μmoli Trolox/g) and Crataegus monogyna (266 μmoli Trolox/g). Cynara scolymus (64 μmoli Trolox/g) and Pinus (56 μmoli Trolox/g) shown the lower values.

Keywords: antioxidant activity, medicinal plants, alcoholic extracts, low pH

INTRODUCTION

Today it is widely accepted that synthetic antioxidants (butylated hydroxyltoluene and butylated hydroxylanisole) must be replaced with natural antioxidants. There are a lot of reasons for this replacement. The natural antioxidants are involved in the prevention or treatment of diseases and in the ageing process; they have antimicrobial, cardioprotective, antimutagenic and anticarginogenic properties [2] [9] [1] [4]. Also, many people believe that the natural antioxidants are safer than synthetic ones [3].

Polyphenols are secondary plant metabolites. They contain one or more hydroxil groups and can be used in human diet. The main classes of polyphenols are: phenolic acids, flavonoids, stilbenes [6]; and lignans [8]. The highest antioxidant activity was measured for ortho-diphenols [5].

The most assessments of the antioxidant activity are made on alcoholic extracts. In human or animals bodies there are lower values for pH. It can affects the antioxidant activity of extracts which are used. Therefore the aim of this paper was to evaluate the antioxidant activity of medicinal herbs in lower pH conditions.


1. Reagents and equipment: All chemicals and reagents were analytical grade or purest quality purchased from Sigma, Merck, Aldrich and Fluka. Deionized water was used. Absorption determination for FRAP and DPPH methods was made using SPECORD 205 spectrophotometer by Analytik Jena.

2. Plant material and sampling: In the present study, 18 medicinal plants were analyzed both for total phenols content and for total antioxidant capacity. These medicinal herbs have been purchased on the local market. The results were compared with experimental variants which used alcoholic extracts [7]. For antioxidant compounds extraction were prepared ethanolic (50%) extracts in ratio 10/20. After 30 minutes all the extracts were filtered and diluted 1/10 with deionized water.

3. Evaluation of total antioxidant capacity (TAC) by FRAP method FRAP method depend upon the reduction of ferric tripyridyltriazine complex to the

ferrous tripyridyltriazine by a reductant at low pH. This ferrous tripyridyltriazine complex has an intensive blue color and can be monitored at 593 nm (Benzie and Strain, 1996).

Reagents: acetate buffer, 300mM/L, pH 3.6 (3.1g sodium acetate 3H2O and 16 mL conc. Acetic acid per 1L of buffer solution); 10 mM/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40


12

mM/L HCl; 20 mM/L FeCl36H2O in distilled water. FRAP working solution: 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3 solution. The working solution must be always freshly prepared. Aqueous solution of known Fe (II) concentration was used for calibration, in a range of 0.1-0.8 mM/L. For the preparation of calibration curve 0.5 mL

aliquot of 0.1, 0.2, 0.4, 0.6, 0.8 M/mL aqueous Fe(II) as Mohr salts solution (1mM) were mixed with 2.5 mL FRAP working solution; FRAP reagent was used as blank. The absorption was read after 10 min. at 25°C and 593 nm.

4. The amount of polyphenolic compounds: It was used the following reagents: 2.0 M Folin-Ciocalteu phenol reagent, Trolox and anhydrous carbonate. The content of total polyphenolic compounds in medicinal herbs and microelements mixture ethanol extracts diluted 1/10 was determined by Folin-Ciocalteu method (1927). For the preparation of

calibration curve 0.5 mL aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 M/mL aqueous Trolox solution were mixed with 2.5 mL Folin-Ciocalteu reagent (diluted ten-fold) and 2.0 mL sodium carbonate solution (7.5%). The absorption was read after 2 h at 20°C, at 750 nm. All determinations were performed in triplicate. Total content of polyphenols in samples in Trolox equivalents was calculated. Squared correlation coefficient (r2) for calibration curve was 0.9706.


The results for total antioxidant capacity (TAC) by FRAP method and total phenols for the studied medicinal herbs are presented in Table 1 and more suggestive in Figures 1 and 2.

Table 1 Total antioxidant capacity (TAC) by FRAP method and total phenols of eighteen medicinal herbs

No. Specification Total phenols

[μmoli Trolox/g]

Total antioxidant capacity – FRAP

[μM Fe2+ ·g-1]

1 Arnica montana 490 115 2 Cynara scolymus 64 45 3 Hippophae rhamnoides 115 169 4 Thy mi herba 227 86 5 Equisetum arvense 144 86 6 Achillea millefolium 109 125 7 Rhamnus frangula 317 131 8 Echinaceae herba 250 134 9 Phoeniculus 118 42 10 Calendula officinalis 173 58 11 Rosa canina 730 362 12 Mentha piperita 157 99 13 Matricaria chamomilla 128 58 14 Malva silvestris 112 61 15 Crataegus monogyna 266 192 16 Taraxacum officinale 115 83 17 Artemisia absinthium 83 38 18 Pinus 58 31

TACFRAP is a measure of the presence in medicinal herbs of the compounds with reducing power.


13

115

45

169

8686

125

131

134

4258

362

99

5861

192

83

3831

0

50

100

150

200

250

300

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400

1

Arnica montana

Cynara scolymus

Hippophae rhamnoides

Thy mi herba

Equisetum arvense

Achillea millefolium

Rhamnus frangula

Echinaceae herba

Phoeniculus

Calendula off icinalis

Rosa canina

Mentha piperita

Matricaria chamomilla

Malva silvestris

Crataegus monogyna

Taraxacum officinale

Artemisia absinthium

Pinus

Figure 1 Total antioxidant capacity (TAC) by FRAP method for medicinal herbs

Among all medicinal herbs the highest TACFRAP value was obtained for Rosa canina (362 μM Fe2+ ·g-1), followed by Crataegus monogyna (192 μmoli Trolox/g), Hippophae rhamnoides (169 μM Fe2+ ·g-1) and Echinaceae herba (134 μM Fe2+ ·g-1). The smallest value was noticed for Pinus (31 μM Fe2+ ·g-1).

0

100

200

300

400

500

600

700

800

1

Arnica montana

Cynara scolymus

Hippophae rhamnoides

Thy mi herba

Equisetum arvense

Achillea millefolium

Rhamnus frangula

Echinaceae herba

Phoeniculus

Calendula officinalis

Rosa canina

Mentha piperita

Matricaria chamomilla

Malva silvestris

Crataegus monogyna

Taraxacum officinale

Artemisia absinthium

Pinus

Figure 2 Total phenols for medicinal herbs

Total phenols determined by Folin-Ciocalteu methods for analyzed samples are presented in Table1 and Figure 2. The highest total phenols values were identified for Rosa Canina (730 μmoli Trolox/g), followed by Arnica montana (490 μmoli Trolox/g) and Rhamnus frangula (317 μmoli Trolox/g). The medium values were noticed for: Thy mi herba (227 μmoli Trolox/g), Echinaceae herba (250 μmoli Trolox/g) and Crataegus monogyna (266 μmoli Trolox/g). Cynara scolymus (64 μmoli Trolox/g) and Pinus (56 μmoli Trolox/g) shown the lower values.

To highlight the differences that occur between alcoholic extracts and alcoholic extracts with low pH we plotted graphically the results.

The differences for total phenols content of alcoholic extracts with different pH are shown in Figure 3. Almost all alcoholic extracts with low pH had a lower content of polyphenols comparatively with the other extracts. The differences range from -20.44% at Equisetum arvense to -72.77% at Pinus. Only two herbs from studied group behave differently.


14

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Arn

ica m

Cynara

s

Hip

pophae r

Thy m

i h

Equis

etu

m a

Achill

ea m

Rham

nus f

Echin

aceae h

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ulu

s

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ndula

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Menth

a p

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icaria c

Malv

a s

Cra

taegus m

Tara

xacum

o

Art

em

isia

a

Pin

us

Total phenols Extract alcoholic EA Total phenols EA and small pH

Figure 3 The differences for total phenols content (alcoholic extracts with different pH)

Total phenols for Malva silvestris have increased with 12% and total phenols for Rosa canina have increased with 98,37%.

In next chart are presented the differences for total antioxidant capacity (TAC) by FRAP method.

20

2

33

16

3 18

0

89

14

7

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58

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a m

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ina

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e h

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oe

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sa c

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nth

a p

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tric

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a c

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lva

s

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tae

gu

s m

Ta

raxa

cum

o

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em

isia

a

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us

Extract alcoholic EA EA and small pH

Figure 4 The differences for total antioxidant capacity (TAC) by FRAP method (alcoholic

extracts with different pH)

According with presented dates it can say that most medicinal herbs have shown lower total antioxidant capacities for extracts with low pH. Calendula officinalis had the same total antioxidant capacity (58) for both types of alcoholic extracts. Cynara scolymus, Hippophae rhamnoides, Rhamnus frangula and Echinaceae herba were 4 medicinal herbs with small differences of total antioxidant capacities. In contrast, total antioxidant capacity of Rosa canina almost doubled (+96.74%). Also, total antioxidant capacity for Malva silvestris has grown very much.


15


The antioxidant potential of medicinal plants is significantly influenced by the pH value. Both total phenolics and total antioxidant capacity by FRAP method of analyzed medicinal herbs have shown lower values for low pH.

For both situations Rosa canina behaves differently. It has shown the highest increasings for total phenols (98.37%) and for total antioxidant capacity by FRAP method (96.74%).

REFERENCES

[1]. Caccetta, R. A. A., Croft, K. D., Beilin, L. J. and Puddey, I. B. 2000. Ingestion of red wine significantly increases plasma phenolic acid concentrations but does not acutely affect ex vivo lipoprotein oxidizability. American Journal of Clinical Nutrition, 71, 67-7 4. [2]. Friedman, M. and Jurgens, H. S. 2000. Effect of pH on the stability of plant phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 2101-2110. [3]. Jayasinhe, C., Gotoh, N., Aoki, T., & Wada, S. 2003. Phenolic composition and antioxidant activity of sweet basil (Ocimum basilicum L.). Journal of Agricultural and Food [4]. Kampa, M., Alexaki, V. I., Notas, G., Nifli, A. P., Nistikaki, A., Hatzoglou, A., et al. 2004. Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action. Breast Cancer Research, 6, R63-R74. [5]. Rice-Evans, C. A., Miller, N. J. and Paganga, G. 1996. Structure- antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933-956. [6]. Scalbert, A. and Williamson, G. 2000. Dietary intake and bioavailability of polyphenols. Journal of Nutrition, 130, 2073S-2085S. [7]. Stef D.S.,Gergen I., Trasca T.I., Harmanescu M., Stef L., Druga M., Biron R., Heghedus-Mindru G. 2010. Screening of 33 Medicinal Plants for he Microelements Content. Scientific Papers: Animal Science and Biotechnologies, 43 (1), 127-132 [8]. Tapiero, H., Tew, K. D., Ba, G. N. and Mathe, G. 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomedicine & Pharmacotherapy, 56, 200-207. [9]. Wen, A. M., Delaquis, P., Stanich, K. and Toivonen, P. 2003. Antilisterial activity of selected phenolic acids. Food Microbiology, 20, 305-311.

ABOUT THE AUTHORS

D.S. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

I. Gergen, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

T.I. Trasca, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

A. Rivis, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

L. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

R. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]


16

THE EVALUATION OF FOOD QUALITY BY USING DIFFERENT METHODS

D.S. Stef, R. Heghedus-Mindru, L. Stef, G. Heghedus-Mindru, M. Cazacu, G.Bujanca, F.

Berbentea

Abstract: The goal of paper was to assess the ice cream quality using both sensorial and non-sensorial properties. Also, it presents an easy sensory evaluation method accessible to consumers without great experience. The study was carried for vanilla ice cream and it used the total quality index. Total quality indicator method takes into account both the sensory properties and physico-chemical properties. It allows you to use the non sensory characteristics making them measurable characteristics. Thus, it is more easily to make a hierarchy to different food kinds based on their quality. The best sample of ice-cream with vanilla was number 2, which was with 19 percent better than control sample and with 37 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3 which was with 5 percent lower than the experimental sample 1 and with 22 percent lower than the experimental sample 2.

Keywords: sensorial, non sensorial, food quality, ice cream, total quality indicator

INTRODUCTION

In the last time, it became very important listening the voice of the consumer. There are many reasons for this: It is necessary in the development or improvement of products; [1] [3-5] [7]; to solve marketing problems; in academic researches. The most popular tools of marketing research are preference mapping techniques [2] [9].

Intensity scaling is frequently used in evaluation of sensory properties. Many experts in sensory evaluation use nine-point scale [6]. Although this method can be easily used by researchers and experts in the field it presents some disadvantages. On the hand this kind of evaluation can be difficult to apply to the final consumer. On the other hand it does not consider the role of non-sensory factors.

Therefore the aim of this paper was to assess the food quality using both sensorial and non-sensorial properties, and to presents an easy sensory evaluation method accessible to consumers without great experience. The study was carried out for vanilla ice cream.


Order to accomplish this work it was used a control sample that was made in the laboratory and three samples from local market. 3 egg yolks, 100 g sugar, 500 ml milk, 250 ml cream and 25 g vanilla were used for making of the control sample.

The evaluation and measurement of quality were done in three stages. The first stage was the evaluation of the sensory characteristics. At the same time the dry matter, the fat and the acidity of samples were measured. Finally, based on sensory evaluation and the laboratory determinations, we calculated the total quality indicator. The methodology for calculating this indicator is presented below.

The simple indices are calculated for each characteristic with one of following relations:

b

aa

X

X or

a

ba

X

X [1]

where: Ia – the analytical indicator of quality; Xa – the estimated value of characteristic; Xb - the standard value (reference) of the characteristic.


17

The simple indices are calculated both for sensorial and chemical characteristics. Further analytical indicators weighted (Iap) are calculated:

pII aap [2] By summing the analytical indicators weighted (indices x weighting factor) results the

total index of product quality. In our case the weighting coefficients were established as it follows: odour - 0.20; taste – 0.20; colour - 0.15; structure and consistency – 0.15; dry matter - 0.10; fats - 0.10 and acidity - 0.10 [8].


The results for tasting of ice cream with vanilla are shown in Table 1. Each sample was assessed by eleven consumers (non experts in the field). The following grades were given: very good, good, satisfactory and unsatisfactory.

Table 1. The results for tasting of ice cream with vanilla

Specification Control Sample 1 Sample 2 Sample 3

Structure and consistency

3 VG + 7 G + 1 S 11 VG 8 VG + 3 G 11 VG

Color 8 VG + 3 G 7 VG + 4 G 7 VG + 4 G 7 VG + 4 G

Odor 7 VG + 5 G 8 VG + 3 G 8 VG + 3 G 8 VG + 2 G + 1S

Taste 9 VG + 2 G 8 VG + 2 G + 1 S 5 VG + 6G 6 VG + 4 G + 1

S

VG: Very Good; .G: Good; S: satisfactory.

Then for each grade (VG, G and S) was given a score using the scoring scale 0 to 1. The next values were used: 1.0 for VG, 0.67 for G, 0.33 for S and 0.1 for US (unsatisfactory). It was calculated the average score for each sensory characteristic (table 2).

Table 2. The average scores of sensory characteristics

Specification Control* Sample 1 Sample 2 Sample 3

Structure and consistency 0.69 0.94 0.91 0.94

Color 0.91 0.88 0.88 0.88

Odor 0.85 0.91 0.91 0.87

Taste 0.94 0.87 0.82 0.81

* (3 x 1.0 + 7 x 0.67 + 1 x 0.33) / 11 Considering these data, it is not clear which was the best sample. The control sample

was the best for odor but the worst for structure and consistency. The samples 1 and 4 had the same result (0.94) for structure and consistency. Also, samples 2 and 3 have shown the same result (0.91) for odor.

Also, the mean scores of the analyzed samples are presented in figure number 1. Neither the tabular results nor the graphical representation clarify the hierarchy of studied samples. This is the main reason to use another method that takes into account both the sensory and the chemical determinations.


18

0.69

0.940.91

0.940.91

0.88 0.88 0.880.85

0.91 0.910.87

0.94

0.870.82 0.81

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Control Sample 1 Sample 2 Sample 3

Structure and consistency Colour Odor Taste

Figure 1. The average score of sensory characteristics

The results of chemical measurements are presented in table 3 and in Figure 2.

Table 3. The results for chemical determinations

Specification STAS Samples

Control 1 2 3

Dry matter, [%] min. 33 32.02 34.08 36.92 35.37

Acidity, [°T] max. 24 15.50 16.20 19.80 18.70

Fat, [%] 5 7.6 10.2 11.5 10.8

33

24

5

32.02

15.5

7.6

34.08

16.2

10.2

36.92

19.8

11.5

35.37

18.7

10.8

0

5

10

15

20

25

30

35

40

STAS Control 1 2 3

Dry matter, [%] Acidity, [°T] Fat, [%]

Figure 2. The results for chemical determinations

According with these data, it can say that the dry matter ranged between 32.02% and 36.92%. The lowest value (32.02%) was registered by the control sample, which did not respect the minimal requirement for this characteristic. The other experimental samples have exceeded the minimum limit with values between 1.08 and 3.92 percentage points.


19

The best for acidity was control sample which registered 15.5°T. This shows that in the process of getting ice cream were used fresh ingredients. It can be explained by the use of fresh milk as raw materials. The second place was occupied by sample 1 (16.2°T), followed by sample 3 (18.7°T).

The fat percentage varied between 5 and 10%. The average value of this parameter was 7.20% and it had with 2.20% percentage points over the limit.

The values of the sensorial characteristics and the chemical composition do not show a clear hierarchy individually of the experimental samples. The total quality index might be a good solution for this problem.

The estimation methodology for total index quality is presented in tables 4 and 5.

Table 4. The calculation of the total indicator of quality

Specification Standard

(Xb) p

Control sample Sample 1

Xa Ia Iap Xa Ia Iap Structure and consistency

0.21 1.40 0.94 0.15 1.02 0.69 0.15 ٭0.67

Colour 0.67 0.15 0.91 1.35 0.20 0.88 1.31 0.19

Odor 0.67 0.20 0.85 1.26 0.25 0.91 1.35 0.27

Taste 0.67 0.20 0.94 1.40 0.28 0.87 1.29 0.25

Dry matter, [%]

min. 33 0.10 32.02 0.97 0.09 34.08 1.03 0.10

Acidity, [°T] max. 24 0.10 15.50 1.54 0.15 16.20 1.48 0.14

Fat, [%] 5 0.10 7.6 0.65 0.06 10.2 0.49 0.04

Total - 1.00 - - 1.18 - - 1.20

*the standard refers to a good sample in sensorial characteristics

Table 5. The calculation of the total indicator of quality

Specification Standard

(Xb) p

Sample 2 Sample 3

Xa Ia Iap Xa Ia Iap Structure and consistency

0.67 0.15 0.91 1.35 0.21 0.94 1.40 0.21

Colour 0.67 0.15 0.88 1.31 0.19 0.88 1.31 0.19

Odor 0.67 0.20 0.91 1.35 0.27 0.87 1.29 0.25

Taste 0.67 0.20 0.82 1.22 0.24 0.81 1.20 0.24

Dry matter, [%]

min. 33 0.10 36.92 1.11 0.11 35.37 1.07 0.10

Acidity, [°T] max. 24 0.10 19.80 1.21 0.12 18.70 1.28 0.12

Fat, [%] 5 0.10 11.5 2.3 0.23 10.8 0.46 0.04

Total - 1.00 - - 1.37 - - 1.15

According with tabular data it can say that the best sample of ice cream was number

2, which was with 19 percent better than control sample and with 37 percent better than standard. On second place stood the experimental sample 1 which was 2 percent better than the control sample and 20 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3 which was with 5 percent lower than the experimental sample 1 and with 22 percent lower than the experimental sample 2.


20


This method (total quality indicator) takes into account both the sensory properties and physico-chemical properties. It allows you to use any non sensory characteristics making them measurable characteristics. Also, it is a lot more easily to make a hierarchy for many kinds of food products rely on their quality.

The best sample of ice cream was number 2, which was with 19 percent better than control sample and with 37 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3.

REFERENCES

[1]. Arditti, S. 1997: Preference mapping; A case study. Food Quality and Preference, 8(5/6), 323–327. [2]. Cooper, L. G. 1983: A review of multidimensional scaling in marketing research. Applied Psychological Measurement, 7(4), 427–450. [3]. Garber, L. L., Hyatt, E. M. and Starr, R. G. 2003: Measuring consumer response to food products. Food Quality and Preference, 14(1), 3–15. [4]. Jaeger, S. R., Rossiter, K. L., Wismer, W. V. and Harker, F. R. 2003: Consumer-driven product development in the kiwi-fruit industry. Food Quality and Preference, 14(3), 187–198. [5]. Kaul, A. and Rao, V. R. 1995: Research for product positioning and design decisions: An integrative review. International Journal of Research in Marketing, 12, 293–320. [6]. Park, J.Y., O‘Mahony, M. and Kwang-Ok Kim. 2007: ‗Different-stimulus‘ scaling errors; effects of scale length. Food Quality and Preference 18, 362–368. [7]. Slater, S. F. and Narver, J. C. 2000: Intelligence generation and superior customer value. Journal of the Academy of Marketing Science, 28(1), 120–127. [8]. Stef D.S. 2007: Food products commodities. Part I, Second Edition, Mirton Publishing house, 218 pp. [9]. Urban, G. L. and Hauser, J. R. 1993: Design and marketing of new products (2nd ed.). New Jersey: Prentice Hall.

ABOUT THE AUTHORS

D.S. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

R. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

L. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

G. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

M. Cazacu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

G.Bujanca, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

F. Berbentea, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]


21

ANGIOTENSIN I CONVERTING ENZYME INHIBITORY ACTIVITY OF

PUMPKIN OIL CAKE 12S GLOBULIN HYDROLYSATES

Ţ. Vaštag, Lj. Popoviš, S. Popoviš, D. Peričin, and V. Krimer-Maleševiš

Abstract: Angiotensin I converting enzyme (ACE) is the key enzyme in the rennin angiotensin aldosterone system regulating blood pressure and its inhibition is considered the first line of therapy for hypertension. Synthetic ACE inhibitors have already used in human therapy, but there has been a new trend towards developing safer, natural ACE inhibitors, mostly peptides from food protein enzymatic hydrolysates.

After cold pressing of pumpkin oil, defatted hull-less pumpkin oil cake (PUOC) 12S globulin (cucurbitin) was utilised as raw material for preparation of protein hydrolysates, containing bioactive peptides. In this study, cucurbitin was hydrolysed with alcalase, flavorzyme and pepsin at optimum reaction conditions (temperature, pH) for each protease, during 60 min and the in vitro angiotensin I converting enzyme (ACE) inhibitory activity of hydrolysates was tested. All hydrolysates regardless of protease used, exhibited ACE inhibition. The apparent IC50 values were 0.0244 mg (0.163 mg/ml), 0.0899 mg (0.599 mg/ml) and 0.0445 mg (0.3 mg/ml) for alcalase, flavorzyme and pepsin hydrolysate, respectively. Based on these results, cucurbitin showed potential as starting material for production of innovative antihypertensive agents.

Key words: Cucurbitin, alcalase, flavorzyme, pepsin, protein hydrolysates, ACE inhibitors. .

INTRODUCTION

Hypertension is one major risk factor for cardiovascular diseases (atherosclerosis, stroke, and so forth). Angiotensin I converting enzyme, ACE (EC 3.4.15.1) is the key enzyme in the rennin angiotensin aldosterone system (RAAS) regulating blood pressure. ACE converts angiotensin I into angiotensin II and inactivates bradykinin [1]. Inhibition of ACE is considered the first line of therapy for hypertension and several synthetic ACE inhibitors (captopril, enalapril, etc.) have already been used in human therapy. However, as they can cause adverse side effects, new trend has been towards developing safer, natural ACE inhibitors [2]. The main recent interest has been directed toward food derived ACE inhibitory peptides and lot of animal and plant proteins have been revealed as sources of ACE inhibitory peptides, including from milk, meat, fish, soybean, sunflower, corn, etc. [3]. Several products containing peptides with ACE inhibitory properties are currently on the market or in development [4]. ACE inhibitory activity of peptides depends on their amino acid composition and sequence, which is related to the specificity of the protease and hydrolysis conditions used for their production. Using optimised procedures ACE inhibitory peptides may be released. Thus, thorough studies on these bioprocesses are fundamental regarding the development of innovative ACE inhibitors.

Besides the most freuqently cultivated oil crops (soybean, rapeseed, sunflower), pumpkin (Cucurbita sp.) seed, especially the hull-less (naked) varieties, has been used for edible oil production in Austria, Hungary, Slovenia and Serbia (province of Vojvodina) for a long time. Pumpkin oil cake (PuOC), the main byproduct of the oil processing, has been mostly used as animal feed despite its components that could be attractive as ingredients in food or pharmaceutical industry [5, 6]. Defatted PUOC has about 65% of proteins, majorly composed of salt-soluble 12S globulin (cucurbitin) and albumins. In order to valorize PUOC, diverse enzymatic processes for modification of PUOC protein isolate and cucurbitin have been developed in our laboratory. The bioprocesses have been aimed at production of functional proteins, either using proteases or enzymes for cross-linking [6, 7]. The improved functionality of hydrolysed PUOC proteins can involve improved techno-functional properties (solubility, emulsifying or foaming activity), as well as bioactivity (antioxidant, ACE inhibitory activity) [7, 8, 9, 10]. Depending on the protease and reaction conditions various bio-functional hydrolysates may be produced.


22

In this study the ACE inhibitory activity of cucurbitin's hydrolysates are demonstrated. The hydrolysates were produced under the optimal reaction conditions (temperature, pH) for three selected food-grade proteases, namely alcalase, flavorzyme and pepsin, at the same enzyme/substrate ratio, during 60 min. In this order, the effect of the type of protease on the properties of hydrolysates can be considered.


Isolation and enzymatic hydrolysis of cucurbitin Cucurbitin was prepared by the procedure described by Peričin et al. [5]. The

enzymatic hydrolysis of cucurbitin was carried out under following controlled reaction conditions; for alcalase and flavourzyme at pH 8.00, temperature of 50°C, and NaCl concentration of 40 mg/ml, and for pepsin at pH 3.00 and temperature of 37°C. The enzyme to substrate (E/S) ratio was 0.02 g /1g for each enzyme. After 60 min, the reaction mixture was heated (100°C, 5 min), centrifuged and the supernatants were used for further analysis. The degree of hydrolysis (DH) was determined by method described by Peričin et al. [6]. The protein content of each analysed sample was determined by method of Lowry et al. [11], using bovine serum albumin as the standard protein.

In vitro assay for ACE inhibitory activity The ACE inhibitory test was performed according to Yoshie-Stark et al. [12]. The

samples were dissolved in 0.2 mol/L potassium phosphate buffer (pH 8.30) was incubated at 37 ºC for 80 min with hippuryl-His-Leu (HHL) in 0.2 mol/L potassium phosphate buffer containing 300 mmol/L NaCl (pH 8.30) and the ACE solution. The final concentrations of the HHL and ACE were 10 mmol/l and 25 mU/ml, respectively. The reaction was stopped by adding 110 μl of 1 mol/L HCl. The hippuric acid (HA) liberated from HHL by catalytic activity of ACE was quantified with reverse-phase high performance liquid chromatography (RP-HPLC). 20 μl of the each sample was injected onto Zorbax Eclipse XDB-C18 column (4.6 Id x150 mm, 5μm, 80Å). The column was eluted with 50% methanol and 0.1% trifluoroacetic acid (in water), with flow rate of 1 ml/min, at 22°C and the absorbance was measured at 228 nm. From the chromatograms, the peak area (correspodening to HA) was integrated, and the amount of HA in the samles was quantified. The ACE inhibition activity calculated as follows (Eqn. 1):

(1)

where A is the amount of HA in reaction without an inhibitor, B is the amount of HA in reaction with potent inhibitor, while A0 and B0 are the respected blanks (where HCl was added in the test tube before the enzyme solution).

Statistical analysis Data were expressed as mean ± standard deviation for triplicate determinations and a

least significant difference test with a confidence interval of 95% was used to compare the means.


The hydrolysis of a protein is usually measured in terms of degree of hydrolysis (DH). Under the reaction conditions used in this study, the final DH in alcalase (AH), flavorzyme (FH) and pepsin hydrolysate (PH) were 26.944±1.1%, 8.33±0.5%, and 18.7037±1.2%, respectively (Figure 1). Different DH values achieved at the same E/S ratio and reaction


23

time indicates that these proteases hydrolysed the same protein at different peptide bonds dictated by their specificity, resulting in different peptide composition of hydrolysates.

26,944

8,33

18,7037

0 5 10 15 20 25 30

Alcalase

Flavorzyme

Pepsin

Degree of hydrolysis, DH [%]

Figure 1. Final DH values of cucurbitin hydrolysates with alcalase, flavorzyme and pepsin.

ACE inhibitory activity of cucurbitin's hydrolysates The in vitro activity of hydrolysates reflects potential in vivo antihypertensive effect as

studies have demonstrated that inhibition of ACE could result in decreased blood pressure in animal models [3]. In the ACE inhibitory assay, HHL acts as the substrate for ACE which catalyses its conversion to hippuric acid (HA) and the dipeptide, histidyl-leucine (HL). All tested hydrolysates exhibited ACE inhibiting activity, which was dependent on their concentration (Figure 2).

Figure 2. The ACE inhibitory activity of cucurbitin hydrolysates vs. protein concentration, for

♦Alcalase hydrolysate (AH); ▲Flavorzyme hydrolysate and ■Pepsin hydrolysate (PH).

y = 17,443ln(x) + 114,76, R² = 0,9205

y = 24,185ln(x) + 125,28, R² = 0,9718

y = 16,6ln(x) + 89,994, R² = 0,9894 0

10

20

30

40

50

60

70

80

90

100

0 0,05 0,1 0,15 0,2 0,25 0,3

AC

E in

hib

ito

ry a

ctiv

ity

[%

]

Final protein concentration [mg]

Log. (Alcalase)

Log. (Pepsin)

Log. (Flavourzyme)


24

The activities were examined for sample amount range from 0.0024 to 0.37 mg, i.e. from 0.016 up to 2.5 mg/ml (in the test tube, based on protein content). Figure 2 shows that logarithmic curves could be fit adequately to represent the ACE inhibitory activity in function of sample amount. The apparent IC50 value of hydrolysates represents the concentration of proteins in the test tube, required to inhibitin 50% of ACE under the described reaction conditions. The IC50 values were determined through established equations (Figure 2), being 0.0244 mg (0.163 mg/ml), 0.0899 mg (0.599 mg/ml) and 0.0445 mg (0.3 mg/ml) (final protein concentration in the test tube), for tested AH, FH and PH respectively. Based on these results AH had the highest activity, followed by PH and FH. These results are similar to inhibitory activity reported for other similar plant protein hydrolysates [10], indicating that cucurbitin hydrolysates can be valuable sources of natural ACE inhibitory peptides.


In this work the in vitro ACE inhibitory activity of cucurbitin hydrolysates were investigated. The results show that cucurbitin, the major storage protein of pumpkin oil cake is a potential starting material for enzyme mediated production of protein hydrolysates containing ACE inhibitory peptides that can be used as ingredients in functional foods or nutraceuticals. However, further studies are necessary to purify and characterise the individual ACE inhibitory peptides within these hydrolysates.

REFERENCES

[1]. Atlas, S. A. 2007. The Renin-Angiotensin Aldosterone System: Pathophysiological Role and Pharmacologic Inhibition. Journal of Managed Care Pharmacy 18, pp S9–S20.

[2]. Acharya, K. R., et al. 2003. Ace revisited: A new target for structure-based drug design. Nature Reviews Drug Discovery 2, pp 891-902.

[3]. Hong, F., et al. 2008. The anti-hypertensive effect of peptides: A novel alternative to drugs? Peptides 29, pp 1062-1071.

[4]. Korho

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