Acta Periodica technologica 2009-40

FACULTY OF TECHNOLOGY NOVI SAD

ACTA PERIODICA TECHNOLOGICA

APTEFF, 40, 1-220 (2009)

ACTA PERIODICA TECHNOLOGICA - Novi Sad (formerly Zbornik radova Tehnolokog fakulteta and Proceedings of Faculty of Technology) publishes articles from all branches of technology (food, chemical, biochemical, pharmaceutical), process engineering and related scientific fields. Articles in Acta Periodica Technologica are abstracted by: Chemical Abstracts, Columbus, Ohio, Referativnii zhurnal Khimija, VINITI, Moscow, listed in Ulrichs International Periodical Directory, and indexed in the Elsevier Bibliographic data bases SCOPUS. YU ISSN 1450 7188 UDC 54:66:664:615 Publisher University of Novi Sad, Faculty of Technology 21000 Novi Sad, Bulevar Cara Lazara 1, Serbia For Publisher Prof. Dr. Zoltan Zavargo, Dean Editor-in-Chief Prof. Dr. Sonja ilas Editorial Board From Abroad Prof. Dr. ivko Nikolov Texas A and M University, Biological and Agricultural Engineering Department, College Station, TX, USA Prof. Dr. Erika Bkssy-Molnr University of Horticulture and Food Industry, Budapest, Hungary Prof. Dr. eljko Knez University of Maribor, Faculty of Chemistry and Chemical Technology, Maribor, Slovenia Dr. T.S.R. Prasada Rao Indian Institute of Petroleum, Dehra Dun, India Prof. Dr. er Karlovi Margarine Center of Expertise, Kruszwica, Poland Dr. Szigmond Andrs Research Institute of Hungarian Sugar Industry, Budapest, Hungary Dr. Andreas Reitzmann Institute of Chemical Process Engineering, University Karlshruhe From Serbia Dr. Ratko Lazarevi, Academician Prof. Dr. Slobodan D. Petrovi Prof. Dr. Erne Ki Prof. Dr. Petar Doki Prof. Dr. Spasenija Milanovi Prof. Dr. Vladimir Srdi CODEN: APTEFF

ACTA PERIODICA TECHNOLOGICA APTEFF, 40, 1-220 (2009)

CONTENTFOOD TECHNOLOGY Biljana R. Cvetkovi and Marija R. Jokanovi EFFECTS OF PRESERVATION METHOD AND STORAGE CONDITION ON ASCORBIC ACID LOSS IN BEVERAGES Gordana R. Dimi, Sunica D. Koci-Tanackov, Duanka J. Pejin, Jelena D. Pejin, Ilija J. Tanackov and Danijela Tuco ANTIMICROBIAL ACTIVITY OF CARAWAY, GARLIC AND OREGANO EXTRACTS AGAINST FILAMENTOUS MOULDS Ljubica P. Doki, Marija I. Bodroa-Solarov, Miroslav S. Hadnaev and Ivana R. Nikoli PROPERTIES OF EXTRUDED SNACKS SUPPLEMENTED WITH AMARANTH GRAIN GRITS Aleksandar Z. Fite and uro M. Vukmirovi REDUCTION OF WHEAT MIDDLINGS USING A CONVENTIONAL AND EIGHT-ROLLER MILLING SYSTEMS Gordana B. Koprivica, Nevena M. Miljenovi, Ljubinko B. Levi and Vjera S. Pribi CHANGES IN NUTRITIVE AND TEXTURAL QUALITY OF APPLE OSMODEHYDRATED IN SUGAR BEET MOLASSES AND SACCHAROSE SOLUTIONS Radomir V. Malbaa, Eva S. Lonar, Spasenija D. Milanovi and Ljiljana A. Kolarov USE OF MILK-BASED KOMBUCHA INOCULUM FOR MILK FERMENTATION Anamarija I. Mandi, Sonja M. Djilas, Jasna M. anadanovi-Brunet, Gordana S. etkovi and Jelena J. Vuli ANTIOXIDANT ACTIVITY OF WHITE GRAPE SEED EXTRACTS ON DPPH RADICALS

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Spasenija D. Milanovi, Mirela D. Ilii, Katarina G. Durakovi and Vladimir R. Vuki TEXTURAL CHARACTERISTICS OF FERMENTED MILK BEVERAGES PRODUCED BY KOMBUCHA Dragan V. Pali and Sophia E. Coetzee PROTOCOL FOR USING PROTEIN SOLUBILITY AS AN INDICATOR OF FULL-FAT SOYBEAN HEAT TREATMENT Dragan V. Pali and Klaas-Jan Leeuw COMPARISON OF THREE IN VITRO METHODS FOR DETERMINING AND PREDICTING THE ORGANIC MATTER DIGESTIBILITY OF COMLETE DIETS FOR RUMINANTS Dragana Pei-Mikulec and Gordana B. Niketi COMPOSITIONAL CHARACTERISTICS OF COMMERCIAL YOGHURT BASED ON QUANTITATIVE DETERMINATION OF VIABLE LACTIC ACID BACTERIA Slaana M. Savatovi, Aleksandra N. Tepi, Zdravko M. umi and Milan S. Nikoli ANTIOXIDANT ACTIVITY OF POLYPHENOL-ENRICHED APPLE JUICE Mirjana A. Vasi, Biserka L. Vujii, Aleksandra N. Tepi, Jelica M. Gvozdenovi-Varga and Zdravko M. umi DIETARY FIBER CONTENT IN SOME DRY BEANS Tanja D. ugi-Petrovi, Nataa M. Jokovi and Dragia S. Savi THE EVOLUTION OF LACTIC ACID BACTERIA COMMUNITY DURING THE DEVELOPMENT OF MATURE SORDOUGH CHEMICAL TECHNOLOGY AND PROCESS ENGINEERING Eva S. Lonar, Miroslava M. Radeka, Sneana B. Petrovi, Andrea S. Skapin, Ognjen Lj. Rudi and Jonjaua G. Ranogajec DETERMINATION OF THE PHOTOCATALYTIC ACTIVITY OF TiO2 COATINGS ON CLAY ROOFING TILE SUBSTRATES METHYLENE BLUE AS MODEL POLLUTANT Nataa Lj. Luki, Svetlana S. Popovi and Jelena Dj. Markovi MATHEMATICAL MODELLING OF FLUX RECOVERY DURING CHEMICAL CLEANING OF TUBULAR MEMBRANE FOULED WITH WHEY PROTEINS Nevena M. Miljenovi, Gordana B. Koprivica, Ljubinko B. Levi, Bojana V. Filipev and Tatjana A. Kuljanin OSMOTIC DEHYDRATION OF RED CABBAGE IN SUGAR BEET MOLASSES - MASS TRANSFER KINETICS

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Slavia S. Puti, Marina R. Stamenovi, Branislav B. Bajeta and Dragana D. Vitkovi DETERMINATION OF TENSION STRENGHT IN THE LONGITUDINAL AND CIRCUMFERENTIONAL DIRECTION IN GLASS-POLYESTER COMPOSITE PIPES Aleksandar R. Stani, Saa I. Jovani, Nikola J. Marjanovi and Zvonimir J. Suturovi THE USE OF L-ASCORBIC ACID IN SPECIATION OF ARSENIC COMPOUNDS IN DRINKIG WATER Marina B. iban, Mile T. Klanja and Mirjana G. Antov TREATMENT OF SUGAR BEET THICK JUICE SPENT WASH BY CHEMICAL AND NATURAL COAGULANTS Ivana M. ijaki, Radmilo R. olovi, Milenko S. Toki and Predrag S. Koji SIMPLE CORRELATIONS FOR BUBBLE COLUMNS AND DRAFT TUBE AIRLIFT REACTORS WITH DILUTE ALCOHOL SOLUTIONS BIOCHEMICAL AND PHARMACEUTICAL ENGINEERING Marija M. krinjar and Nevena T. Nemet ANTIMICROBIAL EFFECTS OF SPICES AND HERBS ESSENTIAL OILS Duanka J. Pejin, Olgica S. Gruji, Jelena D. Pejin, Irena S. Doenovi and Sunica D. Koci-Tanackov THE INFLUENCE OF CARBOXYMETHYLCELLULOSE, XANTHAN AND GUAR-GUM ADDITION IN BREAD DOUGH BEFORE FREEZING ON METABOLISM AND VIABILITY OF Saccharomyces cerevisiae IN MEMORIAM Prof. dr Nikola J. Marjanovi INSTRUCTION FOR MANUSCRIPT PREPARATION

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ACTA PERIODICA TECHNOLOGICA APTEFF, 40, 1-220 (2009)

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FOOD TECHNOLOGY

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C

UDC: 663.86:577.164.2:663.053 BIBLID: 1450-7188 (2009) 40, 1-7 Original scientific paper

EFFECT OF PRESERVATION METHOD AND STORAGE CONDITION ON ASCORBIC ACID LOSS IN BEVERAGES Biljana R. Cvetkovi and Marija R. Jokanovi Global market is flooded with vitamin-enriched foods, mainly beverages. Major vitamins for enriching beverages are the antioxidant vitamins A, C and E. Ascorbic acid is readily oxidized and lost during storage of the beverages, at rates depending on the conditions of storage. This fact is of great importance for the consumer who must know how to store beverages and when to consume them in order to get the maximum benefit of added vitamin C. The objective of this paper was to determine the amount of ascorbic acid lost in beverages applying different preservation methods and storage condition. Beverage was made in laboratory conditions with synthetic L-ascorbic acid added according to the national legislations. After 30 days of storage at 4-8oC ascorbic acid overall loss was from 81.01% to 90.27% in thermally pasteurized samples and from 97.83 % to almost complete loss in samples preserved with sodium benzoate. KEY WORDS: L-ascorbic acid, colorimetric method, beverage, storage INTRODUCTION L-ascorbic acid is largely accepted as additive in human diets because of its antioxidative potential. The richest natural vitamin C sources are fruits and vegetables like pepper, rose hip, citrus fruit, and green vegetables. Fruits and vegetables supply more than 90% of vitamin C in human diets (1). A high recommendation of daily intake for humans has been suggested, since stress in modern life is known to increase the requirement for vitamin C (2). L-ascorbic acid is nutrient that besides its vitamin action is valuable for its antioxidant effect, stimulation of the immune system and other health benefits, such as prevention of scurvy and maintenance of healthy skin, gums and blood vessels. Vitamin C also reportedly reduces the risk of arteriosclerosis, cardiovascular diseases and some forms of cancer. Vitamin C is a generic name for all compounds exhibiting the biological activity of Lascorbic acid (AA). AA is the principal biologically active form but L-dehydroascorbicBiljana R. Cvetkovi, B.Sc., [email protected], Institute for Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad; Marija R. Jokanovi, M.Sc., Assist., [email protected], Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


acid (DHA), an oxidation product, also exhibits biological activity (3). Addition of synthetic ascorbic acid increases content of vitamin C, influences maintenance of colour, flavour and universal stability of the food products (fruit juices, beverages, baby food, etc.) (4). Natural and synthetic L-ascorbic acids are chemically identical and there are no known differences in their biological activities or bioavailability (5). Based on available biochemical, clinical and epidemiological studies, the current recommended daily acceptance for ascorbic acid is suggested to be 100-120 mg/day to achieve cellular saturation and optimum risk reduction of heart diseases, stroke and cancer in healthy individuals (6). As a consequence of the common mans increasing awareness regarding the importance of vitamin C, the global market is flooded with vitamin-enriched foods, mainly beverages. Major vitamins for enriching beverages are the antioxidant vitamins A, C and E. Vitamin C is usually added as ascorbic acid (7, 8). Fortification is a growing trend in soft drinks and in the dairy sector, and played a role in 8% of all new food and drink products introduced in 2003 (9). L-ascorbic acid application in the food industry increases quality and technological properties of food, as well as nutritional value (10). Ascorbic acid is highly sensitive to various modes of deterioration. The main factors that can affect ascorbic acid loss include temperature, salt and sugar concentration, pH, oxygen, light, metal catalysts, initial concentration of ascorbic acid, the ratio of ascorbic acid to dehydroascorbic acid, microbial load and protection by the container (11). The loss of nutritional quality during processing and storage of food has become an important problem. Since the discovery of the basic vitamins and their many forms, efforts have been made to retain them in foods during post-harvest, commercial processing, distribution, storage and preparation. Vitamin C is usually selected as an index of the nutrient quality because of its labile nature as compared to the other nutrients in food (1). The term beverages-soft drink originally applied to carbonated and non-carbonated drinks made from concentrates, although it now commonly refers to almost any cold drink that does not contain alcohol (12). Ascorbic acid added to beverages is readily oxidized and lost during staying, at a rate depending on the conditions of storage. This fact is of great importance to the consumer who must know how to store the beverages and when to consume them in order to get the maximum benefit of vitamin C content. (13). Determination of the nutrient content of foods is becoming extremely important as researchers learn more about the relationship between dietary intake and human health (14). Various methods have been reported in the literature for the quantitative determination of vitamin C in foods or biological fluids. The usual methods include titration (AOAC), colourimetric, spectrophotometry, fluorometry, electrophoresis, and high performance liquid chromatography (HPLC). Objectives of this paper were: a) preparation of non-alcoholic beverages in laboratory conditions with addition of synthetic ascorbic acid and two methods of preservation; b) analysis of the amount of ascorbic acid loss in samples during 30 days under different storage conditions, in closed glass bottles, storage in the refrigerator, and in the dark at room temperature and in a thermostat at 37oC. 2

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


EXPERIMENTAL Materials Beverage preparation. Beverage was prepared by diluting commercial beverage concentrate (Aretol no. 72 408, Celje, Slovenia) in water in a ratio 3:97. Sugar was added like inverted syrup (dry matter content in the final product about 9.5 %), and then citric acid was added to get appopriate sensory characteristics. There were alltogether prepared 12 samples. Ascorbic acid was added in two concentrations: 100 mg/l and 150 mg/l. For thermal pasteurisation were transferred into 200 ml glass bottles, second part of samples were first preserved with sodium benzoate and than transferred into 200 ml glass bottles. Preservation. Twelve samples (with both AA added concentrations) were divided in two parts: a) One part was thermally treated (pasteurisation). Thermal pasteurisation conditions (85oC, 15 minutes) were selected to be the same as in a conventional pasteurisation of industrially produced beverages. b) The other part was preserved with sodium benzoate in concentration of 130 mg /l, in the final product, which is in accordance with national legislations (15). Shelf-life study. Samples of beverages thermally pasteurised or preserved with sodium benzoate were stored in three different temperatures conditions: refrigerator (4-8oC), room temperature (20-22oC), and in a thermostat at 37oC. Samples were evaluated after 30 days of storage, by measuring ascorbic acid content. Method Determination of L-ascorbic acid. Ascorbic acid content was determined using colourimetric method. L-ascorbic acid reduces the tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,-diphenyltetrazolium bromide) in the presence of the electron carrier PMS (5-methylphenazinium methosulphate) at the pH 3.5 to a formazan (MTT-formazan), which is determined by measuring absorbance at 578 nm. Under the conditions stated in this procedure, assay is specific for L-ascorbic acid. The L-ascorbic acid content of these clear solutions was determined without any sample treatment. The detection limit of the method was 0.175 mg/l. Each value was measured in triplicate and averaged with standard deviation. RESULTS AND DISCUSSION According to our results pasteurisation method had high influence on vitamin C content. Thermal pasteurisation gradually decreased L-ascorbic acid content. Ascorbic acid contents immediately after thermal pasteurisation in samples with 150 mg/l and 100 mg/l of added vitamin C, were 37.66 mg/l and 25.84 mg/ respectively (Table 1).

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


Table 1. Average ascorbic acid content with standard deviation and it loss in samples immediately after pasteurisation and preservationSamples 100 mg/l added ascorbic acid 150 mg/l added ascorbic acid Ascorbic acid content (mg/l) Pasteurisation Sodium benzoate preservation 25.84 3.58 71.19 8.02 37.66 2.98 113.40 8.35

According to Blasco et al. (2004), there are two different rates of ascorbic acid degradation observed during the heating process: an aerobic degradation followed by an anaerobic degradation. In the beginning of the heating process oxygen remains in the bottle and therefore aerobic degradation of the ascorbic acid with oxygen in abundance takes place. With prolonged time of heating the atmosphere in the bottle becomes saturated with vapour, so that the oxygen concentration is minimal and the ascorbic acid is degraded anaerobically (16). During the preservation of beverages with sodium benzoate the loss of added ascorbic acid was much lower than in thermally treated samples. Immediately after preservation, the loss of ascorbic acid in samples with sodium benzoate was 24.40 % and 28.81 % for samples with 150 mg/l and 100mg/l added ascorbic acid, respectively.80 70 Ascorbic acid loss (%) 60 50 40 30 20 10 0 Thermal pasteurisation Sodium benzoate preservation28.81 24.4 74.16 74.89

100 mg/l added vitamin C 150 mg/l added vitamin C

Fig. 1. Ascorbic acid loss immediately after the two methods of preservation Table 2. Average ascorbic acid content (mg/l) with standard deviations in the beverages after 30 days of storage at 4-8, 20-22 and 37oCStorage temperature 4-8oC 20-22oC 37oC Pasteurisation AA AA 150 mg/l 100 mg/l 28.37 1.26 2.17 0.30 nd* nd nd nd Preserved with sodium-benzoate AA AA 150 mg/l 100 mg/l 14.6 1.99 0.43 0.08 nd nd nd nd

*nd - not detected; AA- ascorbic acid

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


100 mg/l added vitamin C 120 Ascorbic acid loss (%) 100 80 60 40 20 0 Thermal pasteurisation97.83 81.01

150 mg/l added vitamin C99.57 90.26

Sodium benzoate preservation

Fig. 2. Ascorbic acid loss after 30 days of storage in a refrigerator (4-8 oC) Storage temperature had a great influence on ascorbic acid loss. After 30 days of storage at room temperature (20-22oC) and in thermostat at 37oC ascorbic acid was not detected in any sample. After 30 days of storage at 4-8oC and thermal pasteurisation the overall loss of ascorbic acid was 81.01 % in samples with 150 mg/l added ascorbic acid, and 97.83 % in samples with 100 mg/l added ascorbic acid. In the beverages preserved with sodium benzoate after one month of storage at 4-8oC ascorbic acid overall loss was from 90.27 % in samples with added concentration of 150 mg/l to almost complete loss for samples with 100 mg/l of added ascorbic acid (Fig. 2). Heating method had a definite influence on the retention of ascorbic acid. According to Vikram et al. (2005), by each heating method of orange juice, temperature had a greater influence and the degradation was rapid at higher temperatures (17). The decrease of ascorbic acid concentration to levels unacceptable by declaration or industrial practise often defines the product shelf life. During storage, the vitamin C content gradually decreases at a rate depending on the processing and storage temperature. The more rapid decrease of ascorbic acid concentration at the beginning of the storage can be attributed to the immediate reaction of an amount of ascorbic acid with the dissolved oxygen (18). Degradation of ascorbic acid both by aerobic and anaerobic pathways depends upon many factors such as oxygen, heat, light, storage temperature and storage time. Oxidation of ascorbic acid occurs mainly during the processing, whereas, anaerobic degradation of vitamin C mainly appears during storage, which is especially observed in thermally preserved juices (10). CONCLUSION The decrease of vitamin C content to levels unacceptable by declaration or industrial practise often defines product shelf-life. During storage, the vitamin C content gradually decreases at a rate depending on processing and storage temperature. The more rapid decrease of ascorbic acid concentration at the beginning of the storage can be attributed to the immediate reaction of an amount of ascorbic acid with the dissolved oxygen (18). 5

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


According to Blasco et al. (2004) there are two different rates of ascorbic acid degradation observed during the heating process: an aerobic degradation, followed by an anaerobic degradation. In the beginning of the heating process oxygen remains in the bottle and therefore aerobic degradation of the ascorbic acid with oxygen in abundance takes place. With prolonged time of heating, the atmosphere in the bottle becomes saturated with vapour, so that the oxygen concentration is minimal and the ascorbic acid is degraded anaerobically (16). The experiments have shown that L- ascorbic acid added like additive in non-alcoholic beverage is extremely unstable in water solution. The samples with a lower initial content of ascorbic acid lose it faster than those with a greater content. Ascorbic acid loss was greater in preserved than in pasteurised beverages. It should be recommended that beverage with ascorbic acid added should be consumed after preparation with no long time of storage. ACKNOWLEDGEMENT The authors gratefully acknowledge the financial support from the Ministry of Science and Technological Development of the Republic of Serbia (Project TP-20068). REFERENCES 1. Erentuk. S, Gualaboglu M.S. and S. Gultekin: The effects of cutting and drying medium on the vitamin C content of rosehip during drying. Journal of Food Engineering 68 (2005) 513-518. 2. Olivier Fain: Musculoskeletal manifestations of scurvy, Review, Joint Bone Spine (2004). 3. Lee S. K. and Adel A. Kader: Preharvest and post harvest factors influencing vitamin C content of horticultural crops. Post harvest Biology and Technology 20 (2000) 207220. 4. Del Caro A.: Changes of flavonoids, vitamin C and antioxidant capacity in minimally processed citrus segments and juices during storage. Food Chemistry 84 (2004) 99105. 5. Lee H.S. and G. A. Coates: Vitamin C in frozen, fresh squeezed, unpasturized, polyethylene-bottled orange juice: storage study. Food Chemistry 65 (1999) 165-168. 6. Klimezak I., Malecka M., Szlahta M. and A. Gliszezynska-Swiglo: Effect of storage on the content of polyphenols, vitamin C and the antioxidant activity of orange juices. Journal of Food Composition and Analysis 20 (2007) 313-322. 7. Arya S. P., Mahajan M. and P.Jain: Non-spectrophotometric methods for the determination of Vitamin C. Analytica Chimica Acta 417 (2000) 1-14. 8. Rodriguez-Comesana M., Garcia-Falcon M. S. and J. Simal-Gandara: Control of nutritional labels in beverages with added vitamins: screening of -carotene and ascorbic acid contents. Food Chemistry 79 (2002) 141-144. 9. Prieto P. S., Grande C. B., Falkon G. S. and S. J. Gandara: Screening for folic acid content in vitamin-fortified beverages. Food Control 17 (2006) 900-904. 10. Burdurly H. S., Koca N. and F .Karadeniz: Degradation of vitamin C in citrus juice concentrates during storage. Journal of Food Engineering 74 (2006) 211-216. 6

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940001C


11. Zerdin K., Michael L. R. and J.Vermue: The vitamin C content of orange juice packed in an oxigen scavenger material. Food Chemistry 82 (2003) 387-395. 12. www.wikkipedia.org 13. Kabasakalis V, Siopidou D. and E. Moshatou: Ascorbic acid content of commercial fruit juices and its rate of loss upon storage. Food Chemistry 70 (2000) 325-328. 14. Gokmen V., Kahraman N., Demir N. and J. Acar: Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables. Journal of Chromatography A, 881 (2000) 309-316. 15. Bylaw of quality and other conditions for aditives and their mixtures for food products (Slubeni list SRJ articles no. 56/2003, 4/2002 and 16/2005). 16. Blasco R., Esteve M. J., Frigola A. and M. Rodrigo: Ascorbic acid degradation kinetics in mushrooms in a high-temperature short time process controlled by a thermoresistometer. Lebensm.-Wiss. u.-Technol, 37 (2004) 171-175. 17. Vikram V. B., Ramesh M.N. and S.G. Prapulla: Thermal degradation kinetics of nutrients in orange juice by electromagnetic and conventional methods. Journal of Food Engineering 69 (2005) 31-40. 18. Polydera A.C, Stoforos N.G. and P.S. Taoukis: Comparative shelf life study and vitamin C loss kinetics in pasteurized and high pressure processed reconstituted orange juice. Journal of Food Engineering 60 (2003) 21-23. . . , . , , . - , . ( ) . . , - . 30 4-8 81.01- 97.83% , 90- 99%. Received 17 November 2008 Accepted 26 February 2009

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


ANTIMICROBIAL ACTIVITY OF CARAWAY, GARLIC AND OREGANO EXTRACTS AGAINST FILAMENTOUS MOULDS Gordana R. Dimi, Sunica D. Koci-Tanackov, Duanka J. Pejin, Jelena D. Pejin, Ilija J. Tanackov and Danijela Tuco Inhibitory effect of caraway, garlic and oregano extracts (0.07, 0.1, 0.5, 1 and 2%), against four moulds species was investigated. The caraway extract had the strongest inhibitory effect by inhibiting the germination of Emericella nidulans, Penicillium commune and P. implicatum at the concentration of 0.1% and Aspergillus tamarii at the concentration of 0.5% during 7 days of incubation at 25oC. The extract of garlic only partially inhibited the growth of A. tamarii and P. commune. However, it inhibited completely the growth of P. implicatum and E. nidulans at the doses of 0.5 and 1%. Oregano partially inhibited all mould species, significantly reducing the growth of colonies, especially of E. nidulans (93.3%). KEY WORDS: Spice extracts, antifungal activity INTRODUCTION Moulds highly prevail in nature and frequently contaminate human food. Some of them produce secondary metabolites such as aflatoxins, ochratoxin A, stergmatocystine, which are cytotoxic and carcinogenic, and as such present a potential health hazard for humans (1). Medium moisture food (0.75-0.90 aw), low moisture food (< 0.75 aw) and sour food are especially susceptible to the presence of moulds. The development of moulds on food can be expected in cases when inappropriate sanitary practice is applied in production plants. Moulds occur more frequently than other microorganisms on food products during storage and distribution as a consequence of inadequate conditions. Essential oils extracted from spices and other herbs, as well as their biologically active components, have been intensively investigated for their potential role in the protection of food from microorganisms, especially the foodstuffs with short shelf-life, such as bread, bakery products, cakes, salads, fresh fruits and vegetable, fish, etc. which are the most susceptible to microbial spoilage. Being natural antimicrobial agents, their usageDr. Gordana R. Dimi, Assoc. Prof., [email protected], Sunica D. Koci-Tanackov, M.Sc., Assist., Dr. Duanka J. Pejin, Prof., Dr. Jelena D. Pejin, Assist., Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; Dr. Ilija J. Tanackov, Assoc. Prof., Faculty of Technical Science, University of Novi Sad, Trg Dositeja Obradovia 6, 21000 Novi Sad, Serbia; Danijela Tuco, B.Sc. Etol JVE d.o.o., Bulevar Vojvode Stepe 40, 21000 Novi Sad, Serbia

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


can minimize the application of synthetic preservatives and additives, preserving simultaneously food freshness and sensory quality. Preserving properties of spices and their extracts have been recognized long ago; their residues have been found on old Egyptian mummies (2) and there are evidence of their usage as antiseptic agents (3). Studies have shown that some spices like vanilla do not possess antifungal activity (4) whereas some of them have a stimulating effect (5, 6, 7, 8). This study was aimed at investigating the antifungal potential of caraway, garlic, and oregano extracts against some food-borne fungi. EXPERIMENTAL Materials Commercially available food grade ethanol extracts of caraway, garlic, and oregano were provided from Etol, Celje, Slovenia. Test cultures for antifungal investigations, Aspergillus tamarii, Emericella nidulans, Penicillium commune and P. implicatum were taken from the culture collection of the Laboratory for Food Microbiology, Faculty of Technology in Novi Sad, isolated from food. The cultures were maintained on potatodextrose agar (PDA) slants at 4 C. Preparation of inoculum Prior to the experiment, moulds were cultured on PDA slants for 10 days until fully sporulated. Spores were taken by adding 10 ml of medium which contained 0.5% Tween 80 and 0.5% agar in sterile distilled water (4), scraped with sterile loop and aseptically transferred into sterile test tubes. Spore suspension obtained in this way was adjusted to final concentration of 2106 spores/ml using the hemocytometer, and used for further work. Antifungal test The inhibition of mould growth was determined by performing daily measurements of the radial growth of colonies cultured on PDA medium which contained spice extracts (each plate separately) in the following concentrations 0.07, 0.1, 0.5, 1 i 2% (v/v). For test moulds, PDA plates without any added material were made and used as control plates. The solid plates were inoculated with spore suspension containing 1l (103 spores/ml) in the centre of the medium and were incubated for 7 days at 25oC. Diameter of the growth was determined by averaging the radial growrth of the colony in two orthogonal directions. Each test was run in triplicate. RESULTS AND DISCUSSION Inhibitory concentrations for caraway, garlic and oregano extracts against A. tamarii, E. nidulans, P. commune and P. implicatum are presented in Table 1. 10

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


Table 1. The inhibitory activities of spice extracts against mouldsExtract Caraway Conc. (%) 0.07 0.1 0.5 1 2 0.07 0.1 0.5 1 2 0.07 0.1 0.5 1 2 A. tamarii 12.7 22.2 47.6 100 100 1.6 14.3 25.4 30.1 33.3 12.7 17.5 30.1 47.6 79.4 Inhibition colony growth (%) E. nidulans P. commune P. implicatum 20.0 14.8 11.1 33.3 37.0 77.8 100 100 100 100 100 100 100 100 100 22.2 11.1 11.1 31.1 18.5 33.3 73.3 26.0 100 100 33.3 100 100 59.2 100 11.1 14.8 5.5 15.5 18.5 11.1 31.1 22.2 16.7 48.9 29.6 55.5 93.3 74.1 66.7

Garlic

Oregano

As can be seen from data presented, the caraway extract exhibited the strongest inhibitory activity that was particularly expressed against E. nidulans and both Penicillium species (P. commune and P. implicatum) which did not grow at extract doses over 0.1%. At this level, the growth of P. implicatum was markedly reduced (77.8%). The level over 0.5% was needed to completely inhibit A. tamarii. The garlic extract at 0.5 and 1% concentrations inhibited only partially the growth of A. tamarii and P. commune and completely the growth of P. implicatum and E. nidulans. If compared to A. tamarii, stronger antifungal activity against P. commune was observed in all applied concentrations. The level of growth reduction in the presence of garlic extract for A. tamarii ranged from 1.6 to 33.3% and for P. commune between 11.1 to 59.2%. Although none of the tested species was completely inhibited by the oregano extract, high concentrations were found to significantly inhibit the growth of colonies. The 2% extract inhibited almost completely (93.3%) the growth of E. nidulans. Against A. tamarii and P. commune, the same extract concentration exhibited approximately 70% inhibition rate (79.4 and 74.1%, respectively), whereas P. implicatum was found to be the least sensitive species. The effect of caraway, oregano and garlic extracts on the germination and growth rate of moulds is presented in Figures 1-3. At the concentrations 0.1 and 0.5%, the caraway extract delayed the beginning of germination of A. tamarii by two and three days, respectively, as compared to the control. The appearance of the growth of E. nidulans and P. commune was not under the influence by the increased extract concentration; however, the differences in their growth rate were noticed during the next days. At 0.1% concentration, colonies of P. implicatum became visible only on the sixth day after the inoculation of agar plates.

11

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


Fig. 1. Effect of caraway extract on the growth of moulds

Fig. 2. Effect of garlic extract on the growth of moulds 12

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


The growth rate decline with increase of garlic extract contents in the agar medium was especially pronounced in the case of P. implicatum and E. nidulans, pointing to the greater sensitivity of these spesies (Fig. 2). Higher concentrations did not significantly influence the appearance of A. tamarii, whereas P. commune did not grow in the presence of 2% garlic extract until the fourth day. At lower levels of oregano extract (Fig. 3), the begining of germination was delayed by two days only in the case of P. implicatum, at the concentration of 0.5%. The 1 and 2% concentrations delayed the growth of P. implicatum by two and four days and E. nidulans by two and five days. The growth of A. tamarii and P. commune was delayed only at the concentracion of 2%, for three days. Stronger inhibitory effect on the growth rate of E. nidulans was noticed at the concentrations over 0.1% for A. tamarii and P. implicatum over 0.5% and at P. commune over 1%.

Fig. 3. Effect of oregano extract on the growth of moulds The increasing concentrations of caraway, garlic and oregano extracts caused the absence or delay in germination of tested fungi, showing various inhibitory effects on the growth rate reduction. Caraway was more efficient at lower concentrations, compared to garlic and oregano. Moreover, it was the only extract to inhibit the growth of three species (out of the four tested) during the whole period of incubation (7 days) at 25oC. Previous studies also reported strong inhibitory effect of caraway on Penicillium species. Studies have shown that P. aurantiogriseum, P. corylophilum, P. commune and P. griseofulvum were completely inhibited at 1% dose (8, 9). Antifungal properties of the tested extracts are due to their major constitutive components, carvacrol (from caraway and oregano), limonene (from caraway), thymol (from 13

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


oregano) and sulfur compounds (from garlic) (10, 11). Phenol compounds such as carvacrol, thymol, eugenol, vanillin, geraniol and cinnamaldehyd are known antimicrobial agents (11-17). Phenolic OH-group is very reactive and easily forms hydrogen bonds with active sites in enzymes (6). According to Soliman and Badea (18), caraway showed inhibitory effect on Aspergillus flavus and A. parasiticus at 2000 ppm dose and on A. ochraceus and Fusarium moniliforme at 3000 ppm. Nielsen and Rios (4) showed that esential oils of mustard, garlic and clove are effective in preventing the growth of moulds usually present in bread. Garlic show antifungal activity against certain Aspergillus, Penicillium and Fusarium species (19, 20). Guyenot et al. (3) investigated the protective effect of essential oils of 16 spices against xerophilic fungi from genera Eurotium, Aspergillus and Penicillium, common spoilage organisms in bakery products. It was also reported that oregano extract is capable to completely inhibit Aspergillus parasiticus at the level of 2% in agar medium (6), which is in compliance with our results. Essential oils of cinnamon and oregano proved to be very effective in growth inhibition of Fusarium proliferatum (21). CONCLUSION This study proved that the tested spice extracts can be potentially protective agents against filamentous fungi, frequent contaminants of food. Caraway extract exhibited high efficacy already at 0.5% dose. Garlic was the most effective against E. nidulans and P. implicatum. Oregano exhibited strong inhibitory effect although was unable to completely inhibit the fungal growth. REFERENCES 1. van Egmond, P.H.: Mycotoxins: detection, reference materials and regulation, in Introduction to food- and airborne fungi. Eds. R.A. Samsin, E.S. Hoekstra and J.C. Frisvad, Ponson & Looyen, Wageningen, Utrech, Netherlands (2005) pp.332-338. 2. Silliker, J.H., R.P. Elliott, A.C. Bairol-Barker, F.L. Bryan, J.H.B. Christian, D.S. Clark, J.C. Olson and Jr.T.A. Roberts: Microbial Ecology of Foods. Vol.II, Academic Press, New York, London (1980). 3. Guynot, E.M., J. A. Ramos, L. Seto, V. Purroy, V. Sanchis and S. Marin: Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products. J. Appl. Microbiol. 94 (2003) 893-899. 4. Nielsen, V.P. and R. Rios: Inhibition of fungal growth on bread, by volatile components from spices and herbs, and the possible application in active packaging, with special emphasis on mustard essential oil. Int. J. of Food Microbiology 60 (2000) 219-229. 5. Mabrouk, S.S. and M.A.N. El-Shayeb: Inhibition of aflatoxin formation by some spices. Z. Lebensm. Unters. Forsch. 171 (1980) 344-347. 6. zcan, M.: Inhibitory effect of spices extracts on the growth of Aspergillus parasiticus NRRL2999 strain. Z. Lebensm. Unters Forsch A 207 (1998) 253-255. 7. Boyraz, N. and M. zcan: Antifungal effect of some spice hydrosols. Fitoterapia 76 (2005) 661-665. 14

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


8. Dimi, G. and S. Koci-Tanackov: Antifungal activity of spices extracts on the growth of Penicillium commune and Penicillium griseofulvum, 6th Congress of Medical Microbiology, Beograd, 11-14 June 2008, Book of Abstracts, pp. 335-336. 9. Dimi, G., S. Koci-Tanackov and D. Karali: Inhibitory Effect of Spice Extracts on Growth of Penicillium aurantiogriseum and Penicillium corylophilum, 54th International Meat Industry Conference, Vrnjaka Banja, 18-20 June 2007, Book of Abstracts, p. 89. 10. Baratta, M.T., H.J.D. Dorman, S.G. Deas, D.M. Biondi and G. Ruberto: Chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosmary, oregano and coriander essential oils. J. Essent. Oil Res. 10, 6 (1998) 618-627. 11. Ceylon, E. and C.Y. D. Fung: Antimicrobial activity of spices. J. Rapid. Meth. Autom. Microbiol. 12 (2004) 1-55. 12. Bullerman, L.B., F.Y. Lien and S.A. Seir: Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde and eugenol. J. of Food Science 42 4 (1977) 11007-1116. 13. Hitokoto, H., S. Morozumi, T. Wauke, S. Sakai and H. Kurata: Inhibitory effects of spice on growth and toxin production of toxigenic fungi. Appl. Environ. Microbiol. 39 4 (1980) 818-822. 14. Moleyar, A.V. and P. Narasimham: Antifungal activity of some essential oil components. Food Microbiol. 3 (1986) 331-336. 15. Mahmound, A.L.E.: Antifungal action and antiaflatoxigenic properties of some essential oil constituents. Lett. Appl. Microbiol. 19 (1994) 110-113. 16. Matamoros-Leon, B., A. Argaiz and A. Lopez-Malo: Individual and combined effects of vanillin and potassium sorbate on Penicillium digitatum, Penicillium glabrum, and Penicillium italicum growth. J. Food Protect. 62 5 (1999) 540-542. 17. Moriera, M.R., A.G. Ponce, C.E. del Valle and S.I. Rouza: Inhibitory parameters of essential oils to reduce a foodborne pathogen. LWT 38 (2005) 565-579. 18. Soliman,K.M. and R.I. Badeaa: Effect of oil extrated from some medicinal plants on different mycotoxigenic fungi. Food Chem. Toxicol. 40 (2002) 1669-1675. 19. Mei-chin, Y. and T. Shih-ming: Inhibitory effect of seven Allium plants upon three Aspergillus species. Int. J. Food Microbiol. 49 (1999) 49-56. 20. Benkeblia, N.: Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). LWT 37 (2004) 263-268. 21. Velluty, A., V. Sanchis, A.J. Ramos, J. Egido and S. Marin: Inhibitory effect of cinnamon, clove, lemongrass, oregano and palmarose essential oils on growth and fumonisin B1 production by Fusarium proliferatum. Int. J. Food Microbiol 89 (2003) 145154.

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940009D


, A . , a . -, . , . , . a , (0,07, 0,1, 0,5, 1 2%) . Emericella nidulans, Penicillium commune P. implicatum 0,1% Aspergillus tamarii 0,5% 25C. . tamarii P. commune P. implicatum . nidulans 0,5 1%. , , . nidulans (93,3%). Received 24 July 2009 Accepted 14 September 2009

16

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D

UDC:664.64.016.71.8:664.641.15+664.641.2:664.696 BIBLID: 1450-7188 (2009) 40, 17-24 Original scientific paper

PROPERTIES OF EXTRUDED SNACKS SUPPLEMENTED WITH AMARANTH GRAIN GRITS Ljubica P. Doki, Marija I. Bodroa-Solarov, Miroslav S. Hadnaev and Ivana R. Nikoli Extruded amaranth grain products have specific aroma and can be used as snack food, supplement in breakfast cereals, or as raw material for further processing. Extruded products of corn-amaranth grits blends, containing 20% or 50% amaranth grain grits, were produced by extrusion-cooking using a laboratory Brabender single screw extruder 20 DN. Extrudates with various texture were obtained. During extrusion process starch granules are partially degraded, hence rheological properties were examined. All samples exhibited thixotropic flow behavior. Those samples in which part of the corn grits was replaced with amaranth one had lower viscosity and exhibited lower level of structuration during storage. KEY WORDS Amaranth, extrudate, proteins, starch, rheological, properties INTRODUCTION Amaranth grain has significant nutritional value. Its protein, mineral meters, fat and cellulose percentage are higher compared to cereals (1, 2). Since this plant has similar application as cereals, it is classified as pseudocereal (2). The origin of Amaranthus sp. is from middle and south America, but for the last few decades this cultivar was introduced in European countries as Austria, Poland, Hungary, Serbia and Montenegro (2, 3). Extruded amaranth grain products have specific aroma and can be used as snack food, supplement in breakfast cereals, or as raw material for further processing (4). Sanches Marroquim et al. investigated extruded blends of Amaranth with wheat and outs flour. The optimal combination of these components, to their opinion is 50 ,05or 60 ,04respectively (5). Starch is an important industrial raw material for food products as well as for technical products. Corn starch is relatively easily isolated by wet-milling procedure. AmaranthDr. Ljubica Doki, Assoc. Prof, [email protected], Faculty of Tehnology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; Dr. Marija Bodroa-Solarov, Miroslav Hadnaev, M. Sc. Institute for Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; Ivana Nikoli, B. Sc., Faculty of Tehnology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


starch is interesting because of its small granular size, 12 m, which provides specific functional properties, as good freezethaw stability and resistance to mechanical shear. Amaranth starch content is approximately 63%, while protein content is 15%. The starch has waxy characteristics and specific molecular composition, while proteins have high quality and major content of Samino acids and lysine (6). There is no effective and easy method to isolate the starch from amaranth seed because of its small granules and high protein content. Considering the fact that starch and protein are of high quality, some other methods were developed, like dry milling and separation or extrusion process with the aim to provide products useful for food or nonfood purposes. During extrusion process, properties of native starch granules are modified and pregelatinized starch with changed rheological characteristics is obtained (7). Numerous products with starch undergo thermal treatments during production and application, when starch gels with specific rheological characteristics are formed. Modification of rheological properties can be achieved by mixing different starches. The aim of this work was to investigate rheological characteristics of gels of amaranth and corn grits blends, before and after extrusion process. EXPERIMENTAL Amaranth and corn were acquired from local commercial sources. Grits was obtained by milling whole grain in laboratory mill (Bhler 202, Germany). Blends were prepared in the following ratios: amaranth:corn, 20:80, 50:50 and 0:100, respectively. Particle size distribution of grits is presented in Table 1. Table 1. Particle size distribution of corn and amaranth grits Sample Corn grits Amaranth grits Fractions remained on sieves of particular size, (%) 850 m 650 m 450 m 350 m 250 m 1.2 44.1 50.3 3.7 0.7 0.8 43.0 41.2 9.0 6.0

Grits blends were extruded using a laboratory Brabender single screw extruder 20 DN. Before extrusion, the moisture content of the grits was adjusted to 16%. Such moisture content enables optimal extrusion conditions. It was fed to the extruder under the following conditions Compression ratio 41 Temperature of the first zone 120C Temperature of the second zone 130C Temperature of the third zone 160C Die diameter 3 mm Screw speed 120 rpm The extruded product (flips) was analyzed for moisture (AOAC,1984, 8), bulk density and expansion ratio. 18

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


Extrudate density was calculated. Six products were randomly selected, weighted (m) and measured (L, D), and the density was calculated as follows: 4 m Density = 2 D L

where m is the mass, L length of cooled extrudate with the diameter D (9). Expansion ratio was calculated as the ratio of the diameter of extruded simple to that of the extruded die. Density and expansion ratio are given as average value of six calculations. Statistical Analysis. Analysis of variance (ANOVA) and least significant differences (LSD) were performed by the Statistical Analysis System (Statistical, Tulsa, Oklahoma, USA). Strength, hardness and plasticity were determined on a universal instrument for texture analysis INSTRON 4301. Determination of hardness was performed with head with 1.6 mm diameter running at speed of 60 mm/min. Softness of sample by cutting was examined using knife with 1 mm blade and at head speed of 60 mm/min. Probe for compression with 30 mm diameter was used to examine the force needed to compress extrudate from 1 cm length to 0.5 cm at the Instron head speed of 48 mm/min. The measurements were performed in the six replicates and average value for strength, hardness and plasticity were calculated. The gels for rheological measurements were obtained by heating 6.25% suspension of grits, in a Brabender viscoamylograph up to 95C and then held at 95C for 30 min. Gels were made from grits blends as well as from extruded products milled to grits on a laboratory mill. The obtained paste was cooled to room temperature and rheological measurements of the gels were carried out at 20C. Measurements were carried out right after the cooling (0h) and 24 hours later (24h). Rheometer RheoStress 600HP (Haake, Germany) with measurement set plateplate (PP60Ti), with 60 mm diameter and 1 mm distance, was used. Flow curves were obtained by hysteresis loop method by the following cycle: ramp up 0-500 1/s in 4 min 500 1/s in 2 min constant ramp down 500-0 1/s in 4 min. Micrographs were taken on a scanning electron microscope (JEOLJSM6460LV).RESULTS AND DISCUSSION

Results of the determination of expansion index and density of extrudates are presented in Table 2. The initial moisture level drop from 16% to the final average level of 7.5% for extrudates, was within the expected range of 4-8%, reported by Koeppe (1987) as characteristic for an extruded snack products. The difference in moisture content of extrudates is not statistically important (Table 2).19

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


Table 2. Extrudate propertiesSample 100% corn grits 80% corn : 20% amaranth grits 50% corn : 50% amaranth grits Moisture content (%) (db) 7.6 0.09a 7.4 0.03a 7.6 0.05a Expansion index 4.03 0.21a 2.83 0.11b 1.83 0.18c Density (g/cm3) 0.095 0.02a 0.132 0.01b 0.346 0.04c

Results are mean SD of six measurements a Means with different letters in the same column for each sample are significantly different at the 5% level

Addition of amaranth grits to extrusion blend proportionally reduced extrusion index and increased density of extrudates, due to its reduced expansion properties compared to corn. Reduced expansion resulted in a denser product with smaller air cell diameters as shown in Figure 1.

Fig. 1. Scanning electron micrographs of amaranth:corn 50:50 (A) and corn extrudate (B)

It is difficult to produce expanded products by extrusion cooking of amaranth grain alone, due to its high fat content (6-8% in whole grain). Fat provides a powerful lubricant effect in extrusion cooking and reduced product expansion (11). Flips of 100% corn grits have the highest expansion (4.03) and lowest density (0.095 g/cm3), which provides demanded crispy structure during eating. Blend of 50% corn and 50% amaranth grits resulted in a decreased expansion index (1.83) compared to control 100% corn grits by 2.2 times, but in increased density (0.346 g/cm3 ) by 3.6 times.Table 3. Texture of extrudates measured by Instron 4301Sample 100% corn grits 80% corn : 20% amaranth grits 50% corn : 50% amaranth gritsa

Compression force (N) 13.2 0.23a 20.8 0.32b 24.5 0.15b

Penetration force (N) 3.2 0.09a 5.1 0.11b 11.6 0.08c

Cutting force (N) 19.2 0.20a 18.8 0.18a 18.4 0.14a

Means with different letters in the same column for each samples are significantly different at the 5% level

Table 3 represents the differences in compression, penetration and cutting force (N) for the extrudate with amaranth grits supplement compared to the control with 100% corn 20

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


grits. Increasing the amount of amaranth grits in flips increased the resistance of extrudates, thus the difference in force for compression and penetration compared to corn grits flips is significant. Cutting force which imitates biting, is not statistically much different among treatments. Hardness of flips with 50% amaranth grits represented by penetration force (11.6 N) is by 3.6 times higher than the penetration force for sample with 100% corn grits (3.2N), which is a major difference. Generally, the addition of grits from whole amaranth grain, as a fiberrich raw material in extruded product formulations, resulted in increased density and hardness of the product (12). Figure 2 presents flow curves of gels obtained from corn grits and blends of corn and amaranth grits in the ratios 80:20 and 50:50, determined immediately after the preparation and after 24h. All systems exhibited thixotropic flow behavior. Gels prepared from corn grits had the greatest value of shear stress, i.e. viscosity, and largest thixotropic loop area. The structure is weak and it was brokendown by low share rates. During storage for 24h the system with 100% corn grits is additionally structured and threedimensional gel structure is formed as a result of retrogradation of amylase fractions. Since retrogradation is a process of binding by weak hydrogen bonds that are easily broken, the flow curve for 100% corn grits measured after 24 hours shows a peak characteristic for breaking such structures down at very low shear rates. Gels of examined grits blends had lower viscosity than corn grits and during storage they did not build additional structure. This is a result of replacing part of the corn grits, whose starch contains amylose and amylopectin, with amaranth grits whose starch includes small amount of amylose and high amount of amylopectin fractions (13).B B B120 140 120 100

### ### ###

100

(Pa)

80

(Pa)0 100 200 300 400 500

80 60

60

40 40 20 20 0 0 100 200 300 400 500

0

(1/s)

(1/s)

Fig. 2. Flow curves of the gels from corn grits and cornamaranth grits in the ratios 80:20 and 50:50 determined in 0 h and 24 h

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


In such a way fraction which structurate during time (amylose) is partially replaced by non gelling fraction. Increased amount of amaranth grits decreased viscosity and surface of thixotropic flow curves. From the flow curves, in Figure 2, fitting data in HerschelBulkley equation (14): = 0+ k

n

the values for yield stress 0 and coefficients k and n were calculated. Yield stress decreased with addition of amaranth grits, as part of the corn was replaced. Also, values of yield stress 0 increased for gel samples after 24 hours of storage due to the structuration. Values of viscosity behavior index n for all samples are smaller than 1, which is characteristic for thixotropic behavior.Table 4. Values of yield stress, consistency index and viscosity behavior indexSample 100% corn grits 80% corn : 20% amaranth grits 50% corn : 50% amaranth grits 100% corn extrudate 50% corn : 50% amar. extrudate Yield stress o (Pa) 0h 3.97 1.96 0.72 0 0 24h 4.59 2.75 0.92 0.57 0 Consistency index k 0h 2.78 2.49 2.89 0.30 0.05 24h 10.46 6.75 3.93 12.59. 0.05 Viscosity behavior index n 0h 0.59 0.57 0.52 0.72 0.06 24h 0.38 0.41 0.44 0.21 0.06

120 100 80 (Pa)

100% corn extrudate corn/amaranth 50:50% extrudate 100% corn grits corn/amarnth 50:50%

60 40 20 0 0 100 200 300 400 500

(1/s)

Fig. 3. Flow curves of the gels made from the corn grits and extruded snack, and from 50:50% corn:amaranth grits and extruded snack

Fig 3 presents flow curves of corn grits and grits blends of corn and amaranth in a ratio 50:50, as well as flow curves of obtained extrudated products. Flow curves of gels made from extrudates had lower viscosity than the gels made from corresponding grits. 22

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


Starch granules in extrudates after thermal treatment were partly damaged during extrusion process and they built weak gels. The obtained pastes had a lower viscosity, and they can be classified to the category of pregelatined starch granules.CONCLUSION

Increasing amount of amaranth grits in the extrusion blend causes increase of density and hardness of the extrudated products and decrease in expansion index. When part of the corn grits is replaced with amaranth grits viscosity of gels decreases compared to pure corn grits. Also, extrusion process partially damages starch granules, thus obtained gels of extrudated products have lower viscosity than the initial grits.REFERENCES

1. Saunders, R.M., and R. Becker: Amaranthus: A Potential Food and Feed Recourse in: Advances in Cereal Science (1984) 357-396. 2. BodroaSolarov, M.: Effects of Genotype and Sowing Date on Yield and Yield Components of the Genus Amaranthus L, Ph.D.Thesis, Faculty of Agriculture, University of Novi Sad (2001) 1-113. 3. Kovacs, E.T., MarazSzabo, L., J. Varga: Influence of Variety and Type of Emulsifier for Functional Food Quality and Amaranth Basis, Proceedings of XIV International Congress Cereal Bread 2000 (2001) 223-226. 4. Breene, W.M.: Food Uses of Grain Amaranth, Cereal Food World 36, 5 (1991) 426429. 5. SanchezMarroquan, A., Del Valle, F.R., Escobedo, M., Avita, R., Maya, S., and M. Vega: Evaluation of whole amaranth (Amaranthus cruentus) flour, its air classified fractions, and blends of these with wheat and outs as possible components for infant formulas, J. Food Sci. 51, 5 (1986) 1231-1234. 6. Radosavljevi, M., Jane J., L.A. Johanson: Isolation of Amaranth Starch by Diluted AlkalineProtease Treatment, Cereal Chem. 75, 2 (1998) 212-216. 7. Gonzales, R. J., Torres R. L., De Greef, D. M., Tosi E., E. Re: Brazilian Journal of Chemical Engineering 19, 4 (2002) 391-395. 8. AOAC : Solids (total) and moisture in flour. Air oven method. In Official Methods of Analytic Chemists, 14th edn. (1984) pp. 249. 9. Ding Q., P. Ainsworth, A. Plunkett, G. Tucker, H. Marson: The Effects of Conditions on the Functional and Physical Properties of WheatBased Expanded Snacks, Journal of Food Engineering 73 (2006) 142-148. 10. Koeppe, S. J., Harris, M.A., Hanna, J. H., Rupnow, C. E. Walker, and S.L. Cupett: Physical Properties and Some Nutritional Characteristics of an Extrusion Products with Defatted Amaranth Seeds and Defatted Maize Gluten Meal (80:20 Ratio), Cereal Chemistry 64, 4 (1987) 332-336. 11. Ilo, S., Liu Y., E. Berghofer: Extrusion Cooking of Rice Flour and Amaranth Blends, Lebensm.Wiss.u.Technol. 32 (1999) 79-88. 12. Onwulata, C. I., Konstance R. P., Strange E. D., Smith P. W., V.H. Holsinger: High Fiber Snack Extruded from Triticale and Wheat Formulations, Cereal Food World 45, 10 (2000) 470-473. 23

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940017D


13. BelloPerez, L. A., Colona, P., Roger, P., O. PeredesLopez: Macromolecular Features of Amaranth Starch, Cereal Chem. 75, 4 (1998) 395402. 14. Mezger T. : The Rheology Handbook, Vinsentz Verlag, Hannover (2002) pp.46. . , . -, . .

20 20% 50% . . , , . . . . Received 15 June 2009 Accepted 9 September 2009

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940025F

UDC:664.71-11:664.762:66.012 BIBLID: 1450-7188 (2009) 40, 25-34 Original scientific paper

REDUCTION OF WHEAT MIDDLINGS USING A CONVENTIONAL AND EIGHT-ROLLER MILLING SYSTEMS Aleksandar Z. Fite and uro M. Vukmirovi1 Possibilities for the rationalization of the wheat flour milling process using the eightroller mill on the 1M and 2M passages of the reduction system have been investigated. At the same roll gaps and under the same sieving conditions, the lower flour yield has been obtained using an eight-roller mill compared to the conventional milling system (5-8 %) followed by a higher energy requirements for grinding. By decreasing the roll gap setting and increasing the upper size limit of flour in the process with the eight-roller mill it is possible to increase flour yield and therefore decrease milling energy consumption per unit mass of flour produced without deterioration of flour quality as determined by ash content. With appropriate adjustments of the processing parameters in the eight-roller milling system it is possible to achieve similar milling results to those in the conventional system, while the overall investment, energy and maintenance costs are significantly lower.

KEY WORDS: Wheat flour milling, process rationalization, eight-roller millINTRODUCTION

The objective of the wheat flour milling process is to separate the branny cover and germ of the wheat kernel from the endosperm and achieve as high as possible flour extraction with the lowest contamination of bran and germ that increase the ash content. Breaking the wheat kernel is affected by corrugated cast steel rolls that gradually separate the endosperm, bran and germ. Reduction of relatively pure endosperm to flour is achieved by using smooth rolls. Segregation between the kernel parts occurs in plansifters, where sieves separate particles of different size, and in purifires, where sieves and airflow separate particles of different size, specific gravity and shape (1). Ever since the grinding of grain has been known to the mankind, possibilities and solutions have been sought out in order to simplify the grinding process and make it more efficient. New concepts and ideas only have chance of being successful if the yield as well as the quality of the finished products are not affected and requirements such as reduction of investment, operating and maintenance cost are met (2). The traditionalAleksandar Z. Fite, M.Sc., [email protected], Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; uro M. Vukmirovi, B.Sc., [email protected], Institute for Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

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APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940025F


wisdom in flour milling is that after every grinding step the ground material should be sieved and the undersize material removed before regrinding (3). Over the years the main equipment used in the grinding system has been redesigned to such an extent that it has been possible to multiply the throughputs of these machines but flour process technology has not changed fundamentally since the introduction of the roller mill, the purifier and the plansifter (4). This is the reason why the double grinding of intermediate streams before sieving has been one of the most notable process developments in flour milling (3). Eight-roller mill (a total of 8 rolls in one housing) provides two grinding passages without any intermediate sifting. Compared to a conventional process with the four-roller mill, the introduction of the eight-roller mill into the milling flowsheet offers the following advantages (2, 4, 5-8): fewer pneumatic suction lifts (conveying the stock from the roll to the sifter) resulting in lower material and installation costs lower pneumatic system air requirements resulting in lower energy costs and lower filter surface requirements for cleaning the pneumatic conveying air reduction of sifter surface smaller number of roll stands less spouting and auxiliary components lower space requirements for equipment installation resulting in lower building costs for the new milling plants or increase in grinding capacity within existing limited building space more compact building design makes it easier to keep clean with less area to fumigate with less equipment there is less cleaning and maintenance. On the other hand, twin stage grinding ignores the milling principle that, after grinding, coarse material is separated from the fines and therefore the conditions for controlled milling are less favorable. The decision as to how many double grinding passages can be applied in the flowsheet depends directly on the finished products to be made. It is not always possible to equip installations exclusively with eight-roller mills (4, 8). This is the reason why it is very important to define the passages and optimal roll parameters that allow the introduction of the eight-roller mill into the milling process without deterioration of the yield and quality of finished products. Even though the eight-roller mill has found its place in the modern flour milling process relatively little research has been performed on various factors affecting the milling results using this technique mainly focusing on the front passages of the break system (8-11) rather than the passages of the reduction system. It is evident that when the first and second breaks are combined into a twin passage, the particle size distribution of the stock will not be the same as with conventional single break system (2, 8, 11). There are several disadvantages of using this break system. First, it grinds fine material, coarse middlings, that should not pass to the next break roll, whose function is to separate endosperm from bran. Second, it produces more break flour and fine middlings and less coarse middlings and sizings that can be purified to produce clean middlings and low-ash flour. Third, the capacity of the lower roll is limited because the ground material is lower in density, which increases the volume to the roll (5). Tegeler (8) and Wanzenried (7) 26

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stated that the granulation from flour produced in the eight-roller mill is finer while the flour color is slightly whiter compared to the ones in the conventional process. The research of Zwingleberg (9) and Zwingleberg and Artz (10) showed that appropriate adjustment of the roll parameters is needed regardless of whether the eight-roller mill is used on breakage or reduction passages. Under industrial conditions, only the roll gap settings and feed rate (to a limited degree) can be adjusted during the milling process. The aim of this work was to examine and compare the effect that roll gap changes have on the milling results (degree of particle size reduction, flour release, flour ash and milling energy requirements) obtained using the conventional process and process with an eight-roller mill employed on the 1st and 2nd midds (1M and 2M passages) of the reduction system.EXPERIMENTAL

The samples were obtained from an industrial mill (120 t/day) intercepting the stream (middlings) that would have been sent to the 1M. In this particular mill (having five break, four sizing and six reduction passages) the eight-roller mills are not employed. The samples (50 kg) were separated using the automatic sampler divider (Gompper-Maschinen KG) into 0.5 kg batches and milled on a Variostuhl (model C Ex 2) laboratory roll stand (Miag). Smooth rolls 0.1 m in length and 0.25 m in diameter were used. Table 1 summarizes the experimental range of variables tested. The experiments were designed to compare: a.) conventional milling system the entire stock following 1M was sieved for 3 min on a Bhler laboratory sifter (model MLU-300) and the part of the stock held on the sieve fitted with 150 m bolting cloth was milled on 2M b.) eight-roller milling system the entire stock following 1M was milled on the 2M without intermediate siftingTable 1. Summary of experimental range of variables testedMilling system Conventional Smooth Eight-roller Roll surface Roll gap combinations [mm] 1M-0.10; 2M-0.08 1M-0.10; 2M-0.05 1M-0.08; 2M-0.05 Feed rate [kg/cm/min] 1M-0.20; 2M-0.15* 1.25 1M-0.20; 2M-0.20 5 Differential Fast roll speed [m/s]

*The slower feed rate on 2M corresponds to the amount of flour removed by intermediate sifting of the stock leaving 1M

Sieve analysis of the entire stock following 2M was performed on the above Bhler laboratory sifter for 3 min. For the conventional milling system the sieve openings were 250, 200 and 150 m, along with the bottom collecting pan. For the eight-roller milling system three different stacks of sieves were used. The first stack was the same as that mentioned above. In the second, the sieve with the 150 m bolting cloth was replaced 27

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940025F


with a sieve having 180 m bolting cloth, and for the third stack the sieve openings were 250 and 200 m. Two samples were milled and sieved under the same conditions. The weight distribution among the streams was highly reproducible. Based on the 3 rule, the 99.7% estimated confidence interval for the data (weight percentages) presented in the paper is 0.37%. Flour yield F (%) in the eight-roller and conventional milling systems was calculated from Equations (Eqs.) [1] and [2], respectively.

F (%) =

m2M * 100(%) M

[1]

F (%) =

m1M + m2 M *100(%) M

[2]

The symbols m and M stand for the weights of the flour and native feed, respectively. The subscripts indicate flour following 1M or 2M. The ash content of the total quantity of flour leaving the 2M was determined according to ICC standard method No.104/1 (12). The milling energy consumption during all grinding runs was determined from the wattmeter fitted as an integral part of the Variostuhl laboratory roll stand. Two different power readings were recorded corresponding to operation with (P, kW) and without (P*, kW) the material flow. The milling energy consumption, E kJ/kg, in the conventional and eight-roller milling systems was calculated by Eqs. [3] and [4], respectively.

E= E=

P P2*M P1M P1* M t2M t1M + 2 M m2M m1M ( P1M P1* ) + ( P2 M P2*M ) M (t1M + t 2 M ) m2 M

[3] [4]

Here m (kg) is the mass of flour obtained and t (s) is the time of the grinding run determined by the chronometer. The significance of the differences between milling results obtained using investigated milling systems have been tested by the paired Students t-test.RESULTS AND DISCUSSION

Changes in the particle size distribution of the stocks leaving 2M, brought about by the decrease of the roll gap, followed the same trends in both milling systems. Considering that the 2M feeds were different for the two milling systems, yields of the size fractions of the milling output are not to be compared because the cumulative size distributions (Fig. 1a and 1b) were given relative to the mass of the material milled on the 2M and only serve to show the general trends. By decreasing the roll gap, the quantity of material >200 m (size fractions >250 m and 250/200 m) tends to decrease while the flour yield (200 m). In addition to that, decreasing the roll gap increases the grinding zone size (14) so the grinding action, under predominant compressive forces, is prolonged and also contributes to the greater flour release as a result of increased number of endosperm fractures. Ash is concentrated in the bran, with over half the total in the pericarp, testa and aleurone and the ash content increases from the inner (endosperm) to the outer (bran) 29

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part of the wheat kernel (15). The increase of the ash content of the coarsest fraction of the stock following 2M, while roll gap had no influence on flour ash content (Table 2), proves that bran particles remain intact. Scanlon and Dexter (16) and Scanlon, Dexter and Biliaderis (17), in their studies of the effect of smooth roll grinding conditions on reduction of wheat farina, also reported similar findings.Table 2. Ash content in flour (250 m) of the stock following 2M in the conventional and eight-roller milling systems Ash content (%)dm Conventional system Eight-roller system >250 m 250 m 0.05). The values obtained by laboratory procedures were then regressed against determined in vivo values and the functions for each in vitro procedure for predicting the in vivo OMD of complete diets have been derived. The following equation, shown also in Figure 1, to predict the in vivo OMD from the results of in vitro T&T method has been obtained: OMD (in_vivo) = -17.36 + 0.98 x OMD (in_vitro_T&T), R2 = 0.75; RMSE = 37.59 The regression of the OMD results obtained by Gas production method against the in vivo values, resulted in the following equation, shown also on Figure 2. OMD (in_vivo) = 198.98 + 0.71 x OMD (in_vitro_GP) R2 = 0.21; RMSE = 66.36 The equation, shown also on Figure 2, for predicting the in vivo OMD using Multienzyme incubation (EDOM) method, was as follows: OMD (in_vivo) = 102 + 0.82 x OMD (in_vitro_EDOM) R2 = 0.86; RMSE = 27.30

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UDC:636.085.1/.3:636.085.55:636.2+636.3 BIBLID: 1450-7188 (2009) 40, 79-86 Original scientific paper

Fig. 1. Relationship between the OMD values of compound feeds for ruminants determined in vivo and by in vitro T&T method

Fig. 2. Relationship between the OMD values of complete diets for ruminants determined in vivo and by in vitro GP method

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Fig. 3. Relationship between the OMD values of complete diets for ruminants determined in vivo and by in vitro EDOM method

The organic matter digestibility of complete diets for ruminants in this study was predicted best by EDOM method, closely followed by the two-stage T&T procedure. A low correlation between Gas production and in vivo results (R2 = 0.21) was somehow unexpected, since Palic and Muller (8), investigating the OMD of feedstuffs for ruminants, established an R2 = 0.81 for the relationship between the OMD values determined in vivo and by in vitro Gas production method. The prediction of OMD of compound feeds for ruminants by the use of enzymes have been up to date applied mostly to forages. Enzymatic methods have been much less studied with energy and protein feeds (6), and in those seldom cases, the authors used single-enzyme incubations. Dowman and Collins (12), using incubation with pepsin-HCl, followed by the treatment of 12 complete diets with cellulase, reported a correlation of R2 = 0.87 between in vivo and in vitro values, whereas Aufrere and Michalet-Doreau (6) found an R2 = 0.90 using 24 energy-rich feeds. The procedure of Hvelplund et al. (7) used in this study, might have an advantage over the above-mentioned, as it uses multienzyme incubation and therefore may better mimic the digestion in the animal gastrointestinal tract.CONCLUSION

Although conducted on a small number of samples, the results of this study yielded a clear reference point and showed that the organic matter digestibility (OMD) of complete diets for ruminants can be successfully determined, and the in vivo OMD successfully predicted, using a multi-enzyme incubation procedure. This is important because of the fact that rumen liquor, needed for the in vitro Tilley and Terry and Gas Production techniques, is not always available to analytical laboratories. Further work, with inclusion of more samples of complete diets, is needed to confirm the results of this study. 84

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REFERENCES

1. Barber, G.D., D.I. Givens, M.S. Kridis, N.W. Offer and I. Murray: Prediction of the organic matter digestibility of grass silage. Anim. Feed Sci. Tech. 28 (1990) 115-128. 2. Thomas, P.C. Predicting the nutritive value of compound feeds for ruminants. In: Feedstuffs Evaluation. Eds. W. Haresign and D.J.A. Cole, Butterworths, London (1990) p. 301 3. Tilley, J.M.A. and R.A. Terry: A two stage method for the in vitro digestion of forage crops. J. Brit. Grassl. Soc. 18 (1963) 104-111. 4. Menke, K.H.and H. Steingass: Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28 (1988) 7-55. 5. Jones, D.I.H.and M. K. Theodorou: Enzyme techniques for estimating digestibility. In: Forage Evaluation in Ruminant Nutrition. Eds. D. I. Givens, E. Owen, R. F. E. Axford and H. M. Omed, CABI publishing, New York (2000) p. 155. 6. Aufrere, J. and B. Michalet-Doreau, B: Comparison of methods for predicting digestibility of feeds. Anim. Feed Sci. Tech. 20 (1988) 203-218. 7. Hvelplund, T., M. R. Weisberg and K. Soegaard: Use of in vitro digestibility methods to estimate in vivo digestibility of straws. TSAP Science Conference, Arusha, 3-4 Aug 1990, Proceedings vol. 26, p. 70. 8. Palic, D., H. Muller: Prediction of the in vivo organic matter digestibility of feedstuffs for ruminants using in vitro techniques. Savremena Poljoprivreda 55, 12 (2006) 127-132. 9. AOAC INTERNATIONAL, Official Methods of Analysis, 17th Edition, AOAC INTERNATIONAL, Gaithersburg, MD 20877-2417, USA (2000). 10. Steg, A., J. M. van der Merwe, B. Smits and V. A. Hindle: Prediction of the digestibility of feedstuffs: recent developments. Annual report, ID-DLO, The Netherlands (1987). 11. Payne, R.W., D.A. Murray, S.A. Harding, D.B. Baird and D. M. Soutar: GenStat for Windows (10th Edition), Introduction. VSN International, Hemel Hempstead, UK (2007). 12. Dowman, M. G., F. C. Collins: The use of enzymes to predict the digestibility of animal feeds. J. Sci. Food Agric 33 (1982) 689-696. IN VITRO Klaas-Jan Leeuw

(OMD) in vivo, , in vitro, Tilley Terry (&) (3) , (GP) (4) c - (EDOM) (7). in vivo &, GP EDOM in vitro 85

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684, 716, 685 710 g / (P>0,05). in vivo OMD in vitro . & OMD (in_vivo) = -17,36 + 0,98 x OMD (in_vitro_T&T), (R2= 0,75; RMSE=37,59, GP OMD (in_vivo)=198,98 + 0,71 x OMD (in_vitro_GP), (R2=0,21; RMSE=66,36), EDOM OMD (in_vivo)= 102 + 0,82 x OMD (in_vitro_EDOM), (R2=0,86; RMSE = 27,30). a , in vivo , - , , & GP , . Received 17 June 2009 Accepted 28 Septembar 2009

86

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UDC: 637.146.3:637.04/.05 BIBLID: 1450-7188 (2009) 40, 87-94 Original scientific paper

COMPOSITIONAL CHARACTERISTICS OF COMMERCIAL YOGHURT BASED ON QUANTITATIVE DETERMINATION OF VIABLE LACTIC ACID BACTERIA Dragana Pei-Mikulec and Gordana B. Niketi Yoghurt quality is particularly difficult to standardize because of the many forms, varieties, manufacturing methods, ingredients and consumer preferences that exist. Since these factors will always play an important role, it is unlikely that a uniform yoghurt quality concept will ever emerge, such as has been developed for other dairy products. There are a number of common denominators, however that have bearing on yoghurt quality. Since a number of producers are recognized within the broad category entitled yoghurt. This situation makes yoghurt an interesting, challenging, but also a confusing area to work in. The present investigation was undertaken to isolate from commercial yoghurt the strains involved in its manufacture and determine the characteristics of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus. This study is concerned with the lactic acid bacteria (L.delbrueckii subsp. bulgaricus and S. thermophilus) growth in yoghurt from involving different procedures and with the determination of the number of lactic acid bacteria in dependence of the temperature and acidity in the period of storage. Predominant samples of yoghurt were with 11-107/ml lactic acid lactococci (44.28%).

KEY WORDS: LAB, yoghurt, viable lactic acid bacteria, probioticsINTRODUCTION

One of the first records of yoghurt consumption comes from the Middle East during the times of the Conqueror Genghis Khan in the 13th century, whose armies were sustained by this healthful food. Yoghurt and other fermented dairy products have long been a staple in the diets of cultures of the Middle East, Asia, Russia and Eastern European countries, such as Bulgaria. Yet, the recognition of yoghurts special health benefits did not become apparent in Western Europe and North America until the 20th century, as a result of research done by Dr. Elie Metchnikoff. Dr. Metchnikoff (12, 13) is the first researcher who proposed fermented dairy products with beneficial properties. Conducted research on the health benefits of lactic acid-producing bacteria and postuDr. Dragana Pei Mikulec, [email protected], Senior research fellow, Department of Food Microbiology, Institute of Veterinary Research, Autoput 3, 11050 Novi Beograd, Serbia; Dr. Gordana B. Niketi, Research fellow, JPS Dairy Institute, Autoput 3, 11050 Novi Beograd, Serbia

87

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lated that the longevity of peoples of certain cultures, such as the Bulgarians, was related to their high consumption of yoghurt and fermented dairy products. The benefits of yoghurt depends for live active cultures or living yoghurt cultures. Yoghurt is made by fermenting milk with friendly bacteria, mainly Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus. Yoghurt is a traditional food and beverage in many countries and especially in Serbia. Yoghurt consumption in Serbia has increased during the last decade. Product quality and satisfaction of consumer expectation are discussed since they are essential for the continued successful growth of the yoghurt market. Much emphasis is placed on yoghurt flavor, body, and texture. The specific objectives of the study were: a) to determine the effect of cell lactis acid bacteria L.delbrueckii subsp. bulgaricus and S. thermophilus on growth in yoghurt from a different producers and b) to determine the number of lactic acid bacteria of the temperature and acidity in the period of storage (1,2). Initial counts of Lactobacillus and Streptococcus in the samples of yoghurts were in the range from 8 to 1x106 /g resp. Ratio of Lactobacillus : Streptococcus at the start of the test varied from 1:1 to 1: 2.7.EXPERIMENTAL Yoghurt production

Yoghurt is made by fermenting milk with friendly bacteria, in Serbia mainly with Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus. The milk sugar or lactose is fermented by these bacteria to lactic acid which causes the characteristic curd to form. This process gives yoghurt its refreshingly tart flavor and unique pudding like texture. The yoghurt qualities were judged to be satisfactory without defective taste. The baccilli/cocci ratio in the pre-fermented milk, unstable with free cells, was stabilized when the strains were enterapped. The yoghurt starter cultures play an important role during the production of yoghurt. High cell numbers to about 5-107 C.F.U. ml-1 with a steady bacilli/cocci ratio were present in the effluent milk. The starter culture for most yoghurt production is a symbiotic blend of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in relation 50:50. Although they can grow independently, the rate of acid production is much higher when used together than either of the two organisms grown individually. Streptococcus thermophilus grows faster and produces both acid and carbon dioxide. The format and carbon dioxide produced stimulates growth of lactobacilli. On the other hand, the proteolytic activity of lactobacilli produces stimulatory peptides and amino acids for use by streptococci. These microorganisms are ultimately responsible for the formation of typical yoghurt flavor and texture. Yoghurt that contains live bacterial cultures may help to live longer and may fortify immune system. The yoghurt mixture coagulates during fermentation due to the drop in pH. The streptococci are responsible for the initial pH drop of the yoghurt mix to approximately 5.0. The lactobacilli are responsible for a further decrease to pH 4.0. The following fermentation products contribute to flavor: lactic acid acetaldehyde 88

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acetic acid diacetyl The acid also restricts the growth of food poisoning bacteria. During the yoghurt fermentation some flavors are produced, which give yoghurt its characteristic flavor.Cultured media for the enumeration of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus mixtures

Yoghurt was sampled 1 day after manufacture and the samples were taken in the market. Samples from 2 replicate experiments were processed. There are at present several culture media for the differential enumeration of mixtures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus. The use of these media is important for the control of the relation between these two microorganisms in the yoghurt production starter medium, as well as in the follow-up of both populations during the production and later ripening of yoghurt. The number of lactobacilli was determined on the MRS agar (6) and the number of streptococci was determined on M17 agar medium after 48 h of incubation was investigated, making use of the different morphologies of the colonies as a means of different enumeration. Total bacterial count was determined by direct counting on a microscope glass. Material for the research was 70 samples of yoghurt from 9 different dairy producers. The samples were from Belgrade trade market. As a laboratory control we used the mixed cultures of L.delbrueckii subsp. bulgaricus and S.thermophilus. Cultures enumerated in this study were lactic acid starter cultured used for manufactured of yoghurt. Lactic acid bacteria were enumerated using Elliker (7, 8) and MRS (5, 6) solid agar plates, used for the isolation lactobacilli (3, 4). Reconstituted milk powder was used for the storage lactic acid bacteria in the refrigerator. Strains of lactic acid lactococci were an aerobically transferred three times at 370C for 48 h (BBLGas Pak System). The species designation of isolated was confirmed by Gram stain colonial appearance. MRS agar plate was incubated at 37oC without further adjustment. Plates or tube prepared with MRS agar were placed in plastic bags in anaerobe conditions and then incubated. M17 agar plates were used for the detection of streptococci. Each plate was overplayed with the same solid medium and then incubated at 30-37oC. Duplicate plates were prepared for each medium for the required dilutions (7, 9). Plates were examined after 48 and 72 h. To evaluate the factors that might be responsible for excessive acid development during yoghurt storage, 9 brands of plain commercial yoghurt were purchased from local retail markets and stored at 8, 12, 20oC, and analyzed weekly for 288 hours to monitor changes in acidity (oSH), total viable Lactobacillus and Streptococcus, coliform, yeasts and moulds.Microorganisms

Strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus isolated from commercial products were used. Commercial yoghurt samples were used with no modifications, mixing 1 g of yoghurt in 10 ml saline solution and effective successive dilutions from 10 -2 to 10-6. 89

APTEFF, 40, 1-220 (2009) DOI: 10.2298/APT0940087P


Microorganisms: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus isolated from commercial yoghurt were grown at 43oC in M.R.S. and M17 agar. Isolation and identification: The lactis acid bacteria were isolated by API system for lactobacili and streptococi identification. The API system was incubated at 37oC for 72 h. The various colonies formed were identified. Criteria examined included: Gram reaction, cell morphology, catalasa reaction, growth at 50oC, acid production from lactose, sucrose, trehalose, maltose and manitol, thermoresistence to 63oC, growth in NaCl 2%, hydrolysis of arginine, and growth at pH 9.6.Culture medium and growth conditions

Enumeration of microorganisms: Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus were enumerated by surface spreading 0.1 ml samples on Elliker agar medium and by direct microscopic count on the microscope slade.RESULT AND DISCUSSION

The optimum growth temperature for Lactobacillus delbrueckii subsp. bulgaricus occurred at 45oC and Streptococcus thermophilus has its optimum between 40oC and 45oC. The optimum temperature of the mixed culture was 45oC. The growth of these cultures at different initial pH in Elliker medium shows that the optimum pH for pure and mixed cultures is from 6.5 to 8.0. In Table 1 the results of the chemical a

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Acta Periodica technologica 2009-40