372 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XIII, Issue 4- 2014, pag. 372 - 375 APPLICATION OF TITRIMETRIC TECHNIQUE TO DETERMINE PHOSPHATES IN CHEESES *Mariуa VOROBETS1, Igor KOBASA1, Aureliуa CHEBAN1 1Yu. Fedkovych National University of Chernivtsi, Ukraine, m.vorobets@chnu.edu.ua *Corresponding author Received November 19th 2014, accepted December 29th 2014 Abstract: A method of the titrimetric determination of phosphate (food additives E338 - phosphate acid, E339 - sodium phosphate, E340 - potassium phosphate, E341 - calcium phosphate, E450 - sodi- um and calcium pyrophosphate,) in cheeses has been developed. The research experimental results regarding the phosphate ion content in hard and spread cheeses (in terms of phosphate acid, %) ob- tained by means of the developed methodology are described. The correctness and accuracy of the results obtained by developed technique are confirmed by the definition of phosphates in the studied samples of cheeses by photometric method (by the method according to DSTU ISO 3360: 2008). A comparative analysis of the two methods has been performed. Keywords: food additives, phosphates, hard and spread cheeses, titrimetric method, photometric method 1. Introduction Today, the use of biological and food addi- tives in the food industry has become an integral component technology [1]. To im- provement of the quality of many products and for perfection of the process in the milk industry the phosphates are used. Phosphorus-containing food additives are also used in the production of soft drinks, bakery, confectionery and meat products as stabilizers, acidity regulators, baking pow- der and a latches color. But most of them are used in the production of hard and spread cheeses. Food additives E338 (phosphate acid H3PO4), E339 (sodium phosphate NaH2PO4; Na2HPO4; Na3PO4), E340 (potassium phosphate KH2PO4; K2HPO4; K3PO4), E341 (calcium phos- phate Ca(H2PO4)2; CaHPO4; Ca3(PO4)2) E450 (pyrophosphates Na2H2P2O7; Na3HP2O7; Na4P2O7; Ca2P2O7; CaH2P2O7) have a variety of technological properties: soluble alkali phosphate have a pro- nounced buffer potential to regulate acidi- ty; polyphosphates inhibit growth of gram- positive bacteria, which can significantly increase the shelf life of products; tricalci- um phosphate used in the production of bakery products, special drinks, powdered and condensed milk, ice cream, fish and minced meat, alcoholic beverages, conven- ience foods, breakfast cereals, instant food, confectionery, baking powder, spread cheeses [2]. Phosphates are used as acidity regulator, emulsifier and stabilizer. These salts per- form essential function in the production of cheese, because without salt-fin its produc- tion is impossible [2, 3]. However, phosphates are found in many foods and can cause adverse reactions (al- lergy), especially in children. The result of allergy is an altered mental reaction, im- pulsivity, motor restlessness, impaired concentration, increased aggressiveness Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Mariуa VOROBETS, Igor KOBASA, Aureliуa CHEBAN, Application of titrimetric technique for determination of phos- phates in cheeses, Volume XIII, Issue 4 – 2014, pag. 372 – 375 373 sometimes. Frequent use of food additives E338, E339, E340, E341 in food leads to disturbances in the gastrointestinal tract, vomiting, diarrhea, nausea and aversion to food, weight loss [3, 4]. If a healthy person with food gets a lot of phosphates in her body disturbed calcium metabolism, begins osteoporosis (bones become brittle and rel- atively easily broken). The products with the addition of phosphate are very danger- ous for pregnant women because it is likely to have a child with a variety of disorders in the brain and respiratory system [5, 6]. According to the Committee on the rights of consumers over 80 % of food products, which are sold in the markets of Ukraine, falsified. Therefore, the development and improvement of methods of analysis, in- cluding analysis of the food additives con- tent in the food products is an actual prob- lem nowadays. For researches phosphates (food additives E338, E339, E340, E450, E452, etc.) traditionally are using the pho- tometric method [7]. However, this method requires expensive reagents and instru- ments as well as highly qualified special- ists. Not all laboratories can to have such complex providing. Volumetric analysis is a simple and affordable method, which is often used for studies of liquids. The aim of our investigation was to develop (or to adapt) known the titrimetric method of de- termination of phosphates in milk for re- search phosphates in hard and spread cheeses. 2. Materials and methods The determination of phosphates in hard and spread cheeses by photometric method includes the next points: complete destruc- tion of organic compounds under the ac- tion of sulfuric acid and hydrogen perox- ide; addition of an ammonium molybdate solution in ascorbic acid; measurement of the optical density of blue phosphorus mo- lybdenum complex [8]. The sample was prepared immediately before the definition. Sample of cheese 50±1 g was crushed and stirring until a pasty mass. The sample we put in a Kjeldahl Flask, added concentrated sulfuric acid and were carried out a miner- alization: were heated to form the mini- mum foam by avoiding local overheating and the release of foam from flask; cooled to room temperature; added 3÷4 drops of solution of hydrogen peroxide and heated again. This procedure repeated for as long as the content of the flask will become un- colored and transparent. To remove traces of hydrogen peroxide continued to heat the flask for 30 min, were cooled. The contents of the flask were transferred to a measuring flask of 100 cm3 and volume was adjusted to the mark with distilled water. Using a pipette selected 1 cm3 of obtained mineralize sub- stance and put into measuring flask of 50 cm3 volume; added a mixed solution of sodium molybdate and ascorbic acid. The volume of solution was adjusted to the mark with distilled water. The flask was put in a boiling water bath for 15 min. After cooling, the optical density was measured at the wavelength 820 nm in cu- vettes with optical path length of 10 mm. The titrimetric method is simple, cheap and affordable. For develop a methodology of determining phosphates in cheeses by ti- trimetric method chosen method-prototype for milk. To find the mass of samples cheeses were taken into account: 1) for determination of phosphates in milk for analysis selected 20 cm3 of milk (known [9] that in 100 cm3 of milk is 90÷100 mg of phosphorus, i.e. in 20 cm3 of milk can be determined ≈20 mg of phosphorus); 2) based on acceptable standards: 100 g of spread cheese includes 2074 mg of phosphorus additives. The mass of cheese sample was found by calcu- lation method: ).(1 074,2 02,0100 gx    Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Mariуa VOROBETS, Igor KOBASA, Aureliуa CHEBAN, Application of titrimetric technique for determination of phos- phates in cheeses, Volume XIII, Issue 4 – 2014, pag. 372 – 375 374 The sample preparation was carried out similarly as in the photometric definition. From the prepared samples were taken 1 g of cheese, added 20 cm3 of 0.1 M solution of sodium carbonate. Ashing in a muffle furnace is an integral part of the preparation of samples. The methodology for milk about ashing of samples says nothing. Based on experi- mental studies, we made adjustments. We propose before ashing samples in a muffle furnace to make the melting of the samples during slow heating (sand bath or electric stove), preventing the release of foam to full drying of cheese mass. Then, make the ashing in a muffle furnace at a temperature 600°C for 1÷2 min. Further analysis was performed according to the method for milk: an ash quantitatively transferred to a conical flask, was added 30 cm3 of 0.1 M solution of hydrochloric acid. The solution were evaporated on a sand bath to a vol- ume of ≈15÷20 cm3 (all ash dissolves). According to the method for milk to solu- tion add 2 drops of 0.1 % solution of me- thyl orange indicator. The contents of the flask was cooled and titrated by 0.1 M so- lution of sodium hydroxide to change color to yellow and then by drops add of 0.1 M solution of hydrochloric acid to a neutral reaction (orange color). Our correction: instead of indicator methyl orange to use indicator phenolphthalein. It is necessary to pay special attention to bring the reac- tion to neutral. In liquid was added 20 cm3 neutral with respect to phenolphthalein and methyl or- ange 40 % solution of calcium chloride. The liquid was boiled for 2 min, adding 1÷2 drops of phenolphthalein and titrated by 0.1 M solution of sodium hydroxide to change color from yellow to pink. Again were boiled for 2 min, titrated by 0.1 M solution of sodium hydroxide. This process repeats for as long as after heating, the so- lution remains pink. Then added 2 drops of phenolphthalein and titrated by 0.1 M solu- tion of sodium hydroxide to change the color of the contents of the flask with pink to red. If color for 10 min weakens to add a few drops of titrant. The content of phosphate acid (w, %) were calculated by the formula: %,100 1000 43 43     m ENV w POHNaOHNaOHPOH where NaOHV - the amount solution of alka- li (mean value), which went on titration, cm3; NaOHN - concentration of alkali, mol/dm3; 43POH E - molar equivalent weight of phosphoric acid (32.67 g/mol); m - mass of sample product, g. 3. Results and discussion We carried the experimental determination of phosphate in the samples of hard and spread cheeses of known domestic manu- facturers by the developed methodology. Were studied three samples of hard chees- es and three samples of spread cheeses. To confirm the correctness of the methodolo- gy and the reliability of the results, we conducted determination the content of phosphates in the same samples by photo- metric method according to [7]. The results are shown in Table 1. Table 1 Phosphates in cheeses Number of the sample Phosphates (in terms of phosphoric acid). % Titrimetric method Photometric method Hard cheeses 1. 1.16±0.05 1.19±0.04 2. 1.58±0.02 1.60±0.05 3. 1.09±0.01 1.10±0.015 Spread cheeses 4. 1.80±0.03 1.85±0.02 5. 1.40±0.025 1.37±0.03 6. 1.60±0.03 1.59±0.035 Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Mariуa VOROBETS, Igor KOBASA, Aureliуa CHEBAN, Application of titrimetric technique for determination of phos- phates in cheeses, Volume XIII, Issue 4 – 2014, pag. 372 – 375 375 Comparative analysis of the results ob- tained by the developed method (titrimet- ric) and by the method according to [7] (photometric) show that the value of phos- phates correlate with each other and differ only in the limits of errors. This clearly comparison chart demonstrates in Fig. 1. Fig. 1. The comparison chart of content of phosphates in the samples of cheeses 4. Conclusions Based on the methodology of the titrimet- ric determination of phosphates in milk developed a method for the titrimetric de- termination of phosphates in cheeses. By calculation method found mass of the sam- ples (1 g) made adjustments for sample preparation and analysis. The correctness of the methodology and reliability of the results confirmed by de- termining the content of phosphate in the studied samples photometric method in according to DSTU ISO 3360: 2008 The method of titrimetric determination of phosphates can be used to determine their аs well as in hard and in spread cheeses. 5. References [1]. BULDAKOV A. Food additives. Moscow: «Ut», 240 p. (1996). [2]. E-resource http://dobavkam.net/products [3]. ROSIVAL L., ENGST R. Foreign materials and food additives in foods. Moscow: Light and Food Industry, 264 p. (1982). [4]. SMOLAR V. Toxic effects of food additives. Problems of Nutrition, 5-15. (2005). [5]. SanPiN 2.3.2.1293-2003. Hygiene require- ments for the use of food additives. (2003). [6]. DONCHENKO L., NADYKTA V. Safety of food raw materials and food. Moscow: Food Indus- try, 352 p. (1999). [7]. DSTU ISO 3360:2008. Phosphoric acid and sodium phosphates specifications (including for the Food Industry) (2008). [8]. INIHOV S., BRIO N. Methods of analysis of milk and milk products. Moscow: Food Industry, 156 p. (1971). [9]. Milk and milk products: Normat. Documents: argument. / Ed. V. Ivanova. - L.: NIP Leonorm, - In 3 vols, 402 p. (2000).