BERNDORFER-KRASZNER , E.

TELEGDY KOVÁTS, L.

English

Periodica Polytechnica (Budapest University of Technology and Economics)

EXAMINATION OF THE PROTECTIVE EFFIICT OF TOCOPHEROLS ON PROVITAMIN A (B-CAROTENE) AND VITAMIN A IN l!.NRICHED MARGARINE AND BUTTER By L. TELEGDY KOV .. tTS and E. BERNDORFER-KRASZl'<ER Department of Food Chemistry, Poly technical University, Budapest Received October 3, 1966 Taking into account the chemical properties of tocopherols, 6-hydroxy. chromane derivates, the mode of vitamin E action is probably mostly based on the following two explanations: 1. as intracellular antioxydants they exert a protective effect by inhi-biting the autoxydation of vitamin A in unsaturated fats and similar natural organic substances of vital importance, sensitive to oxygen, such as ascorbic acid; and (or) 2. as electron carriers, they take part in respiration, biological oxydation and other vital processes. Vitamin effect is thus essentially hi-directional and may have an antioxydant effect and an enzyme effect. In this respect the toco-pherols, considered initially as inductive vitamins, are near to the prosthetic vitamins [1]. With regard to food chemistry and industry, the tocopherols have a par-ticularly interesting and significant property: as natural anti-oxydants they inhibit the oxydative rancidity of fats and fatty foodstuffs. A large number of researchers have examined in vivo the protective effect of tocopherols on provitamin A (p-carotene) and vitamin A [2], but only comparatively fe"w analyses have been undertaken to control in vitro this economically important protective effect. In our work we have studied in vitro the antioxydative and protective effect of tocopherols in margarine enriched "with provitamin A (p-carotene) and in butter containing natural vitamin A. Enriched with provitamin A (p-carotene), margarine is a current and up-to-date foodstuff in several countries; its general use is justified by its fine colour similar to that of summer butter and by its biological value. BAUERFEIND et aI. [3] have developed the technology of colouring fatty nutritive substances; they used the vegetable oil suspension of micro-pulverized p-carotene for colouring margarine. KOEHN [4] and BURl'<S et aI. [5] have conducted biological analyses for controlling the degree of conversion in animals. They have found that the effect of p-caro-tene conversion in rats daily receiving 1 mg of tocopherol is as active as that of vitamine A. 12* 514 L. TELEGDY KOVfiTS and E. BERNDORFER KRASZNER The nutritive value of 1>utter is increased by its richness in vitamin A. However, the vitamin A contents of butter shows a considerable fluctuation in the different seasons [6]. It would be an important task to keep the vitamin A content at a uniform level in spring and summer. As antioxydants we have used c;;- and y-tocopherol (Kochlight Lab., Ltd. Bolnbrook, Bucks.) as well as tocopherol concentrate extracted from maize germ oil. The preparation had 0,10% of tocopherol contents, including 80% of y-tocopherol. For examining the behaviour of synergists, we have chosen citric acid, phosphoric acid and selenious acid. Several researchers [7, 8] have recently dealt with the protective effect of selenium compounds on tocopherols, but not with their utilization in food chemistry. We have therefore carried out experiments also to detect the mode of action of selenium in connection with the synergism of selenious acids on thc anti-oxydant effect exerted by tocopherols. Our examinations were of informative character, their utilization in food chemistry is still subject to preliminary toxicological control. The aim of these examinations was to find out in the course of autoxy-dation v,-hich tocopherol extends the induction period longer than any other, , .. -hi ch tocopherol has the most efficient protective effect on /'1-carotene and vitamin A, and finally which synergist increases the anti-oxydative effect most efficiently. 'Ve have studied accordingly the antioxydative properties of tocopherols 1. at autoxydation produced by ultra-violet irradiation, 2. on the application of the Active Oxygen Method [16]. Due to artificial ultra-violet irradiation the fats are a site of processes similar to those presumably taking place in oils, lard or margarine kcpt in unprotected shopwindows. The apparatus used for ultra-violet irradiation has an output of 220 15 W. The samples were placed in Petri dishes of 8 cm diameter, in layers of 8 mms and kept at a distance of 16 ems from the ultra-violet lamp. The temperature during irradiation was of 25 : 2 QC; time of irradiation: 1-3-5 hours. After irradiation, the samples were kept in dark for 24 hours, and the analyses were carried out subsequently. The Active Oxygen Method was applied according to the standards. We have measured the time required for achieving the peroxide value of 62,5 mmol jkg of the test substance under the special circumstances of the method. The length of this time can be taken as an index of resistance to rancidity. In every case we have detcrmined the value of the autoxydation factor too. Our experi-ments included the determination of the peroxide number and of eventual contents in anti-oxydants, metal traces and vitamins (provitamin A, vitamin A and E). '" ~ '-. co ~ () " .. c: " c:; <. tl " ~ 100 80 60 "0 20 PROTECTIVE EFFECT OF TOCOPHEROLS OS PROVlTAJIIN A -con/rol ••••••••.• 100 mg % of selenious acid •• -_ •• 100 mg % of phosphoric acid - - - 100 mg % of cilric acid 5 Irradiation time (hours) 515 Fig. 1. Effect of synel'gents on the !'I-carotene content of enriched margarin during U\'· irradiation 100 ~ 80 ~ <: .. co () 60 " ., c: ~ () '- "0 <;]-" ~ 20 .~. "",,-h '0..: ......... - ··_··5 mg % oC-tocopherol and 50 mg % citric acid ~ .. \ ........ .:::::".::::... " ". _'" - --5 mg '}o cC-/ocopherol and 50 mg 0,0 phf!Jsphoric'acld, \ "a.. , _.- ·-5 mg % cC- tocopherol and 50 mg % selenious aCid . ....... ... •... .............. "~ . 0....... '" \\ ......... "":., '\~ '. . '\. .... :\.. ~ ..... ::\ -control .. .. • .... ·20 mg % oC- tocopherol 3 5 Irradialion time (hours) Fig. 2. Effect of c;(·tocopherol on the p·carotene content of enriched margarin during UV. irradiation The peroxide number was determined according to LEA [9]. The detec· tion of synthetic antioxydants was made with BIEFER'S paper-chromato-graphy [10]. This technique has the advantage of not extracting from the sub-strate the tocopherols present in their natural form. The iron(III) compounds were traced with the thiocyanate method of JACOBS [ll], the copper (II) com-pounds 'vith the dithizone method of MONIER-WILLIA::IIS [12] and the alu-minium (Ill) compounds v .. ith the titrimetric method of JACOBS [ll]. We have 516 L. TELEGDY KOVATS and E. BERNDORFER KRASZNER Table 1 Effect of tocopheroIs and some acid synergents in I Peroxide value mmol/kg in margarin Conc. Acid Conc. Irradiation time Tocopherols rug/lOO g 5)-ncrgents mg/lOO g Originally I 3 5 : - - - - 2.00 30.00 75.00 120.00 50.00 - - 14.00 38.00 70.00 Cl. 20.00 - - 2.00 I 10.00 30.00 55.00 10.00 ! 18.00 46.00 80.00 50.00 20.00 47.00 76.00 Y 20.00 - - 2.00 15.50 '10.50 74.00 10.00 26.00 50.00 84.00 1-50.00 10.00 27.00 68.00 mixture 20.00 - - 2.00 8.00 22.00 60.00 10.00 14.00 33.00 76.00 [ citric 11.00 32.00 40.00 Cl. 5.00 : phosphoric 50.00 2.00 10.00 28.00 35.00 : selenious 8.00 20.00 28.00 ---" ! citric 20.00 38.00 60.00 Y 5.00 phosphoric 50.00 2.00 H.OO 32.00 52.00 i selenious 10.00 24.00 40.00 I citric I 6.00 18.00 30.00 mixture 5.00 phosphorie 50.00 2.00 6.00 12.00 28.00 sel~nious I 5.00 10.00 20.00 worked out a rapid method to be used in fats for the determination of f3-caro-tene, based on the direct extraction of provitamin A by solvents, its isolation from interfering substances by column chromatography and on photometry [13]. Based on the CARR-PRICE reaction, the method of MOOR [14] was used for the determination of vitamin A. The qualitative and quantitative deter-mination of tocopherols in natural organic substances was made with a method of thin-layer chromatography we have developed [15]. The test material was margarine not enriched with vitamins, which we analyzed for possible peroxi-des, tocopherols, f3-carotene and antioxydants and metal traces. Experimental butter "was subjected to the same analyses. Margarine was coloured by crys-talline f3-carotene dissolved in sunflower oil (Light, Lab., Ltd. Colnbrook). f3-carotene has at room temperature an approximative solubility of 0,08 % PROTECTIVE EFFECT OF TOCOPHEROLS ON PROVITAMIN A 517 enriched margarin and butter during lW-irradiation Peroxide va ue mmo./ ~IY m : lo .. content I-'.~".v'"_" o ... content 0 f i\. • ~ "'Vltamm Origi. Irradiation time .: Origi. After hours of irradiation Origi. ,After hours of irradiation nally 1 3 5 nally 1 3 5 nally 1 3 5 0.40 1.S0 17.00 31.50 100.00 40.00 20.00 - 100.00 40.00 12.00 ----I-1- -----O.SO 7.00 14.00 45.00 30.00 8.00 70.00 53.00 24.00 0.40 0.50 1.90 3.10 100.00 50.00 37.00 10.00 100.00 70.00 61.00 32.00 1.00 10.00 22.00 38.00 25.00 5.00 60.00 45.00 10.00 ---LOO 10.00 20.00 40.00 25.00 3.00 60.00 32.00 12.00 0.40 0.85 5.00 10.50 100.00 46.00 30.00 2.00 100.00 64.00 48.00 20.00 1.10 1S.00 28.60 32.00 22.00 -I 52.00 25.00 5.00 -'- ,-0.60 5.00 12.00 60.00 35.00 10.00 62.00 45.00 18.00 0.40 0.50 1.60 3.00 100.00 65.00 40.00 14.00 100.00 66.00 50.00 25.00 1.00 S.OO 19.00 40.00 30.00 8.00 55.00 32.00 S.OO -,-I- :- 1--- j-0.60 2.00 3.00 60.00 45.00 10.00 70.00 65.00 38.00 0.40 0.50 1.S0 2.80 100.00 75.00 65.00 10.00 100.00 72.00 68.00 40.00 0.40 1.50 2.40 80.00 65.00 15.00 80.00 70.00 44.00 i--i-O.SO ,t.OO 7.0 50.00 30.00 5.00 65.00 52.00 22.00 0.40 0.72 3.00 6.50 100.00 54.00 40.00 6.00 J 100.00 67.00 55.00 26.00 0.60 2.50 4.00 60.00 54.00 10.00 70.00 60.00 30.00 - ~-1-050 1.50 2.80 70.00 52.00 20.00 ! 68.00 57.00 30.00 0.40 0.50 1.30 2.50 100.00 70.00 55.00 25.00 100.00 69.00 60.00 35.00 0.40 1.10 1.90 75.00 60.00 30.00 75.00 66.00 4·0.00 i i in vegetable oils. The solution 'was kept in a brown polished glass-stoppered flask, in a refrigerator at : 2 QC protected from light. Before use, its contents in active ingredients was always determined. Margarine was coloured at +40°C, after careful melting and simultaneous introduction of nitrogen gas. Protected from light, the samples were frozen after being mixed with the colouring agent; cut into units required for the examinations and packed into aluminium foil lined with parchment, and kept at a temperature of +2 °C until used. Each of the enriched samples contained about 0.001 % of p-carotene. Enriched with this concentration of p-carotene, margarine had a pleasant colour similar to that of summer butter; and its biological value was significant. We have carried out preliminary examinations in order to find out the optimal concentration of tocopherols and synergents. Tocopherol concentrations 518 L. TELEGDY KOf'ATS and E, BERNDORFER KRASZNER 100 -control ~ 80 ..•••.••.• 20 mg % o-tocopherol _ .. _- 5 mg %o-tocopherol and 50 mg % citric ocid '-c: - -- 5 mg % ;r:: tocopherol and 50 mg % phosphoric acid '" ~ 60 " -,-,-,5 mg % iT-tocopherol and 50 mg % T-setenious acid \.J '" c: '" " 1;0 '-tl \.J ~ 20 3 5 Irradiation time (hours) Fig. 3. Effect of y-tocopherol on the p-carotene content of enriched margarin during UV irradiation 100 -.. 80 .,e. 0 --c: Cl> 60 c: " \.J '" c:~ '10 ~ tl \.J I ~ 20 . . , --- con/rol ":~~ '.\ 0,. •• -_., 5 mg % locopherol mixture and 50 mg % phosphoric acid .. , ....... 20 mg % locopherol mixture '. "-",~, --- 5 mg % tocopherol mixture and50 mg phosphoric acid '~'" "~ ~"c... . -, -' 5 mg % tocopherol mixture and 50 mg % seleniaus acid ", "~' ". ~0, ", '" '. "'" , . '\ ''t:>. ',". '. ,,'\ "-........ .>~~ ......... -b '" 3 5 Irradiation time (hours) Fig. 4. Effect of tocopherol mixture on' the /I-carotene content of enriched margarin during UY irradiation of 50, 20 and 10 mg% were used. In the presence of synergists a tocopherol concentration of 5 mg% was sufficient, The necessary quantities of tocopherol and of synergists dissolved in absolute alcohol were mixed with butter in a nitrogen current, then the sol-vent was carefully eliminated in vacuum at a temperature of 4,0 cC. The pre-pared butter fat (further: butter) samples were irradiated or treated according --. "g z.. c: .!': c: '" Cl '" c: E '" ~ PROTECTIVE EFFECT OF TOCOPHEROLS 0;, PROVITAJVIIS A 100 80 60 40 20 ---control \ - - - 10 mg % oC-tocopherol \". \, .. \ ....... . "--"50mg% • .. •• .. ··20mg% '0. -.....:.:.: .• ····0 .. ..... " '. ...... '''b.. ". .............. " .... ,'. '. " " .... 3 '\. " '. '\. '\.'0 '\. , '\ '0 '\ \ 5 Irradiation lime (hours) 519 Fig. 5. Variation of vitamin-A content of butter stabilized with a-tocopherol during C" irradiation 100 ~ 80 ~ ~ c: ., c: CO '" Cl "<: ·S E: 1;0 '" ~ 20 " 3 --- control -- ._- 10 mg % i'Ftocopherol .. __ .- 50mg% ........... 20mg % 5 Irradiation lime (hours) Fig. 6. Variation of vitamin-A content of butter stabilized with y-tocopherol during FY irradiation to the Active Oxygen Method. In both test substances the changes of (3-caro-tene, peroxide and vitamin A content were controlled. Table 1 shows the effect of tocopherols and synergists in margarine and butter enriched with (3-carotene during ultra-violet irradiation; Table 2. supplies informations under circumstances of AOM; Fig. 1 shows in the presence of different synergists in enriched marga-rine the changes of (3-carotene during ultra-violet irradiation; Figs 2-4 also 520 L. TELEGDY KovATS and E. BER,YDORFER KRASZt,'ER Table 2 Effect of tocopherols and acid synergents in enriched margarin and butter during. Tocopherols Conc. mg 100 g 50.00 Acid synergcnts Cl; 20.00 10.00 ---,-----50.00 Y 20.00 i 10.00 ' 50.00 mixture 20.00 y mixture 10.00 citric 'i.00 phosphoric selenious citric 5.00 phosphoric selenious citric 5.00 phosphoric seleuious AO:JI-treatment Conc. lUg 100 g of mar· gnrin hours 50.00 50.00 47.00 i 56.00 ! i 4·2.00 58.00 78.00 45.00 72.UO 88.00 50.00 80.00 90.00 62.00 Antioxidative index: in mar· in but .. garin ter 1.00 1.00 2.75 1.38 3.75 1.86 2.25 1.04 3.25 1 ~9 . ,-4.50 2.10 2.75 1.19 4 .. 75 1.90 6.00 2.15 3.50 1.47 0.' ,0 content of A·vitamin of butter origi .. after at the nally 15 end 0/ hours ofAmr ,0 100.00 26.50 5.00 100.00 54.00 15.UO 12.00 12.00 32.00 7.00 5.00 17.00 2.00 100.00 48.00 10.00 9.00 55.00 15.00 100.00 57.00 16.00 60.00 20.00 37.00 8.00 100.00! 39.00 9.50 42.00 11.00 50.00 10.00 100.00 52.00 12.00 55.00 16.00 show the changes of p-carotene content in enriched margarine during ultra-violet irradiation in the presence of x-tocopherol, y-tocopherol, tocopherol mixture and synergists; Figs 5-6 show the alteration of vitamin A in the presence of different concentrations of x- and y-tocopherol. Summing up the results: In margarine and butter enriched , .. ith p-carotene the tocopherol concentration of 20 mg% proved to be the' quanti-tative optimum for the prolongation of the induction period under circumstan-ces of both ultra-violet irradiation and AOlVI, irrespective of the sort of toco-pherol, while the qualitative optimum was achieved with a natural tocopherol mixture. During ultra-violet irradiation, synthetic tocopherol also displayed advantageous properties. PROTECTIVE EFFECT OF TOCOPHEROLS OiY PROVITAMI1V A 521 As a synergist (with 5 mg% of tocopherol mixture), selenious acid had the best effect. During ultra-violet 'irradiation, the tocopherol mixture and a combina-tion of tocopherol mixture selenlous acid still preserved 30% of the original !'i-carotene content as in the fifth hour of experiments. If AOM was applied, !'i-carotene content was decomposed in spite of the same protection. During irradiation 1n case of butter treated with AOM, the most effi-cient protection of vitamin A was exerted by x-tocopherol and, in the pre-sence of synergists, by a combination of x-tocopherol and selenious acid; at the end of 5 hours of irradiation, 48% of initial vitamin A contents remained. This observation might explain the outstanding biological and physiological effects of x-tocopherol in vivo equally based on the protecting of vitamin A in the organism. Summary The most probable two explanations of the behaviour of tocopherols in reactions as derived from their chemical character are as follows: 1. As electron carriers tocopherols participate in the biological oxidation, in respiration and in other biological processes. 2. As intracellular antioxidants tocopherols protect unsaturated fats and similar oxy-gen-sensitive organic compounds, e.g. A-provitamines, A-vitamines, ascorbic acid, etc., against autooxidation. From the point of view of food science and industry the most important property of tocopherols is their antioxidative activity which protects lipids and fat foods. As is known, the production of margarine enriched in A-provitamines is of utmost practical importance, how-ever, this is practicable only if the added carotene remains unchanged during storage. Research work to find methods of protecting carotene in enriched margarines and A-vitamines in butter has been carried on. Synthetic and natural tocopherols have been used as stabilizers preventing oxidation in artificial ageing experiments. Tocopherols were found to lengthen the induction periods, protect the A-vitamins and particularly the provitamines, and their chemical derivates did not negatively affect the organoleptic properties of the food. References 1. TELEGDY-Kov.~TS, L.: Valogatott fejezetek az Clelmiszerkemiab6I. Vitaminok. (Selected chapters of food chemistry, Vitamins). Tankonyvkiad6, Budapest, 1962. 2. HARRIs, R. S.- WOOL. 1. G.-:JIARRIAN, G. F.-TIIIMANN, K. V.: Vitamins and Hormones. Academic Press, ::'Iew York. London. 20 (1962). 3. BAUERFEIND, J. C.-RoEos:.'iY, A. D.-SIEMERS, G. F.: Food Eng. 25, 81 (1953). -t. KOEH='i, C. J.: Arch. Biochem. 17, 337 (194.8). 5. BURl'<S,:JI. J.-HAUGE, S. :JI.-QUACKE:.'iBl.'SH, F. W.: Arch. Biochem. 30, 34] (1951). 6. Al'<GLI:.'i, C.-:JLmol'<, J. H.- CHAPMAN, R. A.: Dairy Science 38, 333 (1955). 7. SCHWARZ, K.-FoLTZ, G. M.: Am. Chem. Soc. 79, 3292 (1957). 8. BIER!, J. G.-Al'<DREWS, K. L.: J. Am. Oil Chem. Soc. 40, 365, 1963. 9. LEA, C. H.: Rancidity in Edible Fats. Chem. Pub., :New York, 1939. lO. BIEFER, K. W.: :Mitt. Geb. Lebcnsm. u. Hyg. 53, 243 (1962). 11. JACOBS, M. B.: The Chemical Analysis of Foods and Food Products. IIIrd edition. D. Van ~ostrand Comp., New York, London, 1958. 522 L. TELEGDY KorATS and E. BERSDORFER KRASZNER 12. IIIONIER-WILLIA~IS, G. W: Trace Elements in Food, Chapman and Hall, J;.ondon, 1949. 13. Co-operative of the Institute: :Methodological Exercises in Food Industry (Elelmiszeripari metodikai gyakorlat). Tankonyvkiad6, Budapest, 1965. 14. IIIoon, H.: 1I1itt. Geb. Lebensm. u. Hyg: 44-, 257 (1953). " 15. L,\.SZTITY. R.-l\:I. SZILAS-KELElIfEN-E. BERNDoRFER KRAszNER-F. ORSI-J. VARGA: Elelmezesi Ipar (Food Industry) 20, 163 (1966). 16. LC;CIDL-I.NN, F. X.-MELNICK, D.-lIhLLER, J. D.: J. Am. Oil Chern. Soc. 30, 602 (1963). Prof. Dr. L'iszl6 TELEGDY KOV.~TS dr. Eva BERNDORFER KRASZNER } Budapest, XI. Muegyctem rkp. 3. Hungary Printed in Hungary A kiadasert ifjel az Akademiai Kjad6 igazgntoja Muszaki szerkeszto: Farkas Scindor A kezirat nyomuaba ir!cezetl: 1%7. 1. 16. - Terjedelem: 16,75 (.-\./5) iv, 117 ab::a 67.63365 - Akat1emi~i NyomJa, Budapest - Felc16s vezet6: Bernat Gyorgy 

EXAMINATION OFTHE PROTECTIVE EFFECT OF TOCOPHEROLS ON PROVITAMIN A (β-CAROTENE) AND VITAMIN A IN ENRICHED MARGARINE AND BUTTER

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EXAMINATION OFTHE PROTECTIVE EFFECT OF TOCOPHEROLS ON PROVITAMIN A (β-CAROTENE) AND VITAMIN A IN ENRICHED MARGARINE AND BUTTER

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Periodica Polytechnica (Budapest University of Technology and Economics)