Vitamin C Content of Frozen Orange Juice E. M. NELSONAND H. H. MOTTERN Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C.
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ITRUS fruits, as a class, Orange juice that has been frozen quickly and head space of nitrogen, and (3) differ from most fruits extraction in air and f r e e z i n g permitted to l i p t f y at room temperature shows in that p r a c t i c a l l y all under head space of oxygen. no signiJicant loss of vitamin C. Under the their nutrients exist in the natural The first method c o m p a r e s conditions of this experiment there is no apfruit a s a l i q u i d . T h i s fact with ordinary methods of hanparent advantage f r o m the standpoint of vitamin makes it possible and practical to dling the juice in a container preserve and distribute the juice C preservation in excluding atmospheric oxygen which is not a i r - t i g h t . The of such fruit and collect the other second method compares with during the freezing process. Three preparations portions for the manufacture of commercial methods of hanof orange juice, frozen, respectively, in an atmosvaluable b y - p r o d u c t s . This dling the juice with precautions phere of air, nitrogen, and oxygen, and stored p r a c t i c e , which i s g r o w i n g to exclude a t m o s p h e r i c oxywith the same gas in hermetically sealed conrapidly, has many advantages gen. I n the third method any from the standpoint of economy. changes due to atmospheric oxytainers ut 0" F. (17.8' C.) for a period of 10 The difficulties which have been gen should be increased. m o n t h , all show approximately the same vitamin encountered have been largely C potency as fresh orange juice. in the development of methTESTSON GUINEAPIGS Data concerning the use of 2,6-dichloroods in the preservation of the phenolindophenol as a n index of vitamin C juice-that is, methods which The frozen juice was prewill s a t i s f a c t o r i l y preserve potency are presented and discussed. pared from Florida v a l e n c i a the n a t u r a l f l a v o r a n d full o r a n g e s late i n t h e s e a s o n nutritive value. Particular atand gave the following results tention during the last two years has been given t o the ap- on analysis: plication of quick-freezing methods to orange juice. Ex- Brix 12.4 Brix-acidity ratio 13.3 tensive distribution of frozen orange juice in retail packages Titratable acidity a8 citric acid, % 0.93 pH 3.7 about a year and a half ago called attention to the necessity Ampules of about 50 cc. capacity were prepared from of obtaining fundamental information with respect to the Pyrex test tubes. About 30 cc. of juice were placed in each stability of vitamin C in such juice. Many recent publications, particularly those of Tillmans ampule and the neck sealed in the flame. I n those cases (6) and his co-workers, have provided evidence of the re- where nitrogen or oxygen was used, the appropriate gas ducing property of vitamin C and emphasize the possibility was introduced into the ampules by placing the empty amof determining this vitamin by means of a suitable oxidizing pules in a vacuum desiccator, exhausting, and releasing with reagent. This subject is discussed further by Mottern, the desired gas. The juice that was put into the nitrogenNelson, and Walker (1). 2,6-Dichlorophenolindophenolhas filled ampules had been extracted in an atmosphere of nitroproved to be a promising reagent, having a distinct advantage gen and kept under that gas. Upon filling the ampules, over other commonly used oxidizing agents. This reagent part of the gas was displaced by the juice and the remainder has been used previously in this-bureau for following the oxi- was contained in the head space. The juice was frozen with dative changes that take place in the deterioration of orange solid carbon dioxide within 30 minutes from the time extracjuice (unpublished data). It was found that, when orange tion was begun. About 10 minutes were required for freezjuice was allowed to deteriorate while exposed to air in a ing, after which time the ampules were stored a t 0" F. (- 17.8' flask with a cotton plug, the diminution in reducing ralue (2.). Vitamin C assays were begun a few days after the juice was accompanied by the development of a dark brown color. When air was excluded, the development of the dark color was frozen, and a portion of the frozen juice was retained was delayed, but a direct relationship between change in for a later test to determine the effect of storage. The reducing value and development of color could be established. first vitamin C test was begun in May, 1931, and the second To obtain further information in regard to the oxidative test on the stored material in March, 1932. Both tests changes taking place in frozen orange juice and also to corre- were conducted in exactly the same manner, except that late vitamin C content with reducing value as determined ten animals were used in each group in the first test and by the use of this dye, titrations were carried out on frozen eight animals in each group in the second test. Guinea pigs weighing from 250 to 325 grams were given the followjuice which had been stored under different conditions. The purposes of these biological and chemical investiga- ing basal ration: Parts Parte tions with respect to frozen orange juice were to determine 69 Casein 5 Rolled oata (1) whether vitamin C is damaged during the preparation Autoclaved 25 Sodium chloride 1 alfalfa and freezing process, (2) whether any change in vitamin C content takes place during storage of the frozen juice, One and a half cubic centimeters of each of the frozen and (3) whether atmospheric oxygen influences the sta- orange juice preparations were fed daily to each of the animals bility of vitamin C or oxidative changes in the juice during in three groups. The frozen juice was permitted to liquefy a t room temperature before feeding. One group of animals freezing and storage. Frozen orange juice was prepared by three methods: (1) received the same quantity of fresh juice from oranges which extraction in air and freezing under head space of air, (2) had been reserved from the fruit used in the preparation of extraction in atmosphere of nitrogen and freezing under juice for freezing. These oranges were stored a t 40' F. 216
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INDUSTRIAL AND ENGINEERING CHEMISTRY
(4.4' C.). Another group received no vitamin C. The average survival of the negative control animals in each test was the same, 26 days. The first symptoms of scurvy in the negative control groups were evident before the end of 2 weeks and the animals showed severe scurvy on postmortem examination. Failure of the negative control group of the first test to increase in weight a t the beginning of the experiment is not due to a deficiency of vitamin C . All the animals had been fed a ration rich in vitamin C four or five days before the experiment was begun. The negative control group survived the same length of time in both tests. The broken curves indicate that one animal in each group had died before the end of the fifth week. There was no significant difference in growth rate of the groups fed orange juice, irrespective of whether the fresh juice was fed or juice that had been frozen with exposure to air, oxygen, or nitrogen. Neither was there a significant difference in growth rate between the first tests conducted just after the juice had been frozen and the second test after the frozen juice had been stored for 10 months. At the end of 4 weeks in the first test the average gains of the animals in the groups fed orange juice ranged from 50 to 76 grams. I n the second test the corresponding weights were from 58 to 77 grams. At the end of the fifth week of the first test the average gains ranged from 70 to 97 grams, and in the second test the corresponding weights were 62 to 94 grams. After the fifth week, variations in weight in both tests were great, and numerous deaths occurred. The average weight changes of the groups are shown in Figure 1. Only twenty-one of the forty animals fed orange juice in the first test and 20 of the 32 animals in the second test survived the 8-week test period. All the animals which survived the test showed some evidence of scurvy upon post-mortem examination and, with a few exceptions, the animals which died showed extensive scorbutic lesions. 2,6DICHLOROPHENOLINDOPHENOL AS INDEX OF
VITAMINC POTENCY An approximately 0.01 N solution of 2,6-dichlorophenolindophenol was prepared. Portions (10 cc.) of the juice that had been frozen and stored for about a year were titrated with the dye after the addition of a sufficient quantity of sodium acetate to neutralize the solution. The average value of the titrations of six groups of juice frozen, respectively, with a head space of nitrogen, oxygen, and air, were 7.1, 6.7, and 6.6 cc. With one exception the titrations of the juices given the same treatment did not vary more than 0.1 cc. from the average. Until further evidence is available as to the effect other substances that are present in orange juice may have in reducing the dye used, the above titrations cannot be interpreted accurately. The following experiments were conducted to obtain further information with respect to the relationship between reducing value with 2,6-dichlorophenolindophenoland antiscorbutic properties: The reducing substance from orange juice was prepared in concentrated form according to the following procedure: Orange juice was fermented with yeast a t 30' F. (-1.1' C.) for 24 t o 48 hours. The juice was then just neutralized by the addition of a hot saturated solution of barium hydroxide. The barium hydroxide was filtered off and the filtrate concentrated to about one-tenth of its original volume under reduced pressure. Upon standing overnight, further precipitation of barium citrate took place; this was also removed by filtration. Copper-free lead acetate was added to produce a concentration of about 2 per cent. The solution was made strongly alkaline to bromothymol blue test paper by the addition of dilute ammonium hydroxide. The yellow precipitate was filtered off, washed with water, and extracted with 10 per cent acetic acid. The
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acetic acid extract was separated from the insoluble residue by filtration. This filtrate was made alkaline to bromothymol blue by the addition of ammonium hydroxide. The resulting precipitate after filtration and washing was decomposed with hydrogen sulfide. After filtering off the lead sulfide, the excess of hydrogen sulfide was driven off by distillation under reduced pressure. The concentrated reducing substance -was diluted with water so that 0.5 cc. had a reducing value equivalent to 2 cc. of orange juice as determined by titration with 2,6-dichlorophenolindophenol. The concentrates were prepared as needed a t intervals of about 2 weeks and stored under nitrogen a t 40' F. (4.4"C.), There was no loss in reducing value during storage.
FIGURE1. AVERAGE WEIGHTCHANGES
It has been demonstrated (4, 6 ) that hexuronic acid, which has a high reduction potential, possesses antiscorbutic properties. Glucic acid which had been prepared and described by Nelson and Browne ( 2 ) has a reduction potential similar to that of hexuronic acid, and, as it is also a carbohydrate derivative, experiments were made to determine whether it may possess antiscorbutic properties. Glucic acid was dissolved in water, so that 0.5 cc. contained 2 mg. of the acid. A 0.5-cc. portion of this solution reduced 4.75 cc. of 0.01 N dichlorophenolindophenol, and this quantity was equivalent in reducing value to about 7 cc. of orange juice. Twentyfour guinea pigs, ranging in weight from 240 to 312 grams, were divided into four equal groups. They were fed the scorbutic ration given above, and one group received an additional 2 cc. of orange juice daily. The second group received 0.5 cc. of the diluted orange juice concentrate. The third group received 0.5 cc. of the glucic acid solution, and one group received no vitamin C. On the tenth day of the experiment four animals in the glucic acid group and five animals in the negative control group showed signs of scurvy, and on the eleventh day all the animals in both groups showed swelling and soreness of wrists or stiffness of the hind legs. The glucic acid group survived an average of 23 days, and the negative control group 21 days. Post-mortem examinations showed that all the animals died with severe scurvy. The orange juice and orange juice concentrate groups were discontinued a t the end of 5 weeks, owing to the exhaustion of the available supply of concentrate. Both groups grew a t approximately the same rate and were in excellent condition throughout the experiment. The orange juice group made an average gain of 87 grams in 5 weeks, and the concentrate group 83 grams. There was no macroscopic evidence of scurvy upon. post-mortem examination of the animals.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
DISCUSSION OF RESULTS It is apparent from the biological experiments with frozen orange juice that this juice can be frozen quickly and permitted to liquefy at room temperature with no appreciable loss of vitamin C, and under the conditions of this experiment there was no discernible advantage from the standpoint of vitamin C preservation in excluding atmospheric oxygen during the freezing process. Even after storage for 10 months the juice retained its vitamin C potency, irrespective of whether it was preserved in an atmosphere of oxygen or nitrogen. These facts showed that vitamin C in orange juice is quite resistant to oxidation a t low temperatures and in an acid medium. The variation in vitamin C content of oranges is worthy of further investigation. I n the present studies 1.5 cc. of fresh orange juice did not afford the same measure of protection from scurvy as in previous studies by Nelson and Mottern (3) on normal fruit. The difference in response cannot be attributed to experimental procedure in i o far as the animals, the basal diet, or laboratory technic are concerned. Titrations with 2,6-dichlorophenolindophenol show that there is a measurable difference in the reducing value of frozen orange juice that has been preserved in an atmosphere of nitrogen and air or oxygen. Assuming that this difference not be is due to a per cent loss in vitamin c' it detected by biological experiments. The reducing value as determined by 2,6-dichlorophenol-
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indophenol is not necessarily an accurate measure of antiscorbutic potency. Szent-Gyorgyi @), Zilva (7, 8 ) ) and Svirbeley and Szent-Gyorgyi (4) have published several papers dealing with this point. There may be many substances present in plant material which have some similar reducing properties. While glucic acid and hexuronic acid have similar reducing properties, the former has none of the physiological properties that have recently been attributed to the latter. However, in attempting to eliminate undesirable effects on vitamin C in commercial processes used for the preservation of such products as orange juice, the colorimetric titration may be a very useful guide.
ACKNOWLEDGMENT The glucic acid used was kindly provided by E. K. Nelson.
LITERATURE CITED Mottern, H. H., Nelson, E. M., and Walker, Reed, J . Assoc. OficiaZ Agr. Chem., 15, 614 (1932). (2) Nelson, E. K., and Browne, C. A., J . Am. Chem. SOC.,51, 830 (1929). (3) Nelson, E. M.,and Mottern, H. H., Am. J . Pub. Health, 22, 587 (1932). (4) Svirbeley, J. L., and Saent-Gyorgyi, A., Nature, 129, 690 (1932). ( 5 ) Szent-Gyorgyi, A., Ibid., 129, 943 (1932). (6) Tillmans, J., 2. Untersuch. Lebensm., 64, 11 (1932). Zilva, S. S., Nature, 129, 690 (1932). Zilva, S. S.,Ibid., 129, 943 (1932).
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R E C ~ V EAugust D 10, 1932. Preaented before the Division of hgricultursl snd Food Chemistry at the 84th Meeting of the American Chemical Society, Denver, COIO., August 22 to 26, 1932.
Vitamin C Content of Frozen Orange and Grapefruit Juices LILLIANW. CONNAND ARNOLDH. JOHNSON Research Laboratories of National Dairy Products Corporation, Inc., Baltimore, Md.
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URING the last few years there has been a large increase in the quantity of fruit and fruit juices which have been preserved by freezing. Of special importance in connection with the freezing of such products and t,heir subsequent storage in the frozen condition is the effect on the vitamin content. Frozen orange juice has been marketed for several seasons, but little has been reported concerning its vitamin C content. More recently grapefruit juice has also been marketed in the frozen condition. There are considerable data in the literature on the effects on the vitamin C content of storing oranges, lemons, and other fruits and vegetables a t temperatures slightly above the freezing point. Thus Davy (2) and Delf (3) showed that, for oranges and lemons stored at 2.5" to 5.4" C., the antiscorbutic value was retained unimpaired as long as the fruit remained in good condition. Delf also prepared frozen orange and lemon juice, these juices being stored a t -11" to -14' C. No measurable deterioration in vitamin C content occurred after storage of 17 months in the frozen condition, but after storage for 5 years the antiscorbutic value had decreased about 50 per cent. On the other hand, Koch and Koch (6) found that frozen orange juice lost practically its total antiscorbutic value during storage for 3 months.
MANUFACTURE OE FROZEN JUICE The washed and graded oranges or grapefruit., previously cooled to a temperature of 4 ' to 5" C.; were peeled by a mechanical peeling machine, after which the juice was ex-
pressed by a screw type press, the juice being filtered through a sieve in the bottom of the press and the pulp expelled a t the end. The fruit presses as well as the other equipment with which the juice came in contact were all made of 18-8 chromenickel steel, which metal was found to be least corroded by orange juice and to have the least effect on flavor and keeping quality. The juice immediately after expressing usually contained so much pulp that filtration was necessary in order to remove it. After filtration, the juice was drawn into a vertical direct-expansion ice cream freezer, operated under reduced pressure, where it was frozen, to a slushy consistency. The slushed juice was then discharged into an evacuated hopper connected with the filling machine. As a precautionary measure to prevent the juice from dissolving oxygen as far as possible, nitrogen was introduced to relieve the vacuum and to facilitate discharging the juice into the filling machine and into the containers from the tilling machine. While this measure undoubtedly did not result in completely preventing air from getting into the juice, the manufacturer felt that juice prepared in this way had a better chance of maintaining its quality and vitamin potency during storage than juice not so processed with nitrogen. The slushed juice was finally packed in paraffied cardboard containers which when sealed were conveyed to the sharp room maintained a t a temperature of -24' C. A blast of cold air was directed on the containers until the juice was completely frozen, after which it was stored a t a temperature of -12'to -15°C.
