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G. K. MURTHY Milk and Food Research, National Center for Urban and Industrial Health U. S. Public Health Service, Cincinnati, Ohio Abstract
Ultraeentrifugation and dialysis studies were made to determine the distribution of calcium, phosphorus, and nitrogen in milk treated with ion exchange resins to remove radionuclides. In System I, milk was passed through Dowex 2-X8 ( C l : P : C i t ) resin, acidified to p H 5.35 with citric acid, passed through IR-120 ( C a : M g : K : N a ) resin, and neutralized to p H 6.8 with K O H . In System I I , the same cycle was followed, except that HC1 was used to acidify milk and the milk was neutralized with IRA-68 (P :Cit :OH) weak base anionic resin. A comparison of the test results for treated samples and those for the control sample showed increased concentrations in the casein nitrogen and in acid soluble phosphorus in serum, and a decreased amount of sedimented casein. The amount of calcium and acid soluble phosphorus associated with the sedimented caseinates also decreased. The extent of these alterations depended upon the type of acid used to acidify the milk. The effect of these changes in the ionic balance on the coagulation of milk by rennin and ethanol is briefly discussed. To remove cationic radionuclides (~°Sr, "°Ba, and I~Cs) from milk, it is acidified to p t I 5.3 to 5.4 with eitric or hydrochloric acid and passed through a column containing Dowex ~ 50W-X8 or IR-120 cationic resin in the C a : M g : K : N a cycle. The milk is neutralized to p H 6.6 to 6.7 with K O H (4, 11), Dowex l-X8 ( C I : O H ) , or Dowex 2-X8 ( C I : P : C i t : O H ) anionic resin, depending upon the acidifying agent (8, 9). To remove ~ I , no pretreatment o f milk is necessary, but the milk is passed through a Dowex-2X8 ( C l : P : C i t ) resin column (10). Previous investigations indicated that the best possible integrated system for the removal of all radionuclides was that in which the anionic resin column preceded the cationic resin column. In this integrated system, the milk is Received for publication November 25, 1966. Mention of commercial products does not imply endorsement by the Public Health Service.
passed through a Dowex-2X8 ( C l : P : C i t ) column, acidified to p H 5.3 with 0.75 ~ citric acid, passed through an IR-120 ( C a : M g : K : N a ) cationic resin, and neutralized to p H 6.6 to 6.8 with K O H (5, 6). I f HC1 is used to acidify the milk, alternately IRA-68 (P :Cit :OH) weak base anion exchange resin may be used to neutralize the milk (7). Because of the various treatments applied to the milk, the nature of ionic equilibria may be drastically altered. Since ionic equilibria is one of the important factors that influence the stability of milk proteins, the investigation reported here was undertaken to determine the nitrogen, calcium, and phosphorus distribution between phases of the milk system.
Materials. Milk samples were obtained on the Cincinnati market. The cation resin used was Amberlite IR-120 (H÷), and the anion resins were Dowex 2-X8 (CI-) and Amberlite IRA-68 I~A-68 (Cl). Resin regeneration. Methods for the regeneration of IR-120 and Dowex 2-X8 resins and operation of the columns have been described previously (10, 11). The IRA-68 (C1-) resin was converted into the P :Cit :OH cycle as follows: A charging solution was prepared to contain 5 m ~ of citrate and 100 m ~ of phosphate (Natt2PO,'H20) per liter of solution and the p H of solution was adjusted to 9.0 with 6 ~ NaOH prior to its being made to volume with deionized water. Approximately 100 ml of resin was regenerated with 2,500 ml of solution (8). Milk treatment. Two systems were considered. I n System I, milk was passed through Dowex 2-X8 ( C l : P : C i t ) resin at 0.5 resin bed volume (rbv) per minute, acidified to p H 5.35 with 0.5 ~¢ citric acid, passed through IR-120 ( C a : M g : K : N a ) resin at 0.]25 rbv, and neutralized to p H 6.8 with 1.0 ~[ KOH. In System I I , the same cycle was followed, except that 1.0 ~ HC1 was used to acidify the milk and the milk was neutralized to p H 6.8 with I R A 68 ( P : C i t : O H ) resin by the batch technique (8, 9). Centrifugation. Samples of whole milk were centrifuged at 1,800 rpm for 10 min and al-
TP. and the residual serum and lipid materials were removed by rinsing with deionized water. Methods for determination of p i t and A S P are the same as previously described (10).7+__0. Values for CN were obtained by subtracting values for NCN from those for TN. 6 . When milk is acidified with ttCl.0 mg to 99.0 ---130 ___13 11. free of lipid material.0--+ 3. consequently.8 10.8 rag. When milk is acidified with citric acid. but was dispersed in deionized water with a minimum of NaOH. treated milk showed no significant differences in the TN.7-+ 1.3___3.0+.5 20. The Ca content of samples was determined by the oxalate-permanganate titration method (14).6-+ 1. therefore. were removed with a hypodermic syringe.0-+ 4.0+. 40 rotor in a Spinco Model L preparative ultracentrifuge. Rennet coagulation.2. Dialysis. Values for OP were obtained by subtracting values for A S P from those for TP. Alcohol coagulation.3.3 " Average of seven samples. and sedimented easeinate"--System I Control Analyses Milk Serum Caseinate Milk Resin-treated Serum Caseinate (mg/lO0 ml of milk) Total nitrogen (TN) 559 ___12 b Noncasein nitrogen (NCN) 121 -+11 Casein nitrogen (CN) 438 +-19 Total phosphorus (TP) 92.2 and 36.0-+ 1. and care was taken not to disturb the sedimented syrupy layer above the easeinate fraction. and A S P contents.0 27. Data on nitrogen.4. NO.1 ~[ N a O t t and using five drops of phenolphthalein as indicator.0+. total phosphorus ( T P ) . The skimmilk was removed by siphoning and centrifuged at 4 C for 120 rain and at 105.0 116 -----13 432 __+24 -313 --+24 77.4+.0 Acid-soluble phosphorus (ASP) 62. The TP on an aliquot of the ash solution (1) was men- sured by developing the color as of the ASP.500 g with a no. calcium.9-+ 2. The syrupy layer was carefully removed.0 Total calcium (Ca) 119 +. The caseinate fraction was not washed. Tests were made accordLug to Davies and White (3).0-+ 4.8. NCN. b Ranges in the above values represent standard deviations among milk samples. The nitrogen distribution in milk and its fractions was determined according to Shahani and Sommer (15). 50. When the protein-bound salts are ionized. the Ca ionized from the protein-bound form is complexed with the citric acid and is stabilized as the p H is increased to 6. The supernatants. Results and Discussion Centrifugation results. including the control and resin-treated skimmilk.4-+ 1.8+. The CN in serum increased from 60 to 430 mg (I) and 56 to 90 mg ( I I ) .7+. and total calcium (Ca). possibly exists in a complex with the alkali metals.3 23. except that in System I I the Ca and A S P decreased from 121 and 62. The centrifuge tubes were held inverted. respectively.9 35.6 549 -+-15 119 +--11 430 +-19 92.0-+ 3.0 220.127.116.11. the Ca can bind with the casein and become stabilized upon neutralization to p H 6.0.8 73. TABLE 1 Composition of skimmilk.0 31. All fractions. Analytical methods. Tests were performed as described previously (11). the caseitt in sermn. the Ca ionized from the protein-bound form does not form complexes with the chlorides. When compared with the control sample. and phosphorus distribution are presented in Tables 1 and 2. These decreases in the Ca and A S P resulted from neutralization of acidified milk with the weak base IRA-68 ( P : C i t : O H ) anionic resin (7).0.0+. were analyzed for total nitrogen (TN).1.5 6. J. Sixty milliliters of deionized water were dialyzed against three liters o£ milk at 4 C for 24 hr (2). The observed results were corrected for dilution caused by the addition of acid or alkali.3 53. noneasein nitrogen (NCN). ]>AIRY SCIENCE YOL. Titratable acidity in milk was measured by titrating 10-ml portions against 0.4 181 -+12 -60 --+18 45.6 5.1+. results were expressed as per cent of lactic acid. serum.1. or both. acid-soluble phosphorus ( A S P ) .6 16.3+. in which Ca is adsorbed as a citrate complex (8).810 ~URTHY lowed to stand at ice-bath temperatures for 30 rain to harden the cream layer.0 Organic phosphorus (OP) 31.9 ---390 +-18 48.
but it is believed that the chemical composition would differ similarly. however.8 and J. I n System I I .8 and 34.0 rag. NO. Dialysis studies.8 mg ( I I ) . one may expect the caseinates in treated milk to react differently to certain processing conditions than the caseinates in untreated milk• The TP and A S P in serum increased from 45•7 to 77•6 mg and 35•4 to 53.049 -+. these increases were small. and physical restriction• The resulting calciumeaseinate-phosphate complex will never be the same. Computation of various ratios for the sedinmnted casein and comparison of results with those for the control sample will show that under the experimental conditions of this study less Ca and A S P are associated with the sedinlented casein. 6 . 5 0 .6 to 15.051 ± 0.4. The significance of this fraction can be elucidated by further study• F o r System I I .9 mg and 33. Therefore.~ ~9 • ~ c9 the ability of these to recombine with the casein micelles may depend upon the type of acid added to the milk.001.0 to 47. analysis of this fraction showed different composition than that of the sedimented casein• No analysis of this fraction was made for System I.8 to 16•0 mg ( I ) and 79•1 to 52.9% (I) and 9. Consequently.0 to 90.047 0. respectively ( I ) . Therefore. The CN in the sediment decreased from 390 to 130 mg (1) and from 407 to 318 mg (II). These large increases resulted from the association of OP and A S P with the increased CN content.7 rag. however. results for sedimented casein from treated samples showed decreases of A S P from 27. and decreases of Ca from 73. dialysis of milk treated with anionic resin was discontinued in subsequent trials• Data in Table 3 show that in treated milk the dialyzable Ca and A S P increased by 38. milk samples that had been treated with anionic and cationic resins were dialyzed as described under Experimental Procedure.0 mg ( I ) and 27.o. D A I R Y S C I E I ~ C E "~OL. respectively• This was to be expected since the increase in CN was only from 56. as that originally present in milk.EXCHANGE RESINS AND MILK COMPOSITION 811 ÷l÷l÷l ÷14141 • ° • o ~.0.6 to 36•8 rag. To confirm some of the results observed with cent~Jfugation. increases in TP and A S P were from 43.4 to 5. o o o w. 0.4 mg ( I I ) . the attainment of equilibrium between the dissolved and the complexed forms.003 ( I I ) for the treated samples were comparable to the value for the control sample. except that O P / C N values of 0. N N~ 4dd ¢q 41 4141 2 4. The ultracentrifugate fraction of sedimented syrup increased in the processing treatment. Preliminary data indicated that passage of milk through Dowex2X8 (CI:P :Cit) resin did not significantly affect the p H or the distribution of Ca and A S P of milk or the dialysate.004 (I) and 0.
. the a m o u n t o f 1 • CaC12 r e q u i r e d to p r o d u c e eoagulum c o m p a r a b l e to t h a t in control milk was determined.. Table 4 shows t h a t the t r e a t e d milk ( I ) neutralized to p H 6..6+-2.2+-2.0 402 356 385 166 144 119 162 202 141 202 269 189 327 360 319 395 435 385 323 280 367 ..8O 6..80 6.0% ( I I ) .80 6..8 40. 6.. time pl=[ (% Lavtic) 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 l 2 3 Control Control Control Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Control Control Control None None None Citric Citric Citric Citric Citric Citric Citric Citric Citric Citric Citric Citric None None None tIC1 tIC1 tIC1 None None None KOIt KOtt KOH KOH KOIt KOH KOH KO]:[ KOIt KOH KOtI KOH None None None IRA-68 ( P : C i t :OH) [R.2+-2...A-68 (P :Cit :OH) IRA-68 ( P : C i t :OH) 6.80 6.60 6.20 0. .0 ~¢ per liter) (see) 0.16 0...6 with less CaCI~ a d d e d indicates t h a t in spite o f the excess citrate p r e s e n t .0 0.0.80 6...... .7+.0 7..4 to 6. A f t e r coagulation.3___10..60 6. .00 6. 6.0 0.4± 3.4 to 7....5 3. Coagulatio~ of milk by rennin..8 Ana/yses Control~ 118 +-6 Resin-treateda System I Resin-treatedb System I][ 95..02 7.16 0...16 0.40 6. . b Average of two samples.50 .14 0.0 7.0 with K O H u p o n coagulation o f milk.0 O..16 0..80 6.14 0.0 6.....80 6. an increased a m o u n t of CaC12 was required to yield coagulum similar to that obtained with the control.5 55..9 37.0 is stable to coagulation with r e n n i n and alcohol.15 0.00 7. t h e r e f o r e .16 0.70 .23 O..90 6...7 63.20 .... Analysis o f seven milk samples at 30 C showed TABLE 4 Effect of radionuclide removal process on titratable acidity and coagulation of milk by ethanol and rennin Sample Acid treatment Neutralizing agent pl=[ Titratable acidity Ethanol conc Rennin coagulation CaCh Coag.0 5..40 .26 0.23 0.8 to 7.16 0...0 4.0 67..89 6..0 6.0 5. As p H increased or acidity decreased.62 6...5+-1.6 83..: 50. The increased Ca a n d A S P in the serum o f treated milk a p p e a r to exist in the soluble i n o r g a n i c f o r m (13).4 to 6.0 0.812 MURTYIY TABLE 3 Calcium and phosphorus content of milk and dialysate Sample Calcium Milk Acid-soluble phosphorus Calcium Dialysate Acid-soluble phosphorus Average of five samples. The coagulation time was p r o l o n g e d logarithmically with the a m o u n t o f calcium added to the milk (12).8 to 7.0 9..0 4.. These d a t a agree well with those obtained by c e n t r i f u g a t i o n . the coagulum was allowed to s t a n d f o r 1 h r b e f o r e the n a t u r e o f curd a n d whey was determined. . the calcium-citrate complex is less stable a t p H 6. DAIRY SCIENCE VOL.. .15 .80 7..14 (%) 85 85 82 56 70 54 90 90 80 None None None None None None .14 0. (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 1 Treated ( I I ) 2 Treated ( I I ) 3 Treated ( I I ) J.7+-3..8+--1.5 (mg/lO0 ml of mille) 114 +-__10 10. (ml 1.. 6. 6 ... .0 59.80 0.......14 0.0 0.13 0...7___0.82 6....20 0.6 t h a n a t 6... I n the coagulation tests.0 3. respectively..0 4.0 9.39 6.. .0 2.38 6.26 0. 6. 6.. ..0 8. .0 42.6. P r e l i m i n a r y e x p e r i m e n t s were made to determine the effect of neutralizing milk to p H o f 6. some free calcium is present. 35. Coagulation with renn i n at p H 6. NO.19 ()..16 0.
Gilchrist. F. NO. Complexing of Calcium by Hexametaphosphate. S. A. Dairy Sci. 567. J. Overman. Producer's Creamery Co. Campbell for his interest in the problem and to L. S. the p H d r o p with the control s a m p l e was a b o u t 0.. Ohio. A. and Campbell. B. Dairy Sci. Official Methods of Analysis. Dairy Sci. E. and White. 1964. G. Masurovsky. J. K. R. and Edmundson. 1 mM of Ca m u s t be added f o r every 2 mM excess citric acid p r e s e n t in milk.75 to 1. C. Unpublished studies. J. M. p. 45: 1006. 1958. J. (2) Davies.. I f milk is to be used f o r m a k i n g cheeses or o t h e r milk products. Sta. The Relation Between the Chemical Composition of Milk and the Stability of the Caseinate Complex.. J. 1955. Linlited results were o b t a i n e d f r o m System I I . 131. and EDTA.0 g p e r liter. 1966. E. Neutralization of Acidified Milk with Weak Base Anion Exchange Resin and Composition of Milk. G. A Continuous Ion Exchange Process for Removing Radionuclides from Milk. J. W h e n r e n n i n was added to milk. F. U.. and E D T A in Milk. H. Effects of Complexing Agents on Turbidity and Rennet Coagulation. Illinois Agr. Oxalate. The Use of Ultrafiltration and Dialysis in Isolating the Aqueous Phase of Milk and in Determining the Partition of Milk Con(14) (15) stituents Between the Aqueous and Disperse Phases. calcium. on a m o l a r basis. E.2. 25:256. Edmundson. and Campbell. 1965. K.. J. 1951. 27: 171. Assoc.4 unit. I. O. T. Citrate. Removal of Iodine TM from Milk.5 ml of 1 ~I CaCl~ was r e q u i r e d to o b t a i n s a t i s f a c t o r y coagulum. Murthy. the coagulum was slushy. the caseinates are stable to coa g u l a t i o n with r e n n i n a n d alcohol ( I ) . Shahani.85 to 4. or both. Kaylor for his technical help. whereas. because of acidic reaction o f CaCI~.8 ___ 0. J. 1960. 48:1429.6 8 (P:Cit:0H) resin ( 7 ) . G. Heinemann. This is a t t r i b u t e d to depletion of calcium ( t 5 to 2 0 % ) in milk d u r i n g n e u t r a l i z a t i o n w i t h I R A .. Davies. Dairy Research. a n d the whey was highly turbid. DAIRY SCIENCE VOL. Dairy Sci. E. Dialysis of Milk Containing Complexing Agents. as c o m p a r e d to 1. respectively. Ion Exchange Processes for Removing Radioactive Contaminatio~l from Milk. The Protein and Nonprotein Nitrogen Fractions in Milk. and Craine. Campbell. K. Ohio. Dairy Sci. T. U. Unpublished studies.. Dairy Sci. a n d p h o s p h o r u s d i s t r i b u t i o n between casein micelle a n d the s e r u m p h a s e s is drastically altered d u r i n g p r o c e s s i n g of milk to remove radionuclides.85 g p e r liter f o r u n t r e a t e d m i l k . 1962. 48: 19. J. 6 . A d d i t i o n of 2. E. 47 : 1306. J. Method for Removing Iodine TM from Milk. Dairy Research.. the coa g u l a t i o n time was prolonged. Lebanon. Coagulation by Ethanol. 1966. Murthy.. Kiers. Public Health Service. J. Unpublished studies. The above i n v e s t i g a t i o n s revealed t h a t the nitrogen. M. B. Washington D. H. T. Murthy. S. L. Dairy Sci. Murthy. J. G..0 ml of 1 ~ CaCl~ p e r liter of milk was needed to yield a c o a g u l u m comp a r a b l e to t h a t of the control sample. 1964. Composition of Herd Milk of Brown Swiss Breed. II. T. J. The citric acid c o n t e n t of t r e a t e d milk is 3. 47: 1201. J. G. 47: 1188.. and Sommer. K. K. and Nickerson.... 1964. 1966. D. therefore. D. Complexing of Calcium by Hexametaphosphate. Method for Removing Cationic Radionuclides from Milk. 8th ed. S p o t checks on the p H of the coagulum indicated a d r o p of 0. and Nickerson.. Murthy. Bull. C. its composition o r the processing conditions.3 to 0. Offic.1 to 0. Because of these alterations. Agr. L. S... and White. C.. J. Public Health Service. Cincinnati.EXCHANGE RESINS AND MILK COMPOSITION t h a t 5. 34: 1003.. ~]3 (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) Acknowledgment The author is grateful to Dr. I. Dairy Sci. K. Chemists. Full-Scale System for the Removal of Iodine-131 and Strontium90 from Fluid Milk.. E.. Citrate. J. Murthy. 44: 2158. m u s t be modified to o b t a i n a s a t i s f a c t o r y end p r o d u c t . 50. Missouri. J. Cincinnati. D. (13) References (1) Association of Official Agricultural Chemists. Oxalate. Methods of Analysis. 1965. G. The coagulation times f o r the control a n d the t r e a t e d milks were 585 ± 85 a n d 380 + 30 sec. Removal of Radionuclides from Milk. 1953. J.5 to 3. E. K. Expt. D. J... Odagiri. II. 1961. Odagiri. Lu Milk. R.
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