International Journal of Applied Radiation and Isotopes, 1959, Vol. 6, pp. 156--159. Pergamon Press Ltd. Printed in Northern Ireland
Some Effects of Ionizing Radiations on Liquid Whole Milk and Whey Protein G. G L E W U.K.A.E.A., Technological Irradiation Group, Isotope Division, Wantage Radiation Laboratory, Wantage, Berks Liquid whole milk was irradiated, and changes in flavour, alkaline phosphatase activity and acid soluble nitrogen were examined. Radiation induced ftavour changes became detectable at 20,000 rads whereas 25 Mrads were required to inactivate the enzyme alkaline phosphatase. Preliminary experiments on the effects of combined heat and radiation treatment on phosphatase activity are described. Increased fi'.v, absorption of irradiated solutions of fl-lactoglobulin and radiation induced changes in the eleetrophoretic behaviour of whey proteins were studied.
PROCTOR et al. have been studying the effects of radiation on milk for many years and have published a series of papers on the problems involved, a-5~ They have developed an irradiation procedure by which, it is claimed, sterile milk can be produced, indistinguishable in odour and flavour from fresh milk. Their method consists of irradiating milk in vacuo and removing off-flavour METHODS
compounds as they are formed by simultaneous distillation. This work is of considerable theoretical interest but it is difficult to see how the method can compete with the well-established commercial pasteurization and sterilization processes. This paper will describe some of the results obtained by irradiating milk in sealed containers in an atmosphere of air.
USED IN PRESENT
Electrophoretic pattern of changes in whey protein
Samples of unpasteurized, bulked morning Jersey milk were obtained and treated within a few hours of milking.
These measurements were made utilizing a horizontal electrophoresis chamber, barbiturate/acetate buffer (pH 8.6) and a voltage of 7 V/cm. W h a t m a n No. 3 M M filter paper strips were used as the supporting medium and the protein was stained with 1°7o bromophenol blue in 90% ethanol saturated with HgC12.
Irradiations Electron irradiations were performed in thin-walled 0.5 in. test-tubes revolving in the beam of a 4 MeV linear accelerator. Milk was also irradiated in standard McCartney bottles by y-rays from a Co ~° source.
Estimation of acid-soluble nitrogen
The protein was precipitated with 15% trichloracetic acid (wt/vol) and the tyrosine/ Estimation of alkaline phosphatase activity tryptophan in the filtrate estimated using Milk samples were incubated at 37°C for Folin and Ciocaheu's reagent. Samples were 15 min with disodium phenyl phosphate also digested at pH 2-0 for 15 rain at 25°C buffered at pH 9.7. Phenol liberated was with pepsin (final concentration 0.04%). estimated using FOLIN and CIOCALTEU'S The acid-soluble filtrates were again examined for tyrosine/tryptophan. reagent. ~) 156
Some effects of ionizing radiations on liquid whole milk and whey protein
Flavour tests These tests were conducted on samples of irradiated milk 1-3 hr after irradiation. A RESULTS
Inactivation of alkaline phosphatase by irradiation The enzyme inactivation curve is plotted semi-logarithmically in Fig. 1. The curveis exponential. A d o s e o f approximately 24 Mrads was necessary for complete inactivation. Electron dose rate, within the range 0.75-4.3 Mrads/min did not affect the magnitude of inactivation. The inactivation curve obtained using high dose rate 7-rays (produced by bombarding a platinum target with electrons) at a dose rate of 0.175 M r a d / min was not significantly different from the electron inactivation curve. It is evident that the alkaline phosphatase of milk is extremely radio-resistant and that dose rate, within the range specified, has no effect on the rate of inactivation.
The complementary effects of heat and radiation on alkaline phosphatase A number of workers in the microbiological field have found that radiation sensitizes some micro-organisms to heat treatment. (7,s) It has also been suggested that radiation sensitizes protein to de-naturation by heat. (9't°) Irradiating with Co 6° 7-rays at 40°C gives increased inactivation of the enzyme c o m 3G
modified triangle test was used and fifteen tasters were selected, on the basis of consistency and discrimination of judgement, to take part in the trials.
T h e effect of t e m p e r a t u r e d u r i n g ~,-irradiation on phosphatase activity (doserate: 0-18 M r a d / h r ) Temp.
% Inactivation of e n z y m e i
i i i
pared with irradiation at 20°C (Table 1) (P = 0.015). Table 2 shows the effects of heating at 50°C for 15 min before and after irradiation. Irradiation before heating (P = 0.005) is more effective in sensitizing the enzyme than heating before irradiation. This result is in agreement with results of work on microorganisms in that a synergism exists only when irradiation precedes heating. These results suggest that the inactivation of the enzyme can be significantly increased by the combination of heat and irradiation treatments described, although none of the treatments resulted in complete inactivation. TABLE 2. T h e effect of heating at 50°C before a n d after irradiation on phosphatase activity (Figures in " H e a t e d " a n d " U n h e a t e d " c o l u m n s represent % inactivation)
I Before i irradiation :
5O % ir~ct~vation Fro. 1.
4"9 5'9 10'1 18"6 21"9
6-8 4.7 13.2 22.0 31-3
Irradiation-induced changes in the milk protein Electrophoresis of whey protein. Two series
of experiments were performed. (1) Whole milk was irradiated and the whey protein subsequently prepared °1.01 for electrophoresis. Changes m ~.8' lactalbumin became apparent at about 1.0 Mrad. (2) Dialysed whey was prepared and irradiated prior to electrophoresis. 0.I Changes in ~-lactalbumin commenced at about 0.5 Mrad. In both experiments the e-lactalbumin band had disappeared at 2-0 Mrad. It was expected that the other milk components 20 30 40 10 would protect the whey protein during mg tyrosine/tryptophon / 1DOtal, milk irradiation under conditions described in FIG. 2. experiment (1) and that the whey proteins would be more radio-sensitive when separated from the casein and fat as in experiment (2). many workers and MCLEAN(12) and CARROLL Ultra-violet absorption spectra of irradiated et al. (13) have suggested that a diphenyl-type fl-lactoglobulin. //-Lactoglobulin prepared by linkage between several protein molecules the method of ASCHAVFENBURG and DREW- may occur during irradiation. ALEXANDER REYm) was irradiated with a 7-ray dose of et al. 1141 have suggested that the apparent 3.81 Mrads at two concentrations, 17.3 and increase is due to loss of light by the greatly 32.3 mg/ml in 0.1 N N a O H . There was no increased scattering by large aggregates shift of absorption maxima after irradiation formed during irradiation. Changes in acid-soluble nitrogen before and and Table 3 gives the E values at the two after peptic digestion. Milk was irradiated with absorption maxima. Protein concentration affects the extent of y-rays at levels of 0.025-10.0 Mrads and the change, greater change being observed at tyrosine/tryptophan in the acid-soluble fillower concentration. The compound (or trate estimated using Folin and Ciocalteu's compounds) causing the increased ultra- reagent. Fig. 2 gives the results of this violet absorption is not removed after 20 hr experiment. As the dose increases the prodialysis against running distilled water. In- tein becomes less digestible by pepsin. Flavour studies. Milk treated with doses of creased ultra-violet absorption by proteins and amino acids has been described by 15, 20, 25, 30, 40 and 50 krad/y-rays was presented in the form of a modified triangle TABLE 3. Irradiation induced changes in absorption test. Tasters were asked to score differences
spectrum of fl-lactoglobulin (Protein dissolved in 0.1 N N a O H . Dose: Protein concentration (mg/ml)
1 I ]
282 ml¢ control 105-8 irradiated ~ 181.8 control 109.0 irradiated 134'8
50,000 40,000 30,000 25,000 20,000 15,000
3.854 3.074 2.950 2.047 2.985 0-264
mtt I 289 mtt
107"2 170-1 111.3 132.8
TABLE 4. Some results of ttavour tests on irradiated milk
< < < < <
0.001 0.01 0-01 0.05 0.01 0.8--0.9
Some effects of ionizing radiations on liquid whole milk and whey protein
b e t w e e n samples on a n u m e r i c a l scale f r o m 1 to 10. T a b l e 4 gives the results o f these experiments. At all t r e a t m e n t levels a b o v e 15 k r a d s significant differences b e t w e e n the flavours o f u n t r e a t e d a n d i r r a d i a t e d m i l k w e r e observed. I t a p p e a r s t h a t the dose at w h i c h i r r a d i a t i o n flavour b e c o m e s d e t e c t a b l e is b e t w e e n 15 a n d 20 krads.
p r o b l e m s . T h e present studies are b e i n g u n d e r t a k e n not so m u c h in a n a t t e m p t to achieve a c o m m e r c i a l process, b u t because d a t a o n the changes t h a t take place should yield i n f o r m a t i o n w h i c h m a y be o f use in a p p l i c a t i o n to o t h e r foodstuffs. T h e results p r e s e n t e d h e r e do not, as yet, indicate a n y clear applications for the use o f r a d i a t i o n in p r e s e r v i n g milk.
Conclusions T h e i r r a d i a t i o n o f milk, because o f its c o m p l e x c h e m i c a l composition, poses m a n y
Acknowledgement---The author would like to thank Mr. T. HORNEfor useful discussions during the course of this research.
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7. KAN B., GOLDBLITHS. A. and PROCTOR B. E., Food Res. 22, 509 (1957). 8. KEMPE L. L., Appl. Microbiol. 3, 346 (1955). 9. CLARKEJ.H., Amer. J. Roentgenol. 40, 501 (1938). 10. FRmKE H., J. Phys. Chem. 56, 789 (1952). 11. ASCHAFFENBUROR. and DREWRYJ., Biochem. J. 65, 273 (1957). 12. McLEAN D. L. and GIESE A. C., J. Biol. Chem. 187, 537 (1950). 13. CARROLLW. R., MITCHELLE. R. and CALLANAN M. J., Arch. Biochem. Biophys. 39, 232 (1952). 14. ALEXANDER P., Fox M., STACEY K. A. and ROSEN D., Nature 178, 846 (1956).