TECHNICAL NOTES
We conclude from the evidence that in all
probability 7-, TS-, R- and S-caseins are pieces
of the fl-casein molecule. One uncertainty
should be mentioned. I t has been thought that
all the phosphorus in fl-casein occurs in the
.'c-terminal phosphopeptide, T1, Residues 1 to
25. However, T-casein, which begins at Position
28 in the sequence of fl-casein, is known to
contain one P per molecule. The location of
this atom of phosphorus, presumably as a
phosphorylated amino acid, remains to be
established.
Our conclusion, if substantiated, may have
important imp/ications f o r more understanding
of the biosynthesis of casein micelles, in par-
titular, and of proteins in general.
William G. Gordon, Merton L. Groves, Rae
Greenberg, Susan B. Jones, Edwin B. Kalan,
Robert F. Peterson and Robert E. Townend
Eastern Marketing and Nutrition Research
Division, ARS, USDA, Philadelphia, Pennsyl-
vania 19118
References
(1) Groves, M. L. 1969. Some minor components
of casein and other phosphoproteins in milk.
A review. J. Dairy Sci., 52: 1155.
(2) Groves, M. L., and Gordon, W. G. 1969. Evi-
dence from amino acid analysis for a rela-
tionship in the biosynthesis of "y- and
Simple Method for the Isolation of Riboflavin from Whey
Abstract
A convenient, facile method for the
isolation of riboflavin from whey is de-
scribed. Whey is passed over a column
of the neutral resin Amberlite XAD-2, on
which riboflavin is adsorbed. Recoveries
of the riboflavin with acetone as the eluant
were satisfactory. The method i s also
convenient for removing riboflavin when
it interferes in the analysis of other con-
stituents.
Introduction
Although riboflavin is now readily available
in synthesized form, it is expensive. Therefore,
the need for an inexpensive, facile way to
1 Matheson, Coleman, and Bell, East Rutherford,
New Jersey.
2 Rohm and Haas, Philadelphia, Pennsylvania.
•~Daker Analyzed, Baker Chemical Company,
Phillipsburg, New Jersey.
263
/~-caseins. Biochim. Diophys Aeta, 194: 421.
(3) Peterson, R. F., Nauman, L. W., and Hamil-
ton, D. F. 1966. Amino acid composition of
six distinct types of fl-casein. J. Dairy Sci.,
49 : 601.
(4) Pion, R., Garnier, J., Ribadeau Dumas, B., de
Koning, P. J., and Van Rooyen, P. J. 1965.
Amino acid composition of fl-casein variants.
Diochem. Biophys. Res. Commun., 20: 246.
(5) Ribadeau Dumas, D., Grosclaude, F., and
Mercier, J.-C. 1970. Primary structure of
bovine fl-casein. Isolation and amino acid
composition of the tryptic peptides and of
the peptldes obtained by action of cyanogen
bromide. European J. Dioehem., 14: 451.
(6) Ribadeau Dumas, B., Grosclaude, F., and
Mercier, J.-C. 1970. Location of the sub-
stitution His/Gln differentiating genetic
variants As and A3 in the peptide chain of
bovine fl-casein. C. R. Hcbd. S~anees Acad.
Sci. Paris, 270: 2369.
(7) Ribadeau Dumas, B., Groselaude, F., and
Mercier, J.-C. 1971. Primary structure of
bovine fl-casein. Ordering of the tryptic
peptides and of the peptides obtained by
action of cyanogen bromide. European J.
Diochem., 18: 252.
(8) Rose, D., Drunner, J. R., Ka]an, E. B., Larsolb
D. L., Melnychyn, P., Swaisgood, H. E., and
Waugh, D. F. 1970. Nomenclature of the
proteins of cow's milk: Third revision. J.
Dairy Sci., 53: 1.
isolate riboflavin from dairy products is of
interest. Leviton (1) removed riboflavin by
adsorption on crystallized lactose from whey
concentrates. Koziolowa (2) successfully used
ion exchange resins for the isolation of flavins
in certain food products. We noticed that when
milk was passed over a column of neutral
resin Amberlite XAD-2, a yellowish-green band
formed at the top of the column. This ob-
servation led us to investigate the feasibility of
using this resin for the extraction and recovery
of riboflavin from whey.
Experimental Procedure
A standard solution of riboflavin 1 was pre-
pared by dissolving 10 mg in 250 ml of re-
distilled H20. A column of the resin Amberlite
XAD-22 was prepared by slurrying 4 g of the
resin in redistilled water and pouring into a
glass chromatographic column (1.3 cm id ×
30 era). The column was then washed with
5 to 10 column volumes of water, 5 column
JOURI~AL OF DAIRY SCIENCE VOL. 55, NO. 2
 084 JOURNAL OF DAIRY SCIENCE

volumes of redistilled methanol, 3 and again with TABLE 2. Recoveries using whey plus riboflavin.
5 column volumes of redistilled water. The
column was maintained in water. Riboflavin
Recoveri~ were made of the pure riboflavin added to Recovered Recov-
by diluting 1, 2, 3, and 4 nfl of the standard whey duplicates ered
solution to 10 ml with redistilled water. The (ml) (%)
individual solutions were passed over the column
at about 4 ml/min. Recoveries were made by 0 .155 .160
adding 1, 2, 3, and 4 ml of the standard solu- 1 .240 .235 85
tion to 50 ml of sweet cheese whey and passing 2 .340 .335 83
the solution over the colunm at 4 ml/min. 3 .44 .44 83
A f t er application of the solutions to the resin, 4 .55 .56 84
the column was washed with 5 colunm volumes
of redistilled water or until the eluate was clear. satisfactory. The loss is probably due to binding
The adsorbed riboflavin was then eluted with of added riboflavin by whey protein, thus
30 ml of redistilled acetone) The acetone eluate preventing the riboflavin from being adsorbed.
was evaporated on a steam bath under a stream The method conveniently isolates natural,
of nitrogen, and the residues were dissolved in pure riboflavin. Because the resin is relatively
10 ml of water. The amount of riboflavin was inert and neutral, resin-induced transformations
then determined spectrophotometrically at wave- (2) are minimized. The capacity of the column
length 447 nm (3). was about 1 mg for a 4-g colunm at the desig-
nated flow rate. This could be increased by
Results and Discussion reducing the flow rate.
The results in Table 1 indicate satisfactory The method is economically advantageous.
recoveries of riboflavin by adsorption and de- It can be scaled up thereby utilizing whey as
sorption from the resin. The loss of about 10% a source of riboflavin. The materials are
can probably be attributed to the irreversible relatively inexpensive and can be recovered
adsorption of riboflavin by the column or de- and used repeatedly.
-~ctivation of riboflavin by light. Acetone is the Not only is the method suitable for the
best solvent for removing adsorbed riboflavin. recovery of riboflavin, but it conveniently re-
The column must be moist when the acetone moves riboflavin when it interferes with analyses
is added: if not. poorer extractions are realized. of other milk constituents. In addition, it would
be useful in other areas, e.g., protein extracts,
T.~BLE 1. Recovery of riboflavin. where riboflavin and other flavins interfere with
analyses.
Reading
Dilu- (447 Recovered Recov- C. R. BREWINGTON and D. P. SCHWARTZ,
tions nm) duplicates ered Dairy Products Laboratory, ARS, USDA,
Washington, D.C. 20250
(%)
References
1:10 .125 .115 .115 91
2:10 .255 .225 .220 88 (1) Leviton, A. 1943. Absorption of riboflavin
3:10 .375 .335 .330 89 by lactose. Ind. Eng. Chem., 35: 589.
4:10 .500 .45 .46 91 (2) Koziolowa, A. 1966. Applications of ion
exchange resins in the isolation of flavines
for qualitative and quantitative studies in
However, acetone-water mixtures did not in- food products. Pr. Zakresu Towarozn Chem.
Wyzsza Sak. Ekon. Poanan, Zesz. Nauk.
crease yields. Whereas hot water was an effec-
(Ser. 1) 25: 41.
tive eluant, methanol and ethanol were almost as
(3) Penzer, G. R., and G. K. Radda. 1967. The
efficient as acetone. chemistry and biological function of isoal-
Table 2 shows that when riboflavin was added lorazines (Flavines). London Chem. Soc.
to the whey, the recoveries were lower but Quart. Rev., 21:43.

JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 2