JOURNAL OF DAIRY SCIENCE

VOLUME X X I I
THE

OCTOBER, ]939
RIBOFLAVIN

CONTENT

DAVID B. H A N D

NUMBER 10
OF COW'S

MILK

AND PAUL F. SHARP

Department of Dairy Industry, New York State College of Agriculture,

Cornell University, Ithaca, New York

T h e r i b o f l a v i n c o n t e n t of a b o u t 400 s a m p l e s of m i l k p r o d u c e d i n N e w
Y o r k S t a t e h a s been d e t e r m i n e d . T h e p r e s e n c e of r i b o f l a v i n in c o w ' s m i l k
is of c o n s i d e r a b l e biological significance. R i b o f l a v i n is l i n k e d w i t h biological o x i d a t i o n s ( a n d p o s s i b l y w i t h t h e o x i d a t i o n s o c c u r r i n g in m i l k ) in
t h r e e w a y s : as t h e active g r o u p of a n e n z y m e (12, 9), as a h y d r o g e n a c c e p t e r
(11), a n d as a p h o t o s e n s i t i z e r f o r t h e o x i d a t i o n of v i t a m i n C (7, 4, 2 ) .
T h e a v e r a g e r i b o f l a v i n c o n t e n t of m i l k v a r i e s w i t h the d i f f e r e n t breeds,
t h e J e r s e y b e i n g highest, the G u e r n s e y i n t e r m e d i a t e a n d t h e H o l s t e i n lowest.
On t h e a v e r a g e , H o l s t e i n m i l k c o n t a i n s 33 p e r c e n t less r i b o f l a v i n t h a n J e r s e y
milk. See T a b l e 1. I t is i n t e r e s t i n g t h a t t h e o r d e r of b r e e d is s l i g h t l y difTABLE 1
t~iboflavin content of milk

Barn fed
Milk sample

]No.

analyses
Jersey ..................... [
Guernsey ............... I
Brown Swiss ...... I
Ayrshire ............... ]
Holstein ............... L
Cornell Herd ... I
Composite commercial ........

Pasture fed

Mg. riboflavin per

liter whole milk

~TO.

analyses

Mg. riboflavin per

liter whole milk
Range

Average

1.69
1.42
1.80

24
14
5
10
23
76

1.90-3.40
1.41-2.52
2.00-2.62
1.57-2.38
1.19-2.44
1.19-3.40

2.37
2.04
2.37
1.97
1.64
2.04

1.64

50

1.60-2.27

1.97

Range

Average

25
14
4
9
22
74

1.55-3.42
1.64-2.72
1.80-2.08
1.26-2.08
0.60-2.31
0.60-3.42

2.18
1.91

53

1.34-2.17

1.87

(Analyses on the Cornell herd were made on April 7 and June 8, 1937, and those on
the composite commercial samples on April 4 and June 28, 1938.)
f e r e n t w i t h r e s p e c t to the c a r o t e n e c o n t e n t of m i l k f a t , in w h i c h case the
G u e r n s e y is h i g h e r t h a n t h e J e r s e y .
C o m m e r c i a l m i l k in s u m m e r was f o u n d to c o n t a i n a b o u t 20 p e r c e n t m o r e
r i b o f l a v i n t h a n w i n t e r milk. T h i s i n c r e a s e is a p p a r e n t l y a n effect of t h e d i e t
a n d is t r u e i n g e n e r a l w h e r e the c h a n g e in f e e d is f r o m corn silage to p a s t u r e .
Received for publication March 3, 19~9.
779

780

DAVID B. HAND AND PAUL F. SHARP

The average increase in the riboflavin content of the milk of the Cornell herd
in summer was 13 p e r cent. H o w e v e r the milk of cows receiving molassesgrass silage and phosphoric acid-grass silage was relatively high in winter
and did not increase in riboflavin content when the cows were placed on grass
pasture. Our observations confirm the work of V i r t a n e n and Holmberg
(10) who reported that with phosphoric acid silage the riboflavin content of
the milk remained constant t h r o u g h o u t the year.
A f u r t h e r study of seasonal and individual variation was made on 9 cows
beginning April 13 and extending to J u n e 29, 1938. A total of 164 analyses
were made. Some of the cows were stall fed on molasses-grass silage and
the rest were fed on phosphoric acid-grass silage and numerous samples were
taken during the two weeks period preceding t u r n i n g the cows out to grass
pasture. There was no evidence of a change in riboflavin content when the
cows were p u t out to graze. There was, however, considerable fluctuation
with all the cows throughout the 10 weeks period. The m a x i m u m deviations
of the 9 cows f r o m their individual means were between ~ 15 per cent and
27 per cent. The average value of riboflavin for all analyses for the 2
weeks before going on grass pasture feed was 2.28 and for the 2 weeks after
going on grass pasture was 2.12, indicating no increase. Since the average
deviation in all duplicate analyses was 3.99 per cent, a n y fluctuation less
t h a n this would be questionable with our method. D u r i n g this period of 10
weeks the m a x i m u m deviation of the control solution of riboflavin f r o m its
mean value was 6.0 per cent. The change in feed produced practically
no change in milk production which averaged 33.3 pounds per d a y before
and 32.5 a f t e r the cows were p u t out on pasture.
The riboflavin content of milk varies with the individual cow, as well as
with feed, breed and milk production. Variation among individual cows in
a given season can be more t h a n 500 per cent. The highest single milk
sample observed on grass pasture contained 3.42 mg. per liter and the lowest
contained 0.60 mg. per liter. Variations in individual, commercial, market
milk samples were much smaller and the range was narrower, being from
1.60 to 2.27 mg. per liter. This was to be expected because the commercial
samples were f r o m mixed milk coming from various dairies throughout the
state of New York. The samples were collected f r o m delivery wagons.
Because of the variations due to breed, feed and individual cows, it is
difficult to assign a single value as being the normal riboflavin content of
milk. I n place of a value, Table 1 showing group averages and Figure 1
showing distribution curves for milk samples with respect to riboflavin content are given. Individual variations in samples which are encountered in
most biological work produce a need for a special type of analytical procedure. The methods used m u s t be rapid and simple but need not be extremely accurate. I t is much more significant to have a large n u m b e r of
analyses with an accuracy of 5 per cent t h a n to have a few extremely
accurate results.

781

TItE RIBOFLAVIN CONTENT OF COW~S MILK

The most frequently observed riboflavin content of the milk samples

studied by us was about two rag. per liter. Our results are about 25 per
cent higher than those of Whitnah, Kunerth, and Kramer (13). In our
herd the few Ayrshire cows averaged higher than the Holstein cows, but the
number of Ayrshire cows tested by us (10), is too small to establish this breed
difference. Earlier analyses of the riboflavin in milk in individual samples
are not inconsistent with our results. For example, Kuhn, Wagner-Jauregg,
and Kaltschmitt (5) found 1.00 rag. riboflavin per liter of milk and von
Euler, Adler, and SchlStzer (1) found 2 to 3 mg. per liter. Riboflavin in
I00

u~

90-80--

Commercial

samples

70--

E
c
0

60-5

--

"3

Individual
Cornell

40-u)

cows in
herd

50-C

20--

0,..

I0--

I
I
R,b0flavln
Fro. 1.

in mcj

per

liter

D i s t r i b u t i o n of riboflavin in milk.

milk is reported to be 90 per cent free of combination with protein (5) and
unesterified (6).
The determinations of riboflavin reported here were made by measuring
the intensity of fluorescence of an acetone milk filtrate with a photoelectric
cell and comparing this reading with the fluorescence of uranium glass as a
standard (3). In order to make sure that variations were not due to fluctuations in the instrument, comparative readings were frequently made using
a standard riboflavin solution which remained unchanged during the year.
No relation between riboflavin content of the milk and the development
of oxidized flavor in the dark was observed with the samples studied. A
definite inverse relation was found between riboflavin content and milk pro-

782

DAVID B. HAND AND PAUL F. SHARP

duction, the linear correlation coefficient of which was -0.501 ( ~ 0.058).

This correlation was obtained with 76 cows, 5 different breeds, all receiving
the same type of feed. The relation is even better t h a n the linear correlation
coefficient would indicate because the regression is not linear but skew. The
data yield a correlation ratio of - 0 . 8 3 0 ( 0.024). Since the square of the
correlation ratio is 0.69 it m a y be concluded t h a t two-thirds of the factors
other t h a n feed, which determine the content of riboflavin in milk m a y be
accounted for b y changes in milk production. A correlation of riboflavin
with f a t content has been reported (13) and it has been suggested that riboflavin is concerned in the production of milk fat. A correlation of riboflavin
with f a t content m a y result merely f r o m the inverse correlation with milk
production and thus offer no evidence that riboflavin is active in the production of milk fat. The evidence that there is little or no riboflavin in m a r e ' s
milk (8) would indicate that riboflavin performs no function in the production of fat.
SUMlVfARY

The riboflavin content of about 400 samples of milk was determined by

the measurement of the intensity of fluorescence of an acetone filtrate by a
photoelectric cell. The riboflavin ranged f r o m .60 to 3.42 rag. per liter. A
negative skew correlation of - 0 . 8 3 0 was found between riboflavin content
and milk production. Commercial milk in s u m m e r contained about 20 per
cent more riboflavin t h a n winter milk. Cows, a f t e r being fed in winter on
phosphoric acid grass silage, showed no increase in riboflavin when turned
out on grass pasture.
REFERENCES
(1) EULER, HANS YON, ADLER, ERIC AND SCHLOTZER,ARNULF. Distribution of bound and
free flavin in plants. Zschr. physiol. Chem., 226: 88-94. 1934.
(2) HAND, DAVID B., GUTHRIE, E. S. AND SHARP, PAUL F. Effect of oxygen, light and
lactoflavin on the oxidation of vitamin C in milk. Science, 87: 439-441. 1938.
(3) HAND, DAVID B. Determination of riboflavin in milk by photoelectric fluorescence
measurements. J. Ind. Eng. Chem. Anal. Ed. 11: 306-309. 1939.
(4) HOPKINS, F. GOWLAND. On lactoflavin as a sensitizer in the photocatalytie oxidation
of aseorbic acid. Compt. rend. du Lab. Carlsberg. Set. chim., 22: 226-234.
1938.
(5) KUHN, RICHARD, WAGNEE-JAUREGG, THEODOR AND KALTSCHI~IITT, HANS. On the
distribution of flavines in the plant kingdom. Ber., 67: 1452-1457. 1934.
(6) KUHN, RICHARD AND KALTSCHMITT HANS. On the state of vitamin B.o in cow's
milk. Ber. 68: 386-387. 1935.
(7) ~VIARTINI,EMILIO. Photodynamic action of the flavins on ascorbic acid. Boll. Soc.
Ital. Biol. Sper., 10: 1235. 1934.
(8) RASMUSSEN,RUSSEL A., BOGART,RALPH AND ~/[A~_'NAItD,L . A . Ascorbie acid content
of milk of various species as influenced by ascorbie acid injection and diet. Proc.
Soc. Exper. Biol. Med., 39: 502-505. ]938.
(9) TI-IEORELL, HUGO. The physiological reoxidation of reduced yellow enzyme. Biochem. Zschr., 288: 317-328. 1936.

THE RIBOFLAVIN CONTENT OF COW~S MILK

783

(10) VIRTANEN, ARTTURI AND HOLI~IBElCG, TOIVO. Variations of vitamin B: in milk.

Suoman Kemistilehti l l B , 1-2 (1938) Chem. Abstracts, 32: 3037. 1938.
(11) WAGNER-J-AUREGG,THEODOR AND RUSKA, HEL~UT. Flavines as biological hydrogen
acceptors. Bet., 66: 1298-1302. 1933.
(12) WARBU~G, OTtO AND CHRISTIAn, WALTER. On a new oxidation enzyme and its
absorption spectrum. Biochem. Zschr., 254: 438-458. 1932.
(13) WHITNAH, C. H., KUNE~I~, B. L. AND K~AMER, )/[. M. Riboflavin content of milk
collected in different months and correlated with other constituents of the milk.
J. DAIRY Sc., 21: 593. 1938.