146
222

 

HTHS

A STUDY CF THE STABELITY
OF VITAMIN A IN VARIOUS
COD LN. ER OIL ~ FEED I‘zHXTL’RES

I‘hesés fer the Degree of M. S.
3v§§CWCAN STATE COLLEGE
jcl‘sn R. Lewis

1940

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A STUDY OF THE STABILITY 01' VITAMIN A IN
VARIOUS GOD LIVER OIL -- Fm MIXTURES

by
John Reei Lorie

A THESIS
'Subnitted to the Graduate School of lichigan
state College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE

Department of Chemistry
191:0

TM f5. 11-;
L b”? 4

ACKNOWIEDGMENT

The author wishes to express his appreciation
for the assistance and advice given by Dr. C. A.
Happert, Professor of Chemistry, in the carrying
out of this investigation and the preparation of

thi 5 menu script .

A STUDY OF THE STABILITY OF VITAMIN A IN
VARIOUS GOD LIVER OIL -- FEED MIXTURES .

Introduction

[ith the increase in the knowledge of vitamins during the past
few years, greater emphasis has been placed on their importance in
nutrition. In many cases vitamin supplements such as concentrates
from natural sources or even synthetic preparations are added to
rations to insure an adequate supply in the diet. As an example,
cod liver oil is widely used to increase the vitamin A and D potency
of mixed feeds. In the case of vitamin A. this type of fortification
has not been entirely satisfactory because of the instability of I
vitamin A under practical conditions of use.

Several workers have shown that vitamin A is destroyed during
storage. Holmes. Corbet, and Eartsler (9) found that the vitamin
A of halibut liver oil was completely destroyed in 10 hours when
kept at 96° under an atmosphere of oxygen. They also found that the
vitamin A of halibut liver oil was completely destroyed in 21 weeks
when 10 cc. of the oil was kept in a ho cc. bottle in diffused light
at room temperature. Under these same conditions, the vitamin A of
cod liver oil was destroyed in 6 weeks. Lowen, Anderson, and Harrison
(10) found that the vitamin A of salmon oil decreased from 269 BU/gm.
to 160 BU/gm. or W in 35 days when 50 cc. samples were stored in
125 cc. lrlenmeyer flasks exposed to air at room temperature in dif-
fuse winter light. The vitamin A of halibut liver oil under these
conditions decreased 811$ in 35 days. They also found that 30 cc.

2.

samples of freshly prepared.halibut liver oil stored in 50 cc. Erlen-
meyer flasks exposed to air at room temperature in diffuse spring light
lost 96$ of the vitamin A in 31 days. But in (5) found that samples
of cod liver oil under conditions of commercial distribution did not
lose any appreciable amount of vitamin.A even after they had.been
stored h years.

Other workers have shown that vitamin A added to feeds is destroyed
to a greater extent that the vitamin.A.which.is present in the feed.as
precursors such as the carotenes. Dunn.(h), in l92h, noted that when
cod liver oil was mixed with starch and stored in stoppered bottles
in the dark for 6 months. it lost its anti-xerophthalmic properties.
Harcus (11) observed that when cod liver oil contentrate was mixed
with finely ground compounds such as ferric oxide, magnesium oxide,
calcium carbonate, lactose and others, the vitamin A was destroyed on
'standing. He also found that the vitamin.A.of a mixture of cod liver
oil and the USP vitamin.A deficient ration was destroyed to the extent
of 35% in 10 days. rrcpe and Kemerer (6) found that when fish oils
or their concentrates were added to feed mixtures, 79 - 100$ of the
vitamin.A disappeared after h weeks of storage. Bethke, Record and
Iilder (3) noted that not over 50$ of the added vitamin A from cod
liver oil was destroyed in a casein ration in 6 months. However. in
a similar ration containing meat scraps and dried milk in the place
of casein, approximately 75$’of the vitamin A added.was lost in 6
months. some: and ford (7) mixed cod liver oil with a vitamin A-
deficient ration and stored it for 60 days. On eight-week tests with
chicks, no less of vitamin.A.was detected: however, there was an

indication of some loss when the test period was extended to ten

3.
.weeks. Baird, ningrose and nacuillan (2) reported that Vitamin A from .
fortified cod liver oil. when mixed in the ration to provide respectively
100. 150, 200, and 300 units of vitamin A per 100 grams ration, did not
evidence complete destruction even when the rations were stored in bar-
lap bags at sumer temperatures for 25 weeks. although it underwent
progressive destruction as the storage period advanced.

During some feeding experiments with a commercial dog food, it was
observed that the vitamin A which had been added to the feed during its
mufacture could not be accounted for several weeks later. Chemical
tests of the feed showed that the vitamin A had been destroyed. In view
of the fact that it could be expected that various ingredients of feed
mixtures might show different effects toward the stability of vitamin
A. investigation was carried out to determine. if possible, whether
certain ones might be particularly well suited to preserve the vitamin

A potency.

Experimental

The experimental procedure consisted of adding certain amounts of
cod liver oil to different foods. The oil and feed were mixed by hand
and stored in cloth bags in laboratory lockers. At monthly intervals,
samples were take out and the vitamin A determined by the following
method:

I. Twenty grams of the sample was extracted with ethyl ether in
a onhlet extractor for a minimum of three hours. Abernathy (I) re-
ported that an extraction period of two hours was sufficient to extract
all the vitamin A which was added to a feed. However, we found a

minimum extraction period of 3 hours gave the most satisfactory results.

’4.

2. The ether was then evaporated from the extract by use of a
warm water bath and reduced pressure.

3. The residue was saponified with 10-15 cc. of saturated
alcoholic Ion on a steam bath. It was then cooled and extracted twice
with 100 cc. portions of ethyl ether. The ether extract was washed
with water until free from alkali when tested with phenolphthalein.

’4. The ether extract was then dried overnight using anhydrous
lagdoh. The solution was filtered and the ether evaporated off as
before. ‘

5. The residue was taken up in a suitable volume of chloroform.
Two-tenths cc. of the chloroform solution was mixed with 2 cc. of a
saturated solution of 811013 in 63013 in a l on. cell and the color
determined immediately with a Lovibond tintometer. The results were
calculated as Lovibond blue units per gram.

In order to test the dependability of the method, weighed amounts
of cod liver oil were analysed by the above method and the results
compared with those obtained by the direct determination of vitamin A

in the chloroform solution of the oil. The results are given in

 

 

 

Table I.
Table I
Dependability of Method for the Detenination
of Vitamin A
Sample Clo.‘ BU/gm.
:ms: “
C. l. o. analysed by above method 0.92 1195
c. 1. o. in 6301} (direct determination) 1.27 1117

 

" Clo. A comercial medicinal cod liver oil
" BU/gm. Lovibond Blue Units per gram of oil

5.
The difference in the values obtained is probably within the ex-

perimental error, which indicates that the method is reliable.

In experiment I, the ground Vita Chiba dog food and six other feeds
were mixed with cod liver oil. Kilogram quantities of the feed contain-
ing 5% oil were prepared and a sample was removed for an initial
determination. The rest of the mixture was stored and samples taken
out at monthly intervals for analysis. The results are given in Table
II.

The vitamin A added to the Corn Flake feed was completely destroyed
at the end of one month's storage. The vitamin A of the Vita Cube dog
food underwent a gradual destruction and 75% was lost at the end of
nine months. The other five feeds lost from 31! - 904 in ten months.

preriment 2 was carried out the same as experiment 1 with the
exception that a different series of feeds was used. The results are
given in Table III .

Higher vitamin A values were obtained because an oil was used which
had a greater concentration of vitamin A than the oil used in experimsnt
1. Complete destruction of the vitamin A occurred in one month with Rice
Krispie feed and Ihole [heat Biscuit feed. The amount lost with the
other four feeds varied from 111.65% in seven months.

In experiment 3, two other feed ingredients, bone meal and wheat
germ, were tested in a similar manner. These were mixed with 2 1/233
per cent oil instead of 5% as in the previous experiments. The results
are given in Table IV.

The vitamin A was completely destroyed with the bone meal in one
month and only a trace was left with the wheat germ at the end of five

months.

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3'meer or VITAMIN A. or con LIVER on. mm
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Bone Heal ‘BU/gn mm 0.0

f lost 100.0
[heat Germ Balsa has 1.3.7 30.5 39.6 28.6 1‘”
i lost 0.0 29.0 9.2 33.3 99.0

 

‘ BU'g Lovibond.£1uo unit-
"I a Trace of Elna

6.

It will be noted throughout the tests that there was considerable '
variation in the initial values for vitamin A. This was probably due to
the inherent difficulty in obtaining uniform distribution 6: the cod
liver oil in the feeds, since the mixing was done by hand.

In view of the fact that various cereal products save variable
results and even products from the same grains reacted differently, it
was thought desirable to test the stability of vitamin A in mixtures of
cod liver oil vith the various unprocessed grains.

five different grains were guund finely in a Robert 3111. A mixt-
are of 500 grams containing 2 1/2$ cod liver oil of each grain was pre-
pared, stored and sampled as in the previous uperiments. The results
are given in Table 1.

mm 7

STABILITY 01' VITAMIN A 01‘ DOB LIVER OIL
IEEN mm 1'0 GROUND BM GRAINS

 

 

 

 

0
amp 1“ 0 1 2 _3 fi 5 6
Barley *BU/gn ’41.} 33.5 22.6 1L3 18.2
i 1°.t 1 .1 “5.3 Q0 5509
con 30/9! t$9.5 l11.3 27.5 l$7.9 35.5 27.5
5 lost 16.6 101.5 3.2 23.3 was
Oats Isa/gm 111.3 35.5 22.0 22.0 11.0
% 1°.t Inc]. ”6.8 “6.8 7301‘
Rice BU/gm 12.9 0.0
56 1a.: 100.0
meat 30/9. #1.} 33.0 19.3 25.9 22.0
6 lost 20.0 53.2 37.3 u6.7

 

‘BU : Lovibond Blue Units

7.

The vitamin A was completely destroyed in the case of ground rice
in one month. 'Ehe oats mixture lost 73%. the barley, 56%. the wheat.
W. and the corn, m; of the vitamin A in 6 months.

Since considerable variations with the different feeds occurred
with respect to the destruction of vitanin A, a number of mixtures of
feeds with cod liver oil were tested. Linseed oil meal was mixed with
oatmeal and also with standard niddlings. Iixtures of cod liver oil
with linseed oil meal and standard middlinge had exhibited considerable
stability, whereas that involving oatmeal was not particularly stable.
The cod liver oil was pro-nixed sith the linseed oil meal, since the
latter absorbed the oil readily. Ihen the other feed see added and
aimed thorouchlye ho hundred grams of the nixture was prepared con—
taining 5% of the oil. Another group of mixtures was prepared. in a
similar way except that bone meal was added as an extra ingredient.
Ihe amount of bone meal added was equal to that of each of the other

two feeds. he results are given in the 11.

mm TI
STABILITY 03' VITAMIN 1 MN ADDED 1'0 VARIOUS rm MIXTURES

W W

1.1 seed 011 Real + Oatneal 930/;- 75.6 53.6 n .9 h .5 .5 h .5
n i lost 29.1 .7 32 5 ‘32. 33.5

 

Linseed 011 m1 + Standard BU/gm 73A 51.2 147.9 53.6 h9.5 119.5
Iliddlings i lost 31k? 39.0 31.7 36.9 36.9

Linseed Oil Heal + Oatmeal + BU/gn 77.6 0.0
Bone Ilsa]. 100.0

Linseed Oil Heal + Standard BU/gn 82.5 0.9
Middlings + Bone Heal 100.0

 

‘30 : Lovibond Blue Units

8.
The difference in the amount of’vitamin A lost with the first two

mixtures in five months is perhaps not significant, However, when bone
meal was added, the vitamin A was completely destroyed in one month.
Although the oil was mixed with the linseed oil meal and the bone meal
added as an extra ingredient, the presence of the latter was obviously
responsible for the destruction of vitamin A.

Further experiments were carried out with those feeds in which the
vitamin A was destroyed in one month. Bone meal, ground rice, nice
Krispie feed and corn flake feed were each extracted with ethyl ether
in Soxhlet extractors for 344 hours. The extracted feeds were then mixed
with Si cod liver oil and determinations carried out as in the previous
experiment s. After the feeds were mixed with the oil, they stood over-
night before samples were removed for an initial determination, of the
vitamin A. These results are given in Table VII. The vitamin A was
completely destroyed with the bone meal overnight and in the case of all
the other samples within one week. This shows that the ether extract of
the feed does not contain anything which might accelerate the destruction
of vitamin 1.

Another emeriment was carried out in which linseed oil meal was
extracted with ethyl ether and the oil mixed with the extracted meal was;
in the preceding emeriment. In this case, the vitamin A was completely
destroyed in one month. It say have been destroyed in much less time, but

the sample was not tested until it had been in storage for one month.

Discussion
The results of this investigation show that when cod liver oil is
mixed with different feeds, lurked variations in the rate of destruction

of vitamin A occur. The results shown in Tables 11-? inclusive represent

WWI

STABILITY OF VITAMIN A IEEN mm TO FEEDS PREVIOUSLY

EXTRAGTED IITH mm ETEEB

 

 

 

 

3mm w JILL—
h mm film...—
Bone Meal , 0.0 0.0
Rice (ground 57.8 0.0
Rice Irispies ' 57.8 0.0
corn l‘labes 57.8 0.0

 

‘30 : Lovibond Blue Units
*' Samples stood overnight before first determination.

9.
mixtures of a single ingredient with cod liver oil, except in the case
of the Vita cube dog food. Variations were observed ranging from a loss
of 31$ in 6 nentht to complete destruction in less than one month.

The feeds in which the least destruction occurred were standard
middlings and linseed oil meal. hose which showed the greatest destruct-
ion were bone meal, Corn nabs feed, Rice Krispie feed, ground rice, and
Ihole 'heat Biscuit feed.

Although the vitamin A was destroyed readin when cod liver oil
was mixed with corn Flakes and vhoie lheat Biscuit feed, no such
instability was observed in the case of the ground corn and wheat. It
is interesting that considerable stability was noted with several other
wheat products. however, in the case of the ground rice the vitamin A
was destroyed as rapidly as in the Rice Krispie feed.

No significant difference in the destruction of vitamin A was ob-
served between the addition of oatmeal to linseed oil meal and the
addition of standard middlings to linseed oil meal when the oil was first
mixed with the linseed oil meal. his is interesting in view of the fact
that vitamin A was more readily destroyed when mixed with oatmeal than
with standard middlings.

lhen bone meal was added to the mixtures in which the cod liver oil
had been incorporated with linseed oil meal, the vitamin A was destroyed
very rapidly. Since the oil was mixed with the linseed oil meal and at
least a large amount of it was absorbed by the linseed oil meal, it would
appear that the bone meal catalysed the destruction of vitamin A. Holmes,
and 00th (8) reported that several compounds, which were used in
adsorption columns, caused a catalytic destruction of vitamin A. The
results obtained with bone meal are in agreement with the work of Harcus

(11) who reported very rapid destruction of vitamin A when cod liver oil

10.
'was mixed with various inorganic salts.

It was observed that the ether extract of bone meal, Corn noises,
rice and Rice Krispies did not contain anything which might accelerate
the destruction of vitamin A.

lhen linseed oil meal was extracted with ethyl ether before the
cod liver oil was added, the vitamin A was destroyed in one month and
probably in less time. This shows the presence of a stabilising agent
in the ether soluble fraction of the oil meal. In view of the obser-
vations of Holmes, Gorbet and Hartsler (9) that lecithin acts as an
antioxidant for vitamin A, it is more than likely that this or some
other fat soluble antioxidant is present in considerable amounts in
linseed oil meal and perhaps to a mich lesser extent in certain other
feeds. This would account at least in part for the variations in the
rate of destruction of vitamin A in the various feeds.

These findings are in general agreement with the observations of
Bethbe et al. (3) who reported a loss of 50$ and 75% of the vituin A
in 6 months with two different rations. However, the results of this
study show less rapid destruction of vitamin A than those observed by
l'raps and Kemerer -(6), but more rapid destruction than reported by
Holder and tort (7) and by Baird et al. (2).

This investigation emphasises the importance of the fact that the
addition of vitamin A to a ration during its manufacture does not in-
sure its presence when the ration is used. If cod liver oil is added
to a feed mixture, it should be added to an ingredient which would tend
to preserve the vitamin A. Moreover, the feed should be used within
a reasonable time after the addition of the oil.

11.
9M1

The results of this study show that vitamin A of cod liver oil
is unstable during storage when incorporated in feed mixtures. tide
variations in the rate of destruction of vitamin A were observed with
the different feeds. In the case of Corn Flakes, Rice Hrispies, lhole
Iheat Biscuit, bone meal and ground rice, the vitamin A was completely
destroyed in one week or less. In other foods, there was a progressive
destruction of the vitamin A as the storage period increased.

The rate of destruction observed with some of the cereal products
differed from that of the unprocessed grain. An exceptionti this was
ground rice in which the destruction of vitamin A was the same as that
of nice Krispie feed.

lhen bone meal was added to a feed mixture to which cod liver oil
had been added, the vitamin A was completely destroyed within one
month. This fact indicates that bone meal acts as a catalyst in the
destruction of vitamin A.

The ether extract of certain feeds did not contain anything which
might accelerate the destruction of vitamin A. However, ether extract
of linseed oil meal contained some substance which acted as a stabilis-
ing agent for vitamin A. Apparently, this stabilizer is present to a
greater extent in some feeds than others, which may account in part
for the variations in the rate of destruction of vitamin A which was
observed with the various feeds.

from a metical standpoint, when cod liver oil is to he added
to a feed mixture, it would be best to add it to an ingredient in

which the vitamin A will not be readily destroyed.

(1)

(2)

(3)

(h)

(5)

(6)

(7)

(8)

12.
gagerengeg

Abernathy, II. I. The Use of the Antimony Trichloride Hethod
for the Determination of Vitamin A in reeds, Milk and

Butter. Thesis for u.8. at Michigan State College (1938)

Baird, 1'. 1)., Ringrose, A. T., and HacMillan, M. J.
The Stability of Vitamins A and D in Mixed l'eed Ingredients.
Part II - Vitamin A. real. Sci. 1831041 (1939)

Bethke, a. 11., Record, P. 3., and Iilder, V. E. II. The
Stability of Carotene and Vitamin A in a Kind Ration.
Paul. Sci. 18:179 (1939)

Dunn, L. C. The lffect of Cod Liver Oil in Various Amounts
and Forms on the Growth of Young Chickens. J. Biol.

i... Geo. s. Cod.Liver 011 - Stability of Vitamin.A Content
under Conditions of Comercial Distribution. J. Am.

Pharm. Assoc. 2231085-6 (193”)

Traps, G. 8. and Ismmerer, A. R. Losses of Vitamin A and
Carotene from reeds During Storage. Texas Agr. prt.
Sta. Bull. lo. 557 (1937)

Holder, 3. C. and Ford, 8. K. The Stability of Vitamin A

fimCMLhuOflinmnthm mm.&n
18:3”5 (1935)

Holmes, E. It. and Corbet, R. 3. Catalytic Effects of Porous
Powders on Pure Vitamin.A. J. 3101. Chem. 127:hh9 (1939)

13.
. (9) Hblmes, H. H., Corbet, n. 0., and Hartsler, e. R. Lecithin and

Hydroquinone as Antioxidants for Vitamin A. Ind. and Eng.
Chem. 283133 (1936)

(10) Lowen, I... Anderson, I... and Harrison, H. I. Cereal Hours as
Antioxidants for l'ishery Products. Ind. and Bug. Chem.
29:151 (1937)

(ll) Ilarcus, J. I. The Destructive Action of Finely Divided Solids
on Vitamin A. J. Biol. Chem. 90:50? (1931)

 

 

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