NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
129
excretion, but rather to a failure on the part of the liver cells to assimilate and
elaborate the vitamin when it reaches them in large quantity before it is either
destroyed or passes into general circulation. As the livers of diabetics contained,
according to Wolff and Moore, a very high reserve of vitamin-A due to the inges-
tion of the large quantity of carotene obtained from the vegetable diet, it appears
that, if Green's explanation is considered correct, the activity of liver cells in
storing vitamin-A may vary in different diseases. It was, therefore, felt desirable
to obtain some data on the vitamin-A reserve in the livers of some farm animals,
in health and in disease, and in this preliminary report the results obtained are
given.
METHOD OF VITAMIN-A ASSAY
The Carr-Price method of estimating the vitamin, as modified by Davies
[1933], has been used. Usually 5 grms. of liver was saponified with 10 c.c. of
aqueous 5 to 10 per cent potassium hydroxide, and the unsaponified fraction
twice extracted by means of ether, with the addition of some alcohol for the first
extraction. The ether fraction was finally freed from water by shaking with
anhydrous sodium sulphate and then quickly evaporated. In all essential parti-
culars, the procedure adopted by Davies was used, and the method in our hands
has given quite good results. The tintometer used was the B. D. H. pattern
with artificial light attachment. The results are expressed in Moore's units per
gram of the liver to the nearest significant figure. In all the data given in this
paper, only the blue units are tabulated. The yellow or the red units of the tinto-
metric readings have not been tabulated.
Having decided on the method of assay, it was thought necessary to determine
(1) if there was any difference between the vitamin content of the central and
the peripheral portion of the liver, (2) if decomposition for 4 or 5 days at room tem-
perature would have any effect on the vitamin content, as it was realised that in
some cases it was possible that the liver might not be sampled and put into
potassium hydroxide solution for some days, and (3) if the relation between the
tintometer blue units and the concentration of the unsaponifiable fraction
actually used in the experiment (indirectly giving the amount of original liver)
would be linear. In Tables I, II and III, these results are shown.
From the data given in Table I, it is apparent that there is a slight difference
in the vitamin content of the central and the peripheral portion of the liver, the
value of the central portion being higher and that decomposition for four or
five days (without KOH) does not materially affect the vitamin value (Compare
Davies, loc cit.). The results given in Tables II and III (excepting those of
the goat) are shown graphically in Figs. I and II.
C
130 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
131
132 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
TABLE I
Nature of the sample
Healthy Bull No. 45.
(a) Central portion
.
.
.
.
.
.
(b) Peripheral portion
.
.
.
.
.
.
Healthy Bull No. 26.
(a) Central portion
.
.
.
.
.
.
(b) Peripheral portion
.
.
.
.
.
.
Healthy Goat A.
(a) Central portion
.
.
.
.
.
.
(b) Peripheral portion
.
.
.
.
.
.
(c) Central portion left at room temperature with-
out KOH for 5 days.
(d) Central portion left in the refrigerator for 5 days
as control.
Healthy Goat B.
(a) Central portion
.
.
.
.
.
.
(b) Peripheral portion
.
.
.
.
.
.
(c) Central portion left at room temperature . .
(d) Central portion left in the refrigerator as control
Weight of original
liver represented
by the chloroform
solution of the
unsaponifiable
matter actually
used for the
tintometric
estimation
20 mg.
40 mg.
10 mg.
10 mg.
Blue
units
per
gram
of
liver
(Moore)
400
388
250
200
1,300
1,175
1,325
1,275
1,075
1,000
1,100
1,075
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
133
TABLE II
Relation between concentration of the liver and the blue units
Weight in
mg. of
original
liver
50 . .
100 . .
200 . .
300 . .
400 . .
500 . .
Rabbit A
Tinto-
metric
blue
readings
(Lovibond)
3.2
6.0
9.3
11.3
12.8
14.2
Blue
units
160
150
116
95
80
71
Rabbit B
Tinto¬
metric
readings
2.6
4.8
8.8
10.8
12.2
13.3
Blue
units
130
120
110
90
76
66
Rabbit C
Tinto-
metric
readings
3.7
6.2
11.6
13.5
14.9
16.3
Blue
units
185
155
145
113
93
82
TABLE III
Relation between concentration of the liver and the blue units
Specimen
Healthy Bull
.
.
.
.
.
Goat affected with rinderpest . .
Weight in
mg. of original
liver
10
20
30
40
50
100
200
300
400
20
40
60
80
100
Tintometric
readings
2.0
3.5
4.5
5.8
7.2
11.0
13.8
16.0
17.6
4.7
7.2
9.4
11.2
13.3
Blue units
500
438
375
362
360
275
173
133
110
591
450
390
350
333
It will be observed that in none of these cases is the relation between the con-
centration of liver and the tintometric readings linear, although the unsaponifiable
fraction has been used in the test. [Compare Norris and Church, 1930]. For
obvious reasons, tests could not be carried out at very low dilutions. Our results
are in general agreement with those available in the literature. [Cf. Chakravorty,
Mukherjee and Guha, 1933 ; Datta and Banerjee, 1934, on fish oils]. From these
134 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
data it appeared that for comparative purposes, only the central portions of the
livers should be saponified and that as far as possible, blue units obtained from
the same amount of the original liver should be compared. It has, however,
been found that the latter requirement is almost impossible in practice, because
even among the same species, low amounts of the liver extract of healthy animals
gave measurable Lovibond readings, whereas with the diseased ones, comparatively
much greater amounts of the liver extract had to be used for accurate measure-
ment of the colour. While, therefore, an attempt was made to use the same amount
of the liver for the colour estimation, a second arbitrary standard of colour was
also chosen, namely between 3 and 5 blue of the Lovibond scale (as suggested
by Davies) and suitable amounts of material were used to give a blue colour with
the antimony trichloride reagent within this range. Without these precautions,
especially the weight of the material used, a comparison between the blue units
of animal livers, even of the same species, loses much of its value because of the
great variation in the Moore units per grm. of liver when different amounts of
the liver are used for their determination. In Tables IV, V, VI and VII, the
vitamin-A reserve in the livers of several species of animals under different con-
ditions are shown. The actual amount of liver used in the determination of
the blue units is shown in the tables in every case It must be noted that an
accurate comparison between two or several animals having a very high and
very low vitamin content is not possible under the conditions found in this investi-
gation, and as such only a rough comparison could be made.
TABLE IV
Hill Bulls
Serial
No.
1
2
3
4
5
6
Brand
No.
45
9
26
120
144
955
Condition prior to death or
slaughter
Healthy
.
.
.
.
Do.
.
.
.
.
Do.
.
.
.
.
Do.
.
.
.
.
Do.
.
.
.
.
Experimental T. B. case.
Four lymph glands and
left lung affected.
Feed
Grain,
green
pasture, hay.
Do. .
Do. .
Ten days on a
vitamin
A
deficient
ration;
no
green grass.
Do. .
Grain;
green
fodder, hay.
Weight
of liver
used
in mg.
20
20
20
20
40
100
Blue
units
400
438
250
250
218
33
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
135
TABLE IV—contd.
Serial
No.
7
8
9
10
11
12
13
14
15
16
Brand
No.
414
145
457
22
1162
107
582
156
636
55
Condition prior to death or
slaughter
Experimental T. B. Injected
with 10 mgm. bovine T. B.
culture
subcutaneously
and later intravenously.
Condition good.
Killed
after a year. No post
mortem lesions.
Experimental T. B.
Post
mortem lesions positive.
Condition fair.
Recovered from Rinderpest.
Condition rather poor ; died
of acute gastro-enteritis.
Rinderpest : died on the 14th
day : Coccidiosis as well.
Condition poor.
Rinderpest : died on the 14th
day : Liver icteric.
Rinderpest: died on the 12th
day.
Recovered from rinderpest :
healthy : Condition good.
Rinderpest: destroyed on the
5th day. Condition fair.
Suffering from Theileriasis ;
liver icteric;
Condition
poor.
Animal vaccinated against
Blackquarter.
Reacted
and survived. Subsequent-
ly infected per os with B.
pyocaeneous with negative
result. Destroyed.
Post
mortem. Petecheal areas
in liver; fatty changes,
mild congestion and early
cirrhosis in the liver.
Feed
Grain,
green
fodder, hay.
Do. .
Only hay during
the disease.
Do. .
Grain and hay
Hay . .
Grain, hay and
green fodder.
Hay . .
Grain, hay and
green fodder.
Do. .
Weight
of liver
used
in mgs.
50
50
100
100
100
50
50
50
100
100
Blue
units
225
160
130
80
67
170
215
165
78
50
136 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
TABLE V
Hill goats
Serial
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Brand
No.
Nil
"
"
"
"
. .
55
121
99
122
103
139
137
130
Condition prior to death or
slaughter
Healthy
.
.
.
.
Do
.
.
.
.
Do
.
.
.
.
Do
.
.
.
.
Do
.
.
.
.
Affected with Rinderpest
virus; destroyed on the
5th day : Condition fair.
Recovered from Rinderpest;
died of pneumonia and
debility: Condition very
poor.
Affected with Rinderpest.
Died on the 4th day.
Rinderpest.
Destroyed on
the 5th day.
Recovered from Rinderpest.
Died of penumonia and
debility after about 23 days
of virus inoculation.
Discontinued from Rinderpest
and Ecthyma.
Died of
pneumonia and debility.
Rinderpest.
Destroyed on
the 5th day.
Rinderpest. Died on the 6th
day—peritonitis.
Rinderpest.
Destroyed on
the 18th day.
Feed
Grain and green
leaves.
Do. .
Do. .
Do. .
Do. .
Green leaves ad.
lib.
Do. .
Do. .
Do. .
Grain and green
leaves.
Do. .
Green leaves .
Do. .
Grain and green
leaves.
Weight
of liver
used
in mgs.
20
20
10
20
20
200
100
20
20
20
20
50
20
20
Blue
units
1,125
913
1,150
725
800
75
20
591
388
588
600
165
375
500
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
137
TABLE VI
White Rabbits
Serial
No.
1
2
3
Brand
No.
..
..
..
Condition prior to death or
slaughter
Healthy
.
.
.
.
Do.
.
.
.
.
Do.
.
.
.
.
Feed
Grain and green
vegetables.
Do. .
Do. .
Weight
of liver
used
in mgs.
50
50
50
Blue
units
160
130
185
TABLE VII
Horses
Serial
No.
1
2
3
Brand
No.
364
. .
386
Condition prior to death or
slaughter
Suffered from chronic para-
plegia.
Condition poor;
destroyed.
Animal
suffered
from
" Kumri " ; destroyed.
Foal; 2 months old. Sprayed
intranasally with emulsion
of C. equi. Died of pneumo-
nia.
Feed
Green
fodder,
grain and hay.
Do. .
Milk .
Weight
of liver
used
in mgs.
200
200
300
Blue
units
88
53
50
TABLE VIII
Dairy calf, Holstein Hariana: No. 269 : 2 months old : suffered from
diarrhœa and died. Post mortem observation—Congestion of the intestine. Blue
units on 200 mgs. of liver—41.
DISCUSSION
In considering the foregoing results, it has to be remembered that the data
are not sufficiently numerous to allow any statistical analysis, and any conclusion
which may be drawn is of a tentative nature. It may be recalled here that
Wolff studied more than 900 cases and Moore studied over 300 cases. In one
D
VITAMIN-A IN THE LIVERS OF SOME FARM ANIMALS
139
No other animals have as yet been studied by us, but some references, often
of a qualitative nature, are available in the literature. Thus Rosenheim and
Webster [1927] stated that fats of the sheep, calf and ox contain on the average
as much as ten times the amount of vitamin-A as a good New Foundland cod
liver oil. Wilson [1927] made a comparative study of the vitamin-A content of
extracts of the livers of several animals. Wolff, Overhoff and van Eckelen [1930]
studied rabbit liver and Dann [1932] gives some data showing the passage of
vitamin-A from the mother to the foetus in the rabbit. Moore [1932] has given
some quantitative data on the vitamin-A content of the livers of different breeds
of cattle and has shown that breed has a very important effect in this connection
and Davies has given some data on sheep, ox and pig livers. Some work on the
vitamin-A content of pig liver has been reported by Dunlop [1934, 1935].
Lastly, mention must be made of the interesting investigation of Guilbert
and Hart [1934] on the storage of vitamin-A in cattle and of Guilbert and Hinshaw
[1934] on vitamin-A in poultry livers.
If a comparative study of the storage capacity of the livers of healthy animals
of different species for vitamin-A as given in this paper is made, it is found that
goat liver has a reserve which is more than twice that of the bull liver and six
times that of rabbit liver. Since carotene, which is the main source of vitamin¬
A in these animals, is supplied in fairly high amounts through the green feed,
it is likely that the marked variation in the vitamin-A reserve of the livers in these
three species of animals is due to a difference in the metabolism of carotene by
these species. Whatever the explanation may be, goat liver appears to be a good
source of vitamin-A.
SUMMARY
A study has been made of the vitamin-A reserve in livers of several species
of animals by the antimony trichloride test. It is found that the central portion
of the livers usually contains more vitamin than the peripheral portion, that
reasonable delay in sampling the liver does not produce any noticeable change
in the vitamin-A content and that the relation between the blue units and the
amount of the material used for estimation is not linear at the dilutions studied
in this investigation. There is a possibility that certain diseases, e.g., rinderpest
in bulls and goats, and advanced tuberculosis and Theileriasis in bulls may lower
the vitamin-A reserve, but this requires to be confirmed. Healthy goat liver
contains a much higher amount of vitamin-A than that of bulls, rabbits and
(probably) horses, and appears to be a good source of this vitamin.
In conclusion the authors desire to express their thanks to Mr. J. R. Haddow,
B.Sc, M.R.C.V.S., D.V.S.M., I.V.S., Serologist and Mr. F. Ware, F.R.C.V.S.,
I.V.S., Director, Imperial Institute of Veterinary Research, Muktesar, for their
constant interest in this investigation.
140 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
REFERENCE
Chakravorty P. N., Mukherjee, H. C. and Guha, B. C. (1933). J. Ind. Chem. Soc. 10,
361.
Dann, W. J. (1932). Biochem. J. 26, 1072.
Datta, N. C. and Banerjee, B. N. (1934). Ind. J. Med. Res. 21, 535.
Davies, A. W. (1933). Biochem. J. 27, 1770.
Dunlop, G. (1934, 1935). J. Agric. Sci. 24, 435 ; 25, 217.
Green, H. N. (1932). Lancet. ii, 723.
—
and Mellanby, E. (1928). Brit. Med. J., ii, 691.
Guilbert, H. R. and Hart, G. H. (1934). J. Nutrition, 8, 25.
—
and Hinshaw, W. R. (1934). Ibid., 8, 45.
Harris, L. J., Innes, J. R. M. and Griffith, A. S. (1932). Lancet, ii, 615.
Mellanby, E. (1934). Nutrition and Disease. Oliver and Boyd, Edinburgh.
—
and Green, H. N. (1929). Brit. Med. J. i, 1894.
Moore, T. (1932). Biochem. J. 26, 1.
—
(1932). Lancet, ii, 669.
Norris, E. R. and Church, A. E. (1929, 1930). J. Biol. Chem. 85, 477 ; 87, 139.
Rosenheim, O. and Webster, T. A. (1927). Nature, 120, 440.
Wilson, W. H. (1927). Biochem. J., 21 1054.
Wolff, L. K. (1932). Lancet, ii, 617.
, Overhoff, J. and van Eckelen, M. (1930). Deut. Med. Woch. 56, 1428,
1429.
AN AROMA-PRODUCING LACTIC ACID ORGANISM
ISOLATED FROM INDIAN DAIRY PRODUCTS
BY
N. V. JOSHI, B.A., L.Ag, M.Sc.,
Assistant Agricultural Bacteriologist,
AND
C. S. RAM AYYAR, B.A.,
Third Assistant Agricultural Bacteriologist.
(Received for publication on 12th July 1935).
Empirical natural starters in the form of curds or buttermilk for making
butter continue to be used in the Indian homes and by gowallas, the professional
dairy men in India, even up to the present time. Such starters were also in
common usage in Europe and America till about the year 1890, when after the
rise of the science of bacteriology, a discovery was made independently by Storch
in Denmark, Weigman in Germany and Conn in United States of America that
certain bacteria, which produce lactic acid from lactose in milk, were the causative
agents in the souring of milk or the ripening of cream. The investigations of
Storch led the way to the use of bacterial cultures first in Denmark for ripening
cream in place of the empirical starters consisting of buttermilk from previous
churnings of good butter which were used before. The chief findings of the classi-
cal discovery in the process of cream ripening and the investigations immediately
following it show that different kinds of lactic acid bacteria induce different kinds
of souring : (1) some give a clean sour taste, (2) some produce high acidity, (3)
others give an unpleasant tang, (4) while still others give a mildly acid but strongly
aromatic taste.
The bacterial cultures used as starters were, at first, considered to be pure
cultures of a single variety of the lactic acid organisms. The organism commonly
found in such culture starters is the variety which is predominant in sour milks
and recognised as Streplococcus lactis, (Lister) Lohnis. Orla Jensen has named the
variety occurring in well-ripened cream in dairies as Streptococcus cremoris (Orla
Jensen) and distinguishes it as different from S. lactis on account of some differences
in morphological and cultural characters. Both these organisms, however, produce
the same amount of lactic acid from lactose in milk and are similar in many res-
pects. It was soon realised that pasteurised cream, when inoculated with pure
cultures of any of these lactic acid organisms and ripened, did not always yield a
butter of full aromatic odour and mild acid flavour as was obtained with some
natural starters, i.e., mixed cultures used in certain dairies. This led to the
suggestion that there may be some other organisms responsible for the flavour in
butter from particular dairies which were renowned for their butter. A search was,
therefore, continued by bacteriologists for isolating and securing cultures of
( 141 )
142 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
bacteria, other tann the lactic acid organism, in the dairy starters which produced
butter with full aroma and good flavour, as well as in commercial starters made from
such dairy starters. The discovery that two different types of bacteria are present
in good starters was made in the year 1919 independently in three countries.
Bokehout and De Vries [1919] in Holland, Hammer and Bailey [1919] in U. S. A.,
and Storch [1919] in Denmark found that mixed cultures of S. lactis (Lister) Lohnis
or S. cremoris (Orla Jensen) and another type of organism when inoculated in
pasteurised cream produce the characteristic aroma of good butter. This type of
organism is called S. citrovorus or S. paracitrovorus by Hammer, ' X' bacteria
by Storch and ' Betacoccus cremoris ' by Orla Jensen. Although named differently,
these organisms appear to have the common characteristic of producing aroma
in butter.
While engaged in an investigation of lactic acid organisms in dahi, the prin-
cipal starter used by Indians, we made attempts to find out whether the dairy
products in India contained any flavour-forming organisms, similar to those just
mentioned and whether their activity continues throughout the year or is res-
tricted to any particular season. In this connection we examined several samples
of milk, dahi and butter by plating on purple lactose agar and making pure cultures
from single colonies appearing on the plates. While examining the cultural
characteristics of the several organisms isolated by us we came across one organism
which coagulated milk at 21°C. without any gas and with little acidity after incuba-
tion for 24 hrs. The flavour of the curd produced by this organism was different
from the flavour produced by the usual lactic acid organisms present in dahi.
Hence this organism appeared to combine in it the characteristics of both the lactic
acid producing and aroma-producing organisms, and we considered it likely to be
useful in dairy practice. A detailed study of the cultural and morphological
characteristics of pure cultures of this organism was, therefore, made and its ability
to produce flavour in butter was tested by using it as a starter for ripening
pasteurised cream. The results of this study are included in this paper.
EXPERIMENTAL
(1) Isolation:—Several tubes of sterile milk were inoculated with a small
quantity of each of the following :
(a) Butter of various brands,
(b) Spontaneously soured creams,
(c) Dahi from different localities,
(d) Milk incubated for a few hours after addition of lime juice. The ino-
culated tubes were incubated at 21°C. for four days. Out of these
tubes those which showed smooth coagulation were after necessary
dilution plated on purple lactose agar, and the plates incubated at
21°C.
AROMA-PRODUCING LACTIC AClD ORGANISM
143
From samples of (a), (b) and (d) several strains of streptococci were obtained
of which many proved to be S. lactis (Lister) Lohnis, while a few isolated from
butter samples appeared different. From samples of (c), in addition to streptococci,
another organism similar to the Bulgaricus type was most frequently isolated.
From milk samples in the cold season, strains of streptococci, similar to those
from butter samples, could be only occasionally isolated.
(2) The ability of the different organisms to produce volatile acidity.—Since
lactic acid is odourless, previous investigators had considered the mild acid
flavour in samples of good butter to be due to the formation of some volatile acid
produced during ripening of the cream by the organisms in starters. The fact
that good starters, when inoculated in milk and incubated for a week and then
distilled—after acidifying by sulphuric acid—with steam, gave a large amount of
volatile acid, whilst indifferent starters gave a decidedly lesser amount, appeared
to lend support to the above view. It was further found that the addition of
citric acid to milk or cream considerably increased the volatile acid produced by
the organisms in good starters.
The ability of some of the organisms to produce volatile acid in milk with and
without the addition of citric acid was, therefore, tested several times to find out
whether they possess this quality of good starters. 250 c.c. of milk was used in
each flask for inoculation with the respective organisms and after a week's period
of incubation, the whole quantity of milk was distilled with the addition of 15
c.c. N H 2 S0 4 in a current of steam, and the distillate collected until it amounted
to 1,000 c.c. and titrated with N/10. NaOH using phenolphthalein as indicator.
The results of one typical experiment are given in the following table :—
TABLE I
Volatile acidity produced by different organisms
Origin of the organism
1. S. lactis from soured cream
.
.
.
.
2. Bulgaricus type from dahi
.
.
.
.
.
3. Streptococcus from a sample of good butter . .
4. Streptococcus from milk and lemon juice . .
No. of c.c. of N/10. NaOH
required for neutralization
of distillate (Average of 4
tubes)
Milk
8.0
12.0
24.7
9.5
Milk and
citric acid
(0.40%)
10.0
14.5
43.9
10.5