NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
M. R. SUBRAMANIA SASTRY AND OTHERS
223
debittered seeds on the bullock. Besides, experiments on the goat with raw
powdered seeds with the husk intact and without in any way debittering them
have also been done and found to produce no harmful effects. It is left to
the stock owners to try the results of the latter experiments on bullocks and
other large animals and report their observations. Our findings indicate that
feeding with these seeds is bound to improve their health and general condition.
Investigations on the pharmacolog cal actions of the alkaloid are proceed-
ing and the results will be published in a subsequent paper.
SUMMARY
(i) The alkaloidal content of the lupin seeds received from the Agricul¬
tural Research Station, Nanjanad was found to be 0.1302 per cent.
(ii) The alkaloid lupanine has been isolated from the seeds in yields vary-
ing from 1.5 to 2 gm. from 8 kilogrammes of the material.
(iii) Debittered seeds fed to the goat did not produce any untoward symp-
toms.
(iv) Seeds fed with the husk intact and without being debittered also did
not produce any bad symptoms when given in quantities up to 12 ounces a
day ; but showed satiation on the 4th day after the feed was raised to
1 lb. a day.
(v) 25 to 50 milligrammes of the alkaloid, administered intravenously to
dogs weighing about 10 to 15 lb., caused a definite fall in the blood-pressure and
a dose of 100 to 150 milligrammes (1½ to 2¼ grains) proved lethal and it is
inferred that the alkaloid contained in 60 to 70 lb. or even more of the seeds
would be required to prove lethal to a bullock weighing about 300 to 350 lb.
(vi) The alkaloids of the seeds do not appear to produce any cumulative
effects.
(vii) Debittering of the seeds does not appear to be necessary and they
may, with safety, be fed raw (even with the husk intact)—powdered, crushed
or in any edible form—to the extent of 3 lb. a day for a bullock with an average
weight of about 300 lb. and in proportionately smaller quantities for smaller
animals.
ACKNOWLEDGEMENTS
This work was done at the laboratories of the Research Unit, Medical
College, Madras, under the auspices of the Imperial Council of Agricultural
Research who are financing the Scheme for research into the indigenous drugs
of India used in veterinary practice with special reference to their toxicology.
We are highly grateful for the impetus and the financial help given by the
Council.
Our thanks are due to the Curator, Government Botanical Gardens,
Ootacamund for the supply of the material necessary for the investigation and
also for the description and photographs of the plant.
We are deeply indebted to the Surgeon-General with the Government of
Madras, for affording all facilities for the working of the Scheme, and to the
Director of Veterinary Services, Madras, for the keen enthusiasm he has evin-
ced in the inauguration of the Scheme and for his direction and valuable
advice.
REFERENCE
Wehmer, C. (1929). Die Pflanzenstoffe 1, 526
STUDIES ON VEGETABLE RENNET
BY
RAMJI NARAIN, PH.D., D.Sc.
Second Agricultural Chemist, Punjab
AND
ATAM SINGH, B.Sc.(AG.)
Agricultural Research Institute, Lyallpur
(Received for publication on 21 August 1941)
C HEDDAR cheese is a well-known milk product whose importance lies not
only in its nutritive value but also in its better keeping quality. Of the
other preparations of milk very largely used in India, ghee lacks the milk
proteins of a high biological value much needed in the diet, particularly of
vegetarians ; while khoa and curd cannot be easily preserved over long periods.
Unfortunately, however, the use of cheese prepared by the usual method of
coagulating the milk with rennet derived from the stomach of the calf is held
to be objectionable by a large section of the Indian population. In order to
make such a useful product acceptable to this class of people, a search was
made for a milk coagulating agent of purely vegetable origin which could be
used for the preparation of cheese on a commercial scale.
Knowledge of plants possessing the property of coagulating milk dates from
the sixteenth century. Green [1893] mentions its presence in Gallium varum
the juice of which was described by Methelo in the sixteenth century ' Galium
inde nomen sortitum est suum quod lac coagulet '. Martin [1885] and Baginski
[1882-83] refer to the presence of such an agent in Carica papaya. Lea [1883]
extracted the active principle from the berries of Withania coagulans with
glycerine and sodium chloride solution. Christen and Virasoro [1935] were
able to obtain an active preparation from the flowers of thistle.
A large number of plants has been studied at this Institute, with the object
of selecting one capable of giving a preparation possessing active coagulating
powers. Amongst others the enzyme had been found to be present in Madica¬
go sativa, Lolium perenne, Androprogan schisenan, Ficus palmata, Ficus carica,
Withania somnifora and Withania coagulans. The enzyme from the first three
plants was found to be much less active than from the last four. Again it
was much more active in the latex of Ficus palmata and Ficus carica than in
the juice of these plants, a drop of the latex being sufficient to coagulate
10 c.c. of milk in two to three minutes. The latex from Ficus palmata, however,
imparts a very bitter taste to the curd and the separation of the active rennet
from the latex, owing to the great amount of labour and expense involved in
its collection, was not economical. The latex from Ficus carica, however, be-
sides being highly active, is free from this bitterness of taste. This plant grows
224
226
Studies on Vegetable Rennet
The activity of the product obtained by the three methods of preparation
described above was compared with the following results :
a. Salting out
.
.
.
.
.
b. Dialysis
.
.
.
.
.
.
c. Alcohol precipitation . . .
Activity
at
30°C*.
8200
15000
27000†
26000
Yield
per 100 gm.
of berries
gm.
5.2
1.1
1.9
The product obtained by using the first method, though greater in quantity
owing to its contamination with salt, is not only weak but also possesses an
unpleasant flavour and a bitter taste. The other two methods yield a product
which is more active and almost tasteless. It is obvious, therefore, that the
method of salting out is not satisfactory. Of the other two , one involving
the use of alcohol for the precipitation of the enzyme is simpler in manipula-
tion, takes less time, and gives a higher yield of the enzyme with almost as
much activity as that obtained by the method of dialysis.
Yeshoda [1941] has recently obtained a preparation of vegetable rennet
from Withania coagulans with an activity of only about 11000 at 30°C. The
product obtained by us gave an activity of 26000 at the same temperature.
B. Characteristic properties of the vegetable rennet
(a) The activity of the enzyme was measured at different temperatures
by adding a fixed quantity, viz. one c.c. of the rennet solution to 10 c.c. of milk
brought to and kept at different temperatures and noting the time of coagula-
tion. The results obtained are given below :
Temperature (Centigrade)
30
35
40
50
60
70
75
80
Time
of
coagulation
min. sec.
9—20
5—30
3—30
1—30
0—35
0—25
0—30
No coagula-
tion
* Expressed as parts of milk coagulated by 1 part of the enzyme preparation in 40
minutes at a fixed temperature.
† Calculated on salt free material.
RAMJI NARAIN AND ATAM SINGH 227
It will be seen from the above that the optimum temperature for the
action of the vegetable rennet is in the neighbourhood of 70°C. The activity
increases with a rise in temperature up to 70°C. beyond which it shows a falling
off till 80°C. is reached when it is altogether destroyed. Working with rennets
of animal origin Fleischmann [1876] found the optimum temperature of calf's
rennet to be 41 °C., while according to Waksman and Davison [1926] it is 45°C.
Euler [1910] gives it between 37°C. and 39°C.
Chodat and Rouge [1906] find the optimal temperature for syko-chymase
from Ficus carica to be between 75°C. and 80°C., while for rennet from Witha-
nia coagulans it is about 70°C. Evidently the optimal temperature for vege-
table rennets is much higher than for animal rennets and this fact seems to
have some relationship with the body temperatures of animals and plants.
Plants are exposed to the direct heat of the sun which in a torrid climate rises
to about 60°C. or even more. It has been found that the temperature of the
plant is not very widely different from the temperature of its environment and
the period of the metabolic activity of the plant synchronizes with that of the
photosynthetic activity. The economy of the plant therefore demands that
the chemical reactions going on within its tissues should be able to adjust
themselves in accordance with the temperature of its environment. Similar
adjustment of the activity of the enzymes to changes in environmental con-
dition was noticed by Narain [1918] in his studies of the change of activity
of the oxidizing enzymes in the sugarcane as a result of changes in the reaction
of media.
In order to determine the correlation between the activity of the enzyme
and the temperature of reaction mixture, another similar experiment was
performed under strictly controlled experimental conditions at 5°C. intervals
of temperature. In the case of animal rennet it has been found by Grimmer
et al. [1925, 1928] that between the range 20°-38°C. the duration of coagulation
is a logarithmic function of the temperature, of the following form :—
log (d 1 — a) — log (d 2 — a) = C (t 2 — t 1 )
where d 1 and d 2 are the duration and t 1 and t 2 are the respective temperatures,
a and c are constants whose values are 25 and 0.05 respectively. This rela-
tionship has been found to hold good in the case of vegetable rennet also
between 30°C. - 60°C. The following figures illustrate the application of this
equation to the calculation of the time of coagulation which compares well
with the actual time observed.
Temperature °C. . . . .
Duration observed (seconds) . .
Duration calculated (seconds) . .
30
770
770
35
390
444
40
240
260
45
145
157
50
100
102
55
65
67
60
45
48
(b) Effect of heat on the activity of the enzyme.—It is commonly known that
rennet kept in boiling water for a few minutes is completely destroyed. In
order to ascertain the changes in the activity of the enzyme kept at different
E
228
Studies on Vegetable Rennet
temperatures, a series of experiments was carried out, which gave the follow-
ing results :—
Temperature of heating
°C.
40
50
60
70
80
Kept for 5
minutes
Time required
to coagulate
10 c.c. of milk
min. sec.
6—20
6—45
6—30
7—10
Completely
destroyed
Kept for 10
minutes
Time required
to coagulate
10 c.c. of milk
min. sec.
6—20
6—30
6—35
10—0
Completely
destroyed
It will be observed that inactivation, although to a lesser extent, takes place
even at 70°C. and the enzyme is completely destroyed within 5 minutes at 80°C.
The results reveal a remarkable characteristic of the vegetable rennet that
although 70°C. is the optimal temperature for its activity it is also the tem-
perature at which it is considerably inactivated if kept for 5 to 10 minutes.
As compared with animal rennet it is more resistant to heat, the former
being completely destroyed at 55°C.
(c) Pre-heating of milk.—The temperature at which milk has been kept
before the addition of rennet also exercises considerable effect on its coagula-
tion with rennet. As will be clear from the figures given below activity falls
with an increase in the temperature to which milk is heated, the time of pre-
heating being the same, viz. 8 minutes in all cases.
Temperature at which milk was heated
°C.
Fresh milk
60
70
80
90
100
Time taken to
coagulate 10 c.c.
milk with 1 c.c.
of 0.25 per cent
rennet solution
at 30°C.
min. sec.
6 — 0
6 — 20
7 — 30
20—0
24—0
did not coagu-
late
In this respect the action of vegetable rennet is similar to that of animal
rennet.
RAMJI NARAIN AND ATAM SINGH 229
(d) Influence of the reaction of the medium.—Reaction of the medium
exercises a very pronounced effect on the activity of the enzyme. As a general
rule, the alkaline reaction retards and the acidic accelerates the activity.
A number of authors have found that in the case of animal rennet, acceleration
is either a direct function of hydrogen-ion concentration [ Van Dam, 1908-09
and 1910-11] or a logarithmic function of acidity [Grimmer et al., 1925 & 1928].
The activity of vegetable rennet as affected by different degrees of acidity can
be expressed by the equation :—
log d 1 — log d 2 =C (a 2 - a 1 )
where a 1 and a 2 are degrees of acidity according to Soxhlet and Henkel
(the number of c.c. of N/4 alkali used per 100 c.c. of milk), d 1 and d 2 are the
durations of the time taken for coagulation and C a constant-whose value is
0.03. As would be clear from the figures given below the observed and the
calculated values agree closely:—
Degrees of acidity according to
Soxhlet and Henkel.
Hydrogen in conc. (pH.) . .
Duration observed (seconds) .
Duration calc. (seconds) . .
7.55
6.39
730
723
10.25
6.08
570
604
11.56
5.90
555
548
13.78
5.75
495
474
16.45
5.62
420
395
18.12
5.48
350
350
(e) Effect of salts.—Salts exercise considerable effect on the activity
of the enzyme. Thus the presence in milk of sodium chloride and calcium
chloride in different concentrations has been found to affect the coagulating
power of the rennet added to it as shown by the figures given below. The
amount of milk in each case was 10 c.c. and that of rennet 1 c.c.:—
Sodium chloride
Concentration (Normality)
1.0
0.5
0.25
0.125
0.065
Without sodium chloride
Time
min. sec.
19—0
12—50
10—40
10—20
10—25
10—20
Calcium chloride
Concentration (Normality)
1.0
0.5
0.2
0.1
0.066
0.05
0.033
0.025
Without CaCl 2
Time
min. sec.
6—30
6—5
4—50
4—10
3—45
3—50
4—4
5—30
10—20
E 2
230
Studies on Vegetable Rennet
The data presented show that sodium chloride when present in amounts not
greater than that required to make a 0.125N solution does not affect the
rate of coagulation but higher concentrations of the salt exercise an increas-
ingly inactivating effect. For example, the duration of coagulation is doubled
with milk which is normal with respect to sodium chloride. Calcium chloride
in concentrations upto N/1 does not inhibit the process of coagulation. On
the other hand, it hastens the action of rennet especially in concentrations
between 0.033-0.1 Normal, where the time of coagulation is reduced to about
one-third of that with normal milk. Within this range (.033N-0.lN) the varia-
tion in the amount of calcium chloride has little effect.
(f) Influence of concentration of the enzyme.—Effect of increasing concentra-
tions of the enzyme on the time taken for coagulation is very clearly brought
out by the data presented below. In the case of animal rennet Segelcke and
Storch [1870] had found the product of enzyme concentration M and the time
of clotting T to be constant. The law of Segelcke and Storch holds good in
the case of vegetable rennet as well.
Milk
(c.c.)
10
10
10
10
10
10
10
10
10
10
Quantity of
rennet centi-
gram (M)
1.0
0.8
0.6
0.4
0.3
0.2
0.15
0.10
0.08
0.06
Time
seconds (T)
70
85
110
175
225
360
480
720
895
1200
K = (T × M)
70
68
66
70
68
72
72
72
72
72
(g) Effect of concentration of the substrate.—In order to see if concentration
of the substrate affects the velocity of reaction, an experiment was carried
out with different concentrations of milk, keeping constant the amount of
rennet added, and the time of coagulation noted. As will be seen from the
figures obtained, this rennet behaves similarly to the animal rennet in a similar
experiment by Reichel and Spiro [1905] where the relation was expressed by
the following equation :—
(t—t') M/ V—M=K
RAMJI NARAIN AND ATAM SINGH 231
where M is the volume of undiluted milk, V that of the diluted milk, and t and
t' the time of coagulation of the diluted and undiluted milk respectively.
This is shown by the following figures :—
Milk
(c.c.)
10
10
10
10
10
10
Whey
(c.c.)
0
1
2
3
4
5
Rennet
sol.
(c.c.)
1
1
1
1
1
1
Time of
coagulation
(second)
930
1065
1230
1350
1490
1635
Time
calc. for 10
c.c. of
diluted
milk
. .
968
1025
1038
1064
1090
(t—t') M/
V—M
..
1350
1500
1400
1400
1410
The above figures show that :
(i) The time of coagulation of milk increases with increase in its dilution
with whey which has been previously deprived of all enzymic activity.
(ii) The concentration and not the total quantity of the substrate de-
termines the velocity of coagulation.
(h) Effects of different radiations—(i) A solution of the enzyme in one-
inch layers was exposed for 10 minutes to ultra-violet rays from a mercury
vapour lamp. The solution was kept at a distance of 10 cm. from the source
of light. There was no diminution in its activity. This shows that ultra-
violet rays do not exercise any unfavourable effect on the enzyme.
(ii) Quantities of the rennet solution in thin layers not exceeding 0.5
inch in depth were exposed to lights of different wave length for a period of
three hours. It was seen that direct sunlight, indigo, blue, green, yellow and
red lights, had no influence on its activity.
C. Application of vegetable rennet in making cheddar cheese
As already mentioned, the object of the present investigation was to ob-
tain a vegetable rennet which could be successfully used in cheese making.
Various efforts have been made to achieve this end. Kothavalla and Khub¬
chandani [1940] used a dilute saline extract of Withania coagulans, without
any further treatment for purification. These authors found that keep-
ing all the conditions similar the time taken for coagulation decreased
from 10 minutes to 27 seconds as the temperature was increased from 110°F.
to 160°F. They finally selected 110°F. as the temperature for coagulating
milk as the most suitable one for the preparation of soft cheese. Two cubic¬
centimetres of a 15 per cent extract of the berries in 5 per cent salt solution
232
Studies on Vegetable Rennet
was found to coagulate one pound of milk with an acidity of 0.2 per cent
in 75 minutes. They further found that a larger quantity of the extract pro-
duced an unpleasant flavour in the cheese characteristic of the berries and a
low temperature increased the time of coagulation. To reduce the latter a
larger quantity of rennet was required but it affected the flavour. At a higher
temperature the curd was unsuitable for cheese. For making cheddar cheese
they found that a temperature of 105°F. gave a curd which compared favour-
ably with the one obtained with animal rennet at 86°F.
We have found that 1.2 gm. of the enzyme (prepared by the method
described by us) with an activity of 25,000 is able to coagulate 60 lb. of milk
with an acidity of 0.21 per cent in 45 minutes under the usual conditions of
temperature. The curd obtained is quite firm and free from bitter taste or
unpleasant flavour. During the last four years we have been making cheddar
cheese with this preparation and have encountered no difficulty at any stage
of the process and the final product has been found to be as good as that pre-
pared from animal rennet.
SUMMARY
In order to find out a substitute of vegetable origin for animal rennet for
the preparation of cheese, a number of plants were studied. The dried berries
of Withania coagulans were found to be the best source.
An active preparation of the enzyme free from the bitter taste of the berries
was prepared by extracting these with 20 per cent alcohol and precipitating
the enzyme with further addition of commercial alcohol.
The characteristic properties of the enzyme have been studied. It has
been found that :
(a) the enzyme is very sensitive to changes in the temperature of the
reacting mixtures, the reaction of the medium and the presence
of salts,
(b) pre-heating of milk, depending upon the temperature used, retards
the activity of the enzyme,
(c) the velocity of coagulation varies not only with the concentration
of the enzyme but also of the substrate,
(d) radiations of different wave lengths corresponding to indigo, blue,
green, yellow and red lights as well as direct sunlight have no
influence on the activity of the enzyme,
Using vegetable rennet, cheddar cheese was prepared under the usual
conditions of temperature. The cheese thus obtained was found to be as good
as that prepared with animal rennet.
REFERENCES
Baginsky, A. (1882-83). Z. physiol Chem. 7, 209
Beau, M. (1935). Ergebnisse der enzymforschung IV Band, 182-3
Chodat & Rouge (1906). Centralbt f. Bakt II, 16, 1
Christen, C. and Virasoro, E. (1935). Le Lait, 15, 354
Euler, H. (1910). Allgemeine Chemie der Enzyme, 33
Fleischmann, W. (1876). Das Molkereiwesen Braunshweig.
Green, J. R. (1893). Annals of Botany 7, 112
RAMJI NARAIN AND ATAM SINGH 233
Grimmer, W., Kruger, M. et Hinkelmann, E. (1925 & 1928). Milchwirtsch. Forschg.2
&6
Kothavalla, Z. R. & Khubchandani, P. G. (1940). Ind. Jour. Vet. Sci. & Ani. Husb.
10, 284
Lea, S. (1883). Proc. Roy. Soc. 36, 55
Martin, S. H. C. (1885). J. of Phys. 6, 340
Narain, R. (1918). Agri. Jour. Ind. Special Indian Science Congress Number
Reichel und Spiro (1905). Hofm Beiträge 7, 405
Russel, E. J. 1937. Report on the work of the Imperial Council of Agricultural Research
in applying science to crop production in India, 125
Segelcke und Storch (1870). Ugeskrift der Landmaend
Van Dam, W. (1908-09 & 1910-11). Revue générale du lait 7 & 8
Waksman, S. A. & Davison, W. C. (1926). Enzymes, 200
Yeshoda, K. M. (1941). Current Science 10, 23
Note added on 28 July 1942
The Director General of Supply, Foodstuffs Directorate, New Delhi has recently
(13 August 1942) found the vegetable rennet prepared by the authors to be fit for
the hospital requirements of Defence Services. One tablet weighing 0.4 gm. was able
to set one pint of fresh milk to a firm curd in 40 minutes.
A SEMI-MICRO METHOD FOR ESTIMATING ALLANTOIN
AND ITS EXCRETION IN THE URINE OF
FARM ANIMALS
BY
S. C. RAY, M.Sc., PH.D., A.I.C.
Animal Nutrition Section, Imperial Veterinary Research Institute, Izatnagar
(Received for publication on 22 September 1941)
E XTENSIVE studies on the nitrogenous material catabolized during a
nitrogen-free dietary regime have been carried out. On the basis of
reports of previous work Terroine [1933] concluded that endogenous nitrogen-
metabolism arises from two sources : (1) from body protein catabolism as
represented by urinary metabolites such as urea, amonia and amino-acids, and
(2) from cellular catabolism as represented by urinary metabolites such as
creatinine, creatine, allantoin and other purines. Although it is now well
known that allantoin represents by far the largest purine constituent in the
urine of ruminants, studies on endogenous nitrogen-metabolism in farm ani-
mals have been much handicapped through lack of a satisfactory method for
estimating allantoin in urine.
Published methods for estimating allantoin may be classified as follows:—
(1) Precipitation methods
Wiechowski [1908], Handovsky [1914], Hunter, Givens and Guion [1914],
Champagne and Murot [1931].
(2) Hydrolytic methods
Plimmer and Skelton [1914], Christman [1926], Fosse, Brunel and de
Greave [1929] Allen and Cerecedo [1931] and Fosse, Brunel and Thomas
[1931].
(3) Colorimetric method [Larson, 1932].
In the precipitation method, the titration involved at the final stage of
the estimation lacks a clear end-point. Moreover, the cost of the quantity of
phospho-tungstic acid required for clarifying the urine is prohibitive, when
several samples have to be analysed daily. The hydrolytic methods, also, are
not above criticism, owing to the general disagreement as to the exact course
and nature of the reaction. There is, moreover, the possibility of other urine
constituents liberating the same hydrolytic products as those liberated by
allantoin under the same conditions. Perhaps the most serious objection
against both these methods, as routine procedures, lies in the fact that they are
lengthy and laborious. The colorimetric method is both simple and speedy.
Nevertheless in several cases it has been shown that the ammoniacal copper
234