NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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351
I.—ISOLATION OF A NEW STRAIN OF STR. DIACETYL AROM
INDIAN DAHI
Various species of bacteria were isolated from dahi by the method of
plating on Giltner's litmus lactose agar of the following composition :—
1 . 5 per cent agar,
1 . 0 per cent peptone.
1 . 0 per cent lactose,
2 . 0 per cent azolitmin solution,
500 c.c. meat infusion,
500 c.c. distilled water.
The agar was prepared as ordinary whey agar making the reaction + 1
per cent and adding the lactose and azolitmin just before shaking.
As the aroma bacteria resemble other streptococci in appearance, a sys
tematic classification of these organisms can be based only by their production
of flavour and other physiological properties. The various colonies were
picked out and inoculated into milk tubes with a view to examining the deve
lopment of flavour. Marked variation was noticed in the different cultures.
From the tubes in which a conspicuously fine aroma developed, a new species
of bacteria was isolated. This was plated on Giltner's litmus lactose agar
and a culture from it gave a positive test for diacetyl.
The isolated bacteria, when inoculated into milk, gave a smooth curd
with a pleasing and delicate flavour. The cultural and biological characteris
tics of the bacteria are tabulated below.
Str. diacetyl aromaticus—
Morphology.—spheres 0.8 to 1.3 μ in diameter occurring mostly in
pairs and a few short chains. Gram-positive. Variations in size
occur in different cultures.
Agar colonies.—Small, opaque, slightly raised, irregular edge, with
granular growth inside, occurring as long chains in the colonies
some cells being elongated.
Agar slant.—Beaded and scanty growth with individual colonies.
Gelatin stab.—No growth, gelatin not liquefied.
Litmus milk.—Acid, coagulation, shows reduction of litmus.
Growth in milk.—Curdles the milk within 24 hours at 30°C. with a fresh
butter flavour. Occurs mostly in pairs and a few short chains.
Gives a positive test for diacetyl.
Potato.—Scanty growth, occurring in pairs and chains; some irregular
cells are also observed.
Yeast extract with 2 per cent dextrose and 2 per cent calcium carbonate.—
Very good growth, occurring mostly as long chains.
352
Aroma in Dahi
Indol.—Not formed.
Nitrates.—Not reduced.
Peptone broth.—Poor growth.
Acid.—formed in dextrose, lactose, sucrose, inulin and levulose. No
gas is formed in any of the sugars.
Aerobic facultative—
Ammonia.—Does not produce ammonia from peptone.
Growth in 4 per cent NaCl at 40°C.—Nil.
Growth at pH 9.2.-Nil.
Temperature relationship.—Grows at 21°C., 30°C. and 37°C. Coagula-
tion of the milk and growth is slower at 21°C. than at higher
temperatures.
Optimum temperature.—30°—37°C.
Thermal death point.—65°—70°C.
Source.—Indian ' dahi '
Acidity.—Table I gives the acidity produced at different temperatures.
(Acidity is expressed in number of c. c. of N/10 alkali required to
neutralize 10 c. c. of milk culture).
TABLE I
Time
24 hrs. .
48 „
.
72 „
.
.
.
.
.
.
.
.
.
.
30°C.
3.1
4.5
5.3
37°C.
4.3
5.3
6.1
45°C.
5.1
5.1
4.6
An examination of the cultural and biochemical characters shows that the
organism is different from Sc. lactis and Sc. cremoris. It is a powerful pro-
ducer of acetoin and diacetyl. Sc. lactis has been shown to be ineffective in
oxidizing acetoin to diacetyl in milk cultures [Michaelian, Farmer and Hammer,
1933 ; and Michaelian and Hammer, 1935, 1936]. According to Orla-Jensen
Sc. lactis ferments maltose and dextrin, produces ammonia from peptone,
grows in 4 per cent NaCl at 40°C. or at pH 9.2 [Yawger and Sherman, 1937].
The organism under study behaves very differently. It cannot be classed in
the citrovorus or para-citrovorus groups, since these show in milk a high
volatile acidity which is increased by the addition of citric acid and do not
produce any detectable quantities of acetyl-methyl-carbinol and diacetyl in
R.
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353
milk alone, but do so on the addition of citric acid [Joshi and Ram Ayyar,
1936]. The organism described in this paper does not produce a high volatile
acidity in milk and produces a considerable amount of acetyl-methyl-carbinol
and diacetyl without the addition of citric acid to milk.
The organism can be considered to be a new species, its chief characteris-
tics being the production of aroma, mainly by the formation of diacetyl in the
culture.
II.—PRODUCTION OF AROMA IN MILK CULTURE INOCULATED WITH THE NEW
STRAIN OF DIACETYL AROMATICUS
Experimental.—Estimation of total diacetyl.—[Barnicoat, 1936 ; Schmal.
fuss and Barthmeyer, 1929, 1932].
(Acetyl-methyl-carbinol and diacetyl). Diacetyl is condensed with
hydroxylamine hydrochloride to form dimethyl glyoxime which can be esti-
mated gravimetrically as the nickel compound. It is always desirable to use
an all glass apparatus with no rubber connexions. One litre of the milk cul-
ture, mixed with ferric chloride solution is distilled in a three litre flask on an
oil-bath maintained at 110°C.—120°C. into a receiver containing excess of
diacetyl reagent (two parts of 20 per cent neutral hydroxylamine hydrochloride,
two parts of 20 per cent sodium acetate and one part of 10 per cent nickel sul-
phate)*. For the conversion of all the acetyl-methyl-carbinol to diacetyl
4 to 5 per cent ferric chloride is used. The adapter of the Liebig's condenser
is dipped in the diacetyl reagent in the receiver. The nickel dioxime is formed
in the receiver at the boundary between the reagent and distillate as red pris-
matic needles. Fractions of 50 c. c. of the distillate are collected until all the
diacetyl is distilled. The precipitation of the dioxime is completed by heating
on the water bath at 95°C. for one hour and leaving at the laboratory tempera-
ture for 24 hours. The nickel compound was filtered through a tared sintered
Jena glass crucible (Ig 3 ), washed several times with cold distilled water, dried
at 110°C. and weighed. The weight of the precipitate multiplied by 0.596 is
the weight of diacetyl. The method is quite accurate and trustworthy.
Volatile acids (Hammer's method)—
The volatile acidity was determined by steam-distilling a 250 gm. portion
of the milk culture after the addition of 15 c. c. N/10 sulphuric acid, collecting
the first one litre of the distillate and titrating against standard sodium hydro-
xide. The distillation was performed at such a rate that approximately two
hours were required to secure one litre of the distillate. The results are re-
corded in terms of number of c. c. of standard alkali (N/10) required to titrate
* The hydroxylamine hydrochloride solution should be freshly prepared and the
nickel sulphate filtered from any precipitate before use.
354
Aroma in Dahi
one litre of the distillate. Only a portion of the volatile acid is secured with
this procedure so that the method is of value only for comparative purposes.
Acidity.—The pH determinations were carried out according to Wulffs'
method.
Conditions of maximum flavour development in the culture.—Influence of
yeast extract*.—The influence of yeast extract on aroma production was
studied by culturing the organism in the usual milk culture to which 10 per
cent yeast extract had been added.
Quantitative determinations of the diacetyl content were carried out,
using one litre of the milk culture for each estimation. Table II gives com-
parative figures for the diacetyl content in the cultures, one of which contained
yeast extract (temperature of incubation 28°C).
TABLE II
1 litre of culture (blank)
pH.
5.3
5.1
4.8
4.6
4.6
Time
(hrs.)
24
48
72
96
120
Weight of
diacetyl in
mg.
39.63
64.95
76.88
65.55
38.86
1 litre of culture with the addition of 10
per cent yeast extract to the milk
pH
5.1
5.0
4.8
4.6—4.4
4.6—4.4
Time
(hrs.)
24
48
72
96
120
Weight of
diacetyl in
mg.
53.69
72.71
82.35
70.91
45.17
Yeast extract enhances flavour production. The diacetyl content reaches
the maximum in 72 hours, and decreases after this period. There is a marked
increase in the production of diacetyl in the culture containing yeast extract.
Apart from the improvement in flavour the stimulating effect of the yeast
extract on the growth and development of acid is remarkable.
A marked increase in the diacetyl content is also noticed when the culture
of the aroma bacteria is grown in combination with lactic acid bacteria as
shown in Table III.
The yeast extract was prepared by autoclaving pressed yeast with water. One
lb. of yeast is extracted with water and made up to one litre.
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355
TABLE III
Diacetyl content of mixed cultures of aroma bacteria and lactic acid bacteria.
(pH 4.4 ; period of incubation five days; temperature 28° C.)
Culture
1 litre of standard culture of aroma bacteria
.
.
.
.
.
1 litre of culture of aroma bacteria plus lactic acid bacteria . .
Weight of
diacetyl in
mg.
48.27
58.16
Influence of citric acid and citrates on the production of diacetyl.—The
addition of citric acid (0.2 per cent) to the milk, before inoculating the cul-
ture, does not increase the diacetyl content during the first 48 hours as shown
in Table IV. On the other hand, the addition of sodium citrate (0 2 per cent)
exerts a favourable influence (Table V).
TABLE IV
Effect of citric acid on the production of diacetyl (temperature of incubation
28°C.)
1 litre of culture (blank)
pH
5.1
5.0—4.8
4.4
4.4
Time
(hrs.)
24
48
72
96
Weight of
diacetyl in
mg.
15.68
17.76
19.13
18.89
1 litre of culture with addition of 0.2
per cent citric acid to the milk
pH
4.9
4.7
4.4
4.4
Time
(hrs.)
24
48
72
96
Weight of
diacetyl in
mg.
12.40
14.01
21.87
19.48
Citric acid seems to have an inhibitory influence on the production of
diacetyl in the initial stages. After 72 hours a slight increase in the diacetyl
content is noticed.
356
Aroma in Dahi
TABLE V
Increase in the production of diacetyl by addition of neutral sodium citrate to
milk; (pH 4.2—4.4 ; period of incubation 48 hrs.; temperature 28°C.)
Culture
1 litre of culture (blank) .
.
.
.
.
1 litre of culture with 0.2 per cent sodium citrate
.
.
.
.
.
.
.
.
Weight of
diacetyl in
mg.
29.44
33.90
Table VI incorporates comparative figures for the diacetyl contents of
cultures with 10 per cent yeast extract and neutral sodium citrate (0.2 per
cent). It will be observed that the quantity of diacetyl produced in the cul-
ture containing yeast extract is considerably higher than that in the culture
containing citrate (Table VI).
TABLE VI
Period of incubation 48 hours ; temperature 28°C.
Culture
1 litre of culture (blank)
.
.
.
.
.
.
1 litre of culture with addition of 0.2 per cent sodium cit-
rate to milk.
1 litre of culture with addition of 10 per cent yeast extract
to milk.
pH.
4.4
4.3
4.0
Weight of
diacetylin
mg.
27.49
35.93
52.57
Volatile acidity.—Measurements of volatile acidity in the milk cultures
were carried out after the addition of various quantities of citric acid, citrates,
yeast extract and a mixture of yeast extract and neutral sodium phosphate
R.
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357
to the milk before inoculating the culture. The results obtained are tabulated
in Table VII.
TABLE VII
(250 gm. of milk culture used for each distillation)
Culture
Blank
.......
Addition of 0 . 2 per cent citric acid . .
Blank
Addition of 0 . 2 per cent citric acid . .
Addition of 0 . 3 per cent citric acid . .
Addition of 0 . 4 per cent citric acid . .
Addition of 0 . 2 per cent neutral sodium cit-
rate.
Time
(hrs.)
72
72
24
24
48
96
120
Temp. °C.
37
37
37
37
37
37
30
N/10 alkali
in c.c.
5.6
5.6
5.3
4.4
4.3
3.9
5.2
Citric acid and citrates do not appreciably increase the volatile acid
content of the culture (Table VII). It is interesting to note, however, that
the volatile acidity is increased by the addition of yeast extract, and a mixture
of yeast extract and neutral sodium phosphate to the milk (Table VIII).
TABLE VIII
Culture
Blank
Addition of 10 per cent yeast extract
Ditto
Addition of 5 per cent yeast extract .
Addition of 5 per cent of yeast extract plus
0.2 per cent sodium phosphate.
Blank
5 per cent yeast extract . . . .
10 per cent
„
....
15 per cent
„
....
20 per cent
„
....
Time
(hrs.)
68
68
92
68
68
68
68
68
68
68
Temp. °C.
30
30
30
37
37
30
30
30
30
30
N/10 alkali
in c.c.
4.3
10.9
14.4
4.6
5.7
3.9
6.0
6.2
7.6
8.2
358
Aroma in Dahi
Effect of neutral sodium phosphate.—In another set of experiments neutral
sodium phosphate was added up to 0 02 per cent to the milk before inocula-
tion to find its effect on the flavour and aroma of butter. The aroma of the
resulting butter was superior compared to the blank. Yeast extract (1 to 2
per cent) in combination with phosphate, when added to the milk, gave a
much better sample of butter.
The stimulating effect of phosphate on the fermentation by the aroma
bacteria can be demonstrated by the increase in gas evolution in a culture
medium containing yeast extract, 2 per cent dextrose and 2 per cent calcium
carbonate. Addition of 0.2 per cent to 0.3 per cent phosphate re-starts the
fermentation in a culture when fermentation has stopped in the usual course
after inoculation of the culture of the aroma bacteria. The rate of the re-
action before and after the addition of phosphate is shown in Fig. 1.
FIG. 1. Effect of potassium phosphate on gas evolution by Str. diacetyl aromaticus
in yeast extract.
These studies have shown that the new aroma bacteria gives a fine-
flavoured butter and is a powerful producer of acetyl methyl carbinol and
diacetyl. The fact that the aroma of butter can be considerably improved
by the addition of yeast extract and phosphates seems to open up new
possibilities in the field of cream ripening for butter-making.
R.
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359
SUMMARY
A new species of butter aroma bacteria Str. diacetyl aromaticus has been
isolated from Indian dahi by plating on lactose-litmus-agar. This charac-
teristic type of Streptococcus is a powerful producer of acetoin and diacetyl in
milk culture without the addition of citric acid or other acids and produces
less volatile acidity than the citrovorus group.
This organism is different from Sc. lactis in its biochemical characteristics.
It also differs from Sc. cremoris and Str. citrovorus and Str. paracitrovorus.
Aroma development in the culture is highest at a temperature of 25°-28°C
after 48 to 72 hours' incubation. The maximum amount of diacetyl is pro-
duced in the culture after 72 hours when the pH is about 4.8. Addition of
yeast extract to the milk improves the flavour and the yield of diacetyl. In
association with a lactic acid organism the aroma bacteria gives a higher yield
of diacetyl. Neutral sodium citrate increases the yield of diacetyl to an
appreciable degree. The volatile acid content of the culture is not increased
by adding citric acid and citrates (0.2 to 0.4 per cent) to the milk. Yeast
extract has a pronounced effect in increasing the volatile acidity. A mix-
ture of yeast extract and phosphate increases the volatile acid production.
The flavour of butter is improved on the addition of neutral sodium
phosphate to the milk before inoculating the culture. A mixture of phosphate
and yeast extract gives better results. Phosphates have a stimulating effect
on the production of gas by Str. diacetyl aromaticus.
This species of Streptococcus would appear to have great possibilities in
butter-making.
ACKNOWLEDGEMENT
The author wishes to thank Dr V. Subrahmanyan and Mr B. N. Banerjee
for their kind guidance and help throughout this work.
REFERENCES
Barnicoat, C. R. (1935). Analyst 60, 656
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Boekhout, F. W. J. and Ott de Vries, J. J. (1919). Zentr. Bakt. II & 49, 373
Davies, W. L. (1934). Analyst 59, 46
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–––––––––––––(1937). C. A. 31, 3164
Hammer, B. W. and Bailey, D. E. (1919). Iowa Agric. Expt. Sta. Res. Bull 55,
and subsequent Bulls.
––––––––––––– (1920). Iowa Agric. Expt. Sta. Res. Bull. 63, 59
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Proc. World Dairy Cong. 2, 985
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Proc. 24th Ann. Conv. Int. Assn. Milk Dealers, Lab. Sec-
tion, 21
360
Aroma in Dahi
Joshi, N. V. and Ram Ayyar, C. S. (1936). Ind. J. Vet. Sci. and Anim.
Husb. 6, 141
Kluyver, A. J. (1933). J. Soc. Chem. Ind., 369-T
Knudsen, S. and Sorensen, A. (1929). Zentr. Bakt., II. 79, 75
––––––––––––– (1931). J. Dairy Res. 2, 137
Michaelian, M. B. (1932). Iowa State College J. Sci. 6, 455. (Abstract of
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Farmer, R. S. and Hammer, B. W. (1933). Iowa Agric. Expt. Sta. Res.
Bull. 155. Expt. Sta. Rec. 69, 103
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Orla-Jensen, S., Orla-Jensen, A. D. and Spur, B. (1926). J. Bact. 12, 333
Ritter and Christen , (1935). Landw. Jb. Schweiz. 49, 749
Ruehe, H. A. (1937). C. A. 31, 4735
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Storch (1919). Forsog laboratoriets, 102 de Beretning
Schmalfuss, H. and Barthmeyer, H. (1928). Ztschr.f. Physiol. Chem. 176, 282
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Van Niel, C. B. (1927). Biochem. Ztschr. 187, 472
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Kluyver, A. J. and Derx, H. C. (1929). Biochem. Ztschr. 210, 234
Van Beynum, J. and Pette, J. W. (1937). C. A. 31, 169
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DAMAGE TO HIDES CAUSED BY CATTLE TICKS IN
INDIA
BY
B. N. SONI, B.Sc. (PB.), B.Sc. AGRI. (EDIN.)
Imperial Veterinary Research Institute, Mukteswar
(Received for publication on 24th July 1939)
(With Plate XXVII)
INTRODUCTION
THE Hides Cess Enquiry Committee, in their Report published in 1929-
30, remarked as follows :—
' A large body of opinion holds that next to the Warble-flies the parasites
which cause most damage, both direct and indirect, to hides and skins are
ticks. They are found both among cattle and sheep.' The committee fur-
ther stated: ' It has not been found possible to estimate the extent of the
damage caused by ticks to hides and skins
We cannot give separate
figures in respect of tick damage, but we are convinced that the total loss must
be enormous, and even the damage to hides and skins perhaps amounts to
several lakhs of rupees per annum.' Some evidence in confirmation of the
foregoing statements was obtained by the present writer when, during August
1938, he visited certain well-known tanneries at Cawnpore. Here a good
number of tanned hides showing tick marks were observed and there was a
general complaint from the tanners regarding this damage which was causing
an enormous monetary loss to them. Specimens of so-called ' ticky ' hides
showed tiny sores more or less healed and breaking the smoothness of the
grain. Again, in October 1938, at the suggestion of Mr. F. Ware, Animal
Husbandry Commissioner with the Government of India, the writer inter-
viewed Mr. J. Bleeck, a member of the Hides Cess Enquiry Committee
and a representative of the firm of European shippers of hides in
Calcutta. This gentleman was of the opinion that the enormous damage
caused by ticks to hides and skins in this country demanded immediate
attention. In addition to providing some useful information regarding
tick damage in India, which will be summed up later in this article,
Mr. Bleeck supplied the writer with some pamphlets dealing with the
subject of tick damage to hides and skins occurring in countries outside India.
Extracts from these pamphlets, as reproduced below, will show the importance
of such damage, and, indirectly, its bearing upon the hide industry in India
which, according to the Hides Cess Committee Report, produces about one-
third of the total production of hides and skins in the world, while, among the
chief stock raising and hide producing countries, she is in a position to spare
a big surplus for export. Thus, Messrs John Miller & Co., Chicago (cited in
Damage to Hides caused by Cattle Ticks published by the proprietors of
( 361 )
362
Damage to hides caused by Cattle Ticks in India
Cooper Cattle Dips) observe : ' Cattle ticks have a very deteriorating effect
upon hides and calf-skins, particularly calf-skins. We do not buy many
Southern hides or skins on account of the ticks, but when we do get some here
we are obliged to sell them for No. 3 stock at about one-third less price than
good Northern stock free from ticks. This does not apply so much to heavy
hides for sole leather purpose but for all light hides and calf-skins, it renders
them altogether useless, for all kinds of leather. It would be greatly to the
advantage of all concerned, both hide dealers, tanners and cattle raisers, if
something could be done to destroy ticks.' Messrs. Emery & Co., Chicago
(cited in the same work), write : ' Ticky hides or Southern hides do not sell
for as much money as the Northern hides. These ticky hides when unhaired
are all spotted and make a very poor leather and most tanners refuse to buy
any Southern hides at any price.' Mr. J. M. Bond, Chicago, remarks : ' In
regard to cattle ticks, I would say that they damage the hides so badly
that very few of them can be sold in this market and when they are sold,
the price is from 2 to 2½ cents lower than the price of our Northern and
Western hides. This reduction is largely due to tick damage, though partly
to poor take-off. I handle very few Southern hides on account of ticks.'
Considering the ecological conditions of India which are very favourable
to the increase of tick population, the writer is of the opinion that the tick
problem is probably not less serious in India than it is in the United States of
America, where an enormous amount of money is spent every year upon the
control of these pests. A large percentage of the chrome leather now pro-
duced in India is finished with the grain left on so that all imperfections and
tick marks on the grain side show very plainly. Formerly leather for uppers
was made from bark-tanned stock and was buffed and the grain removed.
For this leather tanners could use cheap hides that were covered with imper-
fections and tick marks. The situation today, as it has been explained, is
very different, as there is an increasing demand for grained leather, and for
this large proportions of Indian hides will not be available until the tick is
eradicated.
EXPERIMENTAL OBSERVATIONS ON TICK DAMAGE
' The injury caused to hides by the perforations made in the skin
by the maggots of the Warble-fly are familiar to most cattlemen. They
are large and local (on the back) and form conspicuous blemishes that at
once discount the value of the hide stock so affected. The smaller but much
more numerous pit marks made by ticks are less familiar to them because less
obvious, but are recognized by purchasers of hides as by no means negligible
blemishes, not only because they are punctures in the hide substance, but by
reason of the permanent stains that surround each puncture on the finished
leather.'.—Damage to Hides caused by Cattle Ticks.
The blemishes caused by tick bites are variously described by experts
in handling hides as ' abrasion upon the grain of the hide', ' break in the grain
Ind. Jour. Vety. Sei. & Anim. Husb. Vol. IX, Part IV ]
PLATE XXVII
FIG. 1
Portion of raw hide showing tick bites
FIG. 2
Tick marks on a piece after it had been tanned
FIG. 3
Tick marks on finished leather