Indiall J. Dairy Sci., 30, J, 1977, pp 30-35
RAPID DETECTION AND ESTIMAnON OF EXTRANEOUS
GLUCOSE IN MILK AND EFFECTS OF THE ADULTERANT ON CERTAIN PHYSICO·CHEMICAL
PROPERTIES OF BUFFALO MILK
N .K. ROY an d S.B. MfTTAL
Natiollal Dairy Research Institute. Kamal, India
Rccctved
00
2.S October. J915
ties of buffalo milk and pr(Xedures for storage
of samp les were identical to those reported
earlier (Mitta] and Roy, I 976a). Glucose of
L.R. grade was used for a\l studies. The
evolved methods were based on the principle of
determination of monosaccharides in presence of disaccharides in blood (Tauber and
Kleiner. 1932-33). Six sets of buffalo milk,
pure as well as mixed with glucose at different
levels of 0·5, 1·0,2·0 and 5·0% wi", were used
for these studies unles5 otherwise mentioned.
INTRODUCTION
Glucose, like urea and ammon ium sulphate
(Mittal and Roy, 1976a and b) when added to
milk for unscrupulous purpose of masking
the effects of watering (Anonymous, 1972)
poses problem for its rapid detection through
a platform test or estimation . Some rapid
methods of detection involve costly and uncommon reagents like glucose oxidase and redox
indicators (Bagnulo et al., 1968; Amariglio,
1966; Dorier and VereBe, 1962; Carvajal and
Dorbecker, 1957i , while other methods were
complicated with lengthy procedures often involving sophisticated instruments (Madrid
Vicente, 1972; Reineccius et al., 1970; Valen·
tinis and MaUioni, 1970; Nakanishi lind Tokita,
1958; Ramchandra et ai., 1955). The same is
true for various recommended methods for the
estimation of glucose in milk (Vuillaume,
1952; Anantakrishnan and Herrington, 1948;
Ramsdell, 1945). Consequently, investigations were undertaken to evolve a platform test
to detect, and a rapid colorimetric method to
estimate extraneous glucose in buffalo mi lk.
Further, inftuence of glucose addition on some
physico-chemical properties of milk was a Iso
studied. The details of methods evolved and
otber result. are presented here .
Reagents requir< d
(A) Modified Barfoed's reagent: Prepared
according to Tauber and Kleiner (1932-33)
using lactic acid instead of acetic ncid.
(B) Phosphomolybdic acid ,,'agent : Prepared according to Folin and Wu as described
by Oser (] 965).
(C) A. cetate buffer: IN sodium acetate and
IN acetic acid mixed in equal volumes, having
pH 4·75.
Detection." To one ml of milk with glucose
taken in a test tube I ml of reagent A was
added, a nd the mixture heated for 3 min in
boiling water bath . After cooling under
running tap water for 2 min, I ml of reagent
B was added to the turbid mixture when a deep
blue colou r was developed. The colour developed under identica l experimentation in a
control sample of pure milk was very pale blue:
MATERIALS AND METHODS
Buffalo milk samples, the methods used
for the study of the physico-chemical properI
2
N.K. Roy and S.B. Mittal
and similar to that of diluted reagent A. Alternatively, a protein free filtrate, prepared by
mixing equal volumes of milk and acotate
buffer. heating in boiling water for 3 mio and
filtering. could be used in place of milk as such.
In the latter case the blue colour formed witb
adulterated samples was morc prominent.
ESlima(ion: To 0·2 ml af protei n frce
filtrate from adulterated milk (prepared as
described above) was added 1·8 ml disti lled
water and 2 ml reagent A. and the mixture
heated in boiling water far a min jmum of 4 min.
After cooling for 2 min. 3 ml of reagent B was
added and contents mixed ,md filtered through
a dry Whatman No. 42 filter paper. The
tiltrate was collected in a colorimeter cell after
discarding first one ml , and its absorbance
measured in a photoelectric colorimeter, using
red filte r against a blank prepared identjcally
from a pure milk sample. The concentration
of glucose in the sample was determined with
tbe belp of a standard curve prepared with milk
samples containing known amou nts of added
glucose. The blue colour was stable from
its development upto 24 hr.
RESULTS AND DISCUSSION
Detectioll : Twenty five trials for detection
ofeJ<traneous glucose in m;lk in 9 concentrnt;ons
ranging from 0·0 I to l·O % (w/v) following the
procedure described in Ihe procedure section
established the efficiency of Ihe developed
method. Direct use of adulterated milk led to
rapidity and simplicity in such detection. although the use of acetate buffer precipitated
filtrate gave more sensiti~e results. It may be
noted tha t use of other protein precipitants
like tungstic acid, trichloro-acetic acid in place
of a cetic acid did not give any characteristic
colour. The intense blue colorat;on in presence
of added glucose was distinctly different
from
that
developed
in the control
sample, which was merely pale blue resembling
very dilute copper salt solution. Forty nine
more trials, in which several variations in
different facrors like strength of the reagents,
heating periods, etc., were adopted confirmed
reliability of t he evolved method. Strict
adherence to the details of the procedure and
use of lact ic aci d in place of acetic acid for preparat;on of modified Bnrfaed's reagent were
imperative to detect differentially the extraneou s glucose fwm natural one in milk. Other
adulterants like sucrose, urea and ammonium
sulphate did n ot interfere with the test. Interfere nce due to presence of fructose could be
eliminated by its differential detection with
Selivanol'f's test.
Estim ation : Resu lts from six trials, each in
dupl;cate , for es timation of glucose in mille
were statistically analysed to arrive at the
equation No. I.
... (1)
Y=258'6 X-3'35
wherein Y and X correspond to Klett reading
and concentration of glucose in gilitre. Very
good fitness of the observed absorbances on the
regressi on line givon by equation No.1 established the validity of Beer-Lambert's law for the
developed colour due to extraneous glucose
in the range of 0·0 l to 0·16 %. Recovery of
added gluco.e was 103 %, in the range of
96-11 0 %. The accuracy of the method,
calculated ,tatistically, was within -+. 2 %.
The recovery percent waS slightly on the higher
s;de and might have been due to the little amount
of glucose natura lly present in the milk. Rapidity 'in the development of the colour, instantaneously with the addition of phosphomolybdic acid reagent, and stability of the colour
even after 24 hr were additional advantages of
the procedure standardised.
InOuence of glucose on physico-chemical properties of buffalo milk
pH: The pH of buffalo milk, 6·869 at 30'C
for normal average, increased due to addition
of glucose up to 5 ~~ (Graph A in Fig. I), As is
evident from the slope of the different sections
of this graph, and from the statistical
Extraneous Glucose in Milk
Fig. 1 A
ReJatfop betWeeli tollCentration of extraneous
gloc.se aDd pH of OOIf.I. milk al30OC.
analys is in lI,e Table, such pH increase of milk
due to glucose addition was non-significant
of the concentration of glucose, unlike the
earlier reported effects of urea (Mittal and
Roy, 1976a) and ammonium sulphate (Mittal
and Roy, 1976b). Glucose might have interfe red with ti,e hydrogen ion activity of milk
merely as an inert diluent in adulterated milk.
Storing milk with added glucose at room temperature led to considerable lowering in pH of
milk, as dcscTibed through Graphs B to E in
Fig. 1. The control sample suffe red relatively
less decrease in pH than the adulterated samples,
whicll obviuusly was due to more development of acidity in the glucose containing milk
from rapid niicrobi.l growth.
Fig.lBloE
b.vlD~ different
«MIcmtrafions of added glllCose On storage at
22-2SoC.
Cbanso, in pH of buffalo milk
'0
1:&300
GLUCOSE ('1-1100-...1)
2<0 30 4·0
5-0
Electrical CO"dflcU1l1~e: The electrical conductance of norma! buffalo milk, 2·899
millimhos.icm at 30"C increased with addition
of glueo,e to milk (Fig. 2, Graph A). Such increase was ~ignificant only at P=0·05 in the
limited study up to 2 %concentration of glucose.
In fact, there was hardly any further increase
in electrical conductance of mill<: corresponding to 1·0 and 2·0 ~~ of glucose ove r tbat duc to
0·5 ~,~ glucose in milk. The effect of storage
o CONTROL
,,- 0·5 /.
"'-1'0/
• -:;0;'.
.q.-~H"'. CONCN·
r
•
4
I
I
STORAGE TIME CH><)
TABLE
Variance rlttios for the effect
or glucose 00
pbysJco·cbem~1
properties of buffalo milk at 3OOC.
---------------~---------------------------Variance ratio (F)
Sc. No.
Source of
var.iation
pH
L
Between samples
~.
Between concentra-
15'16""
1·38(; NS
Electric.1
conductance
19·80"
3·866'
Density
7'000·"'"
31O-0 U
Vjscosity
13·1S"
9 137"
Surface tension
S3·59"
0·0200 l':S
tiOD of glucose
3.
Due: to storage
period
4.
lnteration between
storage period and
concentration
101'1 ~, .
0·7598 NS
600-7' '"
Oo)1MiO ]'o;S
"'Significant at p ,,"-O·OS.
13·01' •
0·0220 NS
4·974"
0·2261 NS
4
NK. Roy and S.B. Mirtal
Fig. 1 A
Iaftueaee of extraoeoms glucose on electrial
.""d_otu..., or _010 milk at JOOC.
Fla:. 1 B 10 E
Effect
or stoNae OD tbe tlectrical eonducbtn~e
or bulrolG
0
OS
mllk taI.WIIl! added gl......
GLU:OSE (tlt· .. -..,.J ')
1·
2·0
2,~
5~
significant (Table). Increase in milk density
due to glucose was. however. not exactly
proportional to the molar concentration of the
added solute , but WaS greater at lower concendensity
tration. Moreover, rise in milk
due to glucose was greater than that due to
UTea (Mittal and Roy, 19700) and lower than
that due to ammonium sulphate (Miltal and
(Roy, 1976b) and was not in molar proportions.
,"0
.....
~
t
~
...,~
~
.....
'~.
"
QCONTROL
e _O-s, t
z
8
~
...
~
O-"S'
... _2· 1-
i!I- Ec"l,n,.CcNC-No·
~~
. ,
4
~E~c..J
of milk on its electrical conductance was very
similar on the pure milk and adulterated
samples. Such jnference is supported by the
actual findings (Fig. 2, Graphs B to 0), as well
as the non· significant variance ratio for inter·
action between storage period and concentration of glucose (Table). The electrical
conductance was,
however,
significantly
dependent on the storage period (F_600·7).
There was a sudden and sharp rise in conductance after storing for 12 hr and the same
was obviously due to the development of acidity during storage after such period (Fig. I).
Density: The average density at 30·C
tor normal bulfalo milk was 1·0315 gl ml, in the
range of 1·0309 to 1·0325 g/ml. Addition of
glucose increased the milk density to 1·0338
(1·0331 to 1'()346), 1·0356 (1·0352 to 1·0369),
and 1·0392 (1·0383 to 1·0405) corresponding
to 0·5, 1·0 and 2·0 % of glucose (ranges given
in parenthesis). Such density rise due to
adulteration with glucose WaS statistically
Vtscosity: n,e viscosity average (and
range) of norma l buffalo milk at 3O"C was
1·638 (1·578 to 1·683) cpo It increased sharply
to 1·665 (1·619 to 1·71 5) and 1·702 (J.638 to
1·791) due respectively to addition of 0·5 and
1·0% glucose, while 2·0 % glucose increased it
furt.her (Fig. 3). although less markedlY to
\·734 (1 ·674 to ! ·811). Influence of adulter.
rants on milk viscosity has been controversially
reported earlier. While Puri el al. (1963)
could not observe any change in milk viscosity
due either to starch or sucrose, Prakash and
Ram (1950) claimed that addition of starch
to milk could be detected from changes in
viscosity produced. There was also a fairly
marked increase in viscosity of milk. both
norma! and adulterated, on storing for 10 hr.
Unlike in adulteration with urea (Miltal and
Roy, J976b) there was greater rise in viscosity
of milk during storage with high concentration
of the adulterant. as is evident from the
increased slopes of Graphs 0 and E and as compared to those of Band C (Fig. 3). The greater
extent of bacterial degradation of glucose in
milk samples with higher glucose content
was
obviously
responsible
for
such
geater increase in viscosity. In fact, after
about 12 hr of storing the samples were too
viscous for measurement with Ostwald viscometer. Marked increase in viscosity due to
development of acidity in milk was reported by
Taperoouse and Vuillaume (1934) . Variations
in viscosity of milk with glucose concentrations
and storage time were statistically very significant (Tab!e,.
Extraneous Glucose in Milk
5
Fig. 3 A
Fig. 4 A
Variation iD viscosity of huffillo miJk (,OD11lirtit'!g
added glucose.
Effect of extraneous Klucose 0111 surface
t ...lon of bnIf.l" ndlk.
Fig. 3 B to E
Fig. 4 B 10 E
Elroct of .torago 00 v!scosity of buffalo milk
contRilling added glDcolie.
Varladon ill surf.<e teo.lon dnriDg stongo
of bulfalo mllk bovlog added glucose.
GUICOSE Ct/t•• .....tj
GUJCOSl C1IIM ......... )
10-0
1-'50
10
o
I B2S ;:.O_.......:O:,:~'--_..;'.O=--~.;t·rS_--'20r----.
11100
•
~
~
~450
"'
~
~77S
z
~
e4... ·5
"
~
~"!2I.o
~
1·750
CONTROL.
CI-O·Sl
<:I
•
·"~IOY.
1'725
c.
'<i.'
~
>- 1·700
I
....
~
•
STOI.lGE TJME- (Hr.J
1·'75
QCOHHoL
1·"50
+-.2>01'
-¢' -S·T- 'lJ'.o. CONCIJ
"-O·SX
... _1·0;1'
",-aOl
<I>- y ...CONCI<·
of either pure or adulterated samples caused
significant lowering in the surface tension,
irrespective of concentration of glucose in the
samples, as evident from variance ratios presented in the Table. However, addition of glucose could prevent to a certain extent the lowering in surface tension of milk during storage.
Alcohol test and clot on boiling test.' Study
witb 7 sets of samples of normal as well as
adultered milk revealed that addition of glucose
up to 2 % had no noticeable in.f!uence on the
··quality of milk, either in fresh or stored conditions, as judged by both alcohol 'and clot
on-boiling tests.
Surface tension,' Surface tension of normal
buffalo milk at 30·C decreased. but not significantly (Table) with increased concentrations
of glucose added to milk, (Graph A in Fig. 4).
The ranges for the individual samples were
considerably overlapping, namely, 40·31 to
SUMMARY
48·39,41·48 to 48·44, 41·39 to 48·02 and 40·82
to 47·92 for pure milk and milk adulterated
A platform test was evolved to detect extrawith 0·5, )·0 and 2·0 % glucose, respectively.
Mahajan and Mathur (1949) reported that addi- neous glucose iu buffalo milk. The method
tion of starch or other similar adulterants did consisted of mixing 1 ml milk with 1 ml modinot affect the surface tension of milk. Storing fied Barfoed's reagent, heating for 3 min in
N.J(. Roy tpUI S.B. Mittal
6
boiling water and cooling under tap water
fOT 2 min.
Addition of I ml phosphomolybdic
acid reagent to this mixture immedi ately deve.
loped a deep blue colour indicating the presence
of extraneous glucose in milk. This test could
detect the adulterant up to 0·01 %, even in
presence of sucrose, lactose , urea or ammonium
sulphate.
A simple and rapid colorimetric procedure
to estimate extraneous glucose in milk, with an
accuracy of ±2 %, was also evolved, employing
same reagents.
Study on physico-chemical properties of
buffalo milk containing added glucose revealed
that the adulterant caused significant increase
in density, viscosity (at P=O·OI ) and electrical
conductance (at P_O·05) of fresh and stored
buffalo milk. On the contra ry pH and surface
tension of buffalo milk increased not-signi/i.
canUy. Addition of glucose also did not
affect the quality of milk as judged through
alcohol test and clot·on-boiling test.
Anonymou, (1972) Communication from participants
in " Short course trainiDj on quality control in
dairy produclS manuf~1912" at NatiOllal
Dairy ReS(arch Illstitute. Kamal
Ba""ulo. R .. Moraantini. M., Acundo, G.D. and Caiola.
C. (1968) Lall•. , 42. 791 .
carvajal, S. and Dorbecter, F . (1957) Cicncia (Mex.)
l7, 33. (Dairy Sci. A(,sIT ., 11, 1718. (959).
Dorier, P. and Verene, L. (1962) Ann. Falsi/. Expert.
Chim., paris. 55, 177. (DaIry Sei. Abstr., 25,
285, 1963).
Ro.,.
Madrid Vincente, A. (1972)
esp. !..<1M., 83, 19.
(Dairy Sci. Abslr., 34, 2853, J912).
Mah;tjan. LD. and MaIllUT, O .C. (1949)
18, 204.
ew-r.". SCi.,
Mittal, S.B. and Roy. N.K. (19760) Inc(jan J. Dairy
Sci., 19~ 71.
Min •• ,
s.n.
and Roy, N.I(. ( 197661 I1rdlan J . D<J1r7
Sci., 29, 283.
Nakanishi , T. and 101<ilO, F. (J958)
Res., 9, 37.
Oser,
Tahak. J. Agrl.
B.L. (1965)
Editor, Hawk', phy$lolDgiad
.hem/Slry (TMH Ednn). Tata Mc·Graw HID
Publishing Cowpany, Limitc4, New Delhi.
Prakash, O. and Ram. (1950) Curr••t Sel., 19,
~.
Puri, B.lt. Sat Parka' h and Totaj.. K.K. (1963)
ACKNOWLEDGEMENTS
The authors express their thanks to Dr. N. C.
Ganguli, Head of Division, Dr. P.G . Nair,
Principal, and Dr. D. Sundaresan, Director of
the Institute for their kind interest in the
work.
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S. ((966) Lo"., 46, 11.
~I.krisbnan,
C.P. and Heriinaton, B.L. (1943)
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Indi<Jn J. Da,ry Sci. , 8, 83.
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Ramsdell, G.A. (1945) J. Dairy &i., :18, 671.
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P.O.
Tapcrnouse, A. and Vuillaume, R. (\934) LaU., 10&,
449.
Tauber. H. and Kleiner, I.S.· (1932.33). J. BioI. Ch<M.,
99, 24~.
.
Valentini., G. and ~atti.oni, . R . . (1m) '.' im1utlT~
AIl_nl, Pinerolo, 9, 57 (Dairy Sci. Almr:, 32,
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