Kumar, P;gmrnt of COW milk
Studies on the new green pigment of cow milk.
I. Characterization
M. ARUN KUMAR',
N.N.
OASTUR and M.V. BHAn'
Natio'nal" Dairy Research Insr!tute , Adugod i. Bangllo re. India
1. Introd uction
h is known that pigmentation in milk varies fro m species to Spe ·
cies. Cow milk is dch In rat s-oluble carotenoid s, whilst Eoat, sheep
and buffalo milk are almost devoid of this pig ment. Amongst the
wa.ter solUble pigments, riboflavin is prese nt in the milk of all the
.abo\le species.
Bile p j gment~, biliverdin alld bili..-ub in have bee n ideMified in tne
milk and btJl~r!u (..-om buffalo milk (4). While study ing the bile
pjg~nls of bufb:lo milk, DANtrL (5) observed the ~aci.tioll o f a
neW green pigment wi th the ,~in fract ion of fresh cow milk. This
green pigment which can be extracte d from caSein wit h .acetone by a
similn procedure used fOJ biliverdin extract ion fro m the buffa.lo
milk, is. dininct from the bile pigment, bilillel'din , Identified tn frt:ih
burf;lio milk. The prelicnt piper describ es a st udy on th~ new f3t
soluble green p(gment of "fresh cow mille
2. Marer"Is and method.
Pooled fresh cow milk ..,niples were o buined from the
Institute's herd which compri'!ied indigenous and exot ic
breeds.. Tetrame[hylsila .l e and deuterated chloroform were
obtained from Sigma Chemical Co., U.S.A. All other reagent'
were of analytical grade.
2.1 Ex rraction and purification of the green pigment of
cow milk
As the concentration of the green pigment was very lo w
It 0 1'&/1),
the pigment extracted from 20 I of milk per da y
was. stored at refrigerated temperatures an d thc proces.s rc·
pcated till 5 mg pigment from 600 I of milk was <olIected
s.pread over a period of nearly a month .
Citric acid precipitated casein was prepared from 20 I of
fresh skim mil k and the pigment wa, ex trac ted by soaking
the material in 3 I of acetone ovcrn ight. The acetone extract
was filtered, concentrate d (by vacuum d istillation) and the
pigment tran,ferred to ethyl ether by ex tracting the concentrated aqucou:s. acetone layer 3 times wilh 1 I of ('"!her.
This ether extract o f the green pigment waS evaporated to
dryness, esterified and purified as described for biliverdin (9).
2.2 Criteria of purity
The homogeneity of the e'terified green pigment was
confirmed by thin layer chromatograph y on silica gel G
plate, (20 em x 20 em) using the so lvent syuem benzene :
petroleum ether (60-80 0C); methanol, in the ratio of
9: 5: 1, described by ROOIGER and O'CARRA (15) for bil.
pigment separation. Biliverdin dimethyl .ster was ,potted
~s a reference compound. The plate, were developed by as·
cending chromatography In closed chambers lined internally
with filter paper sawrated with the solvent system till a run
",f 16 em was obbined. The pigment was washed 011 the
'ilia gel with .. rbon IA:tuchloride 3 times to remove all
trace, of the development solvents to minimize impurity
1 DepA.rtment of Bioche mistry. College of Fhhtrlti, Milngalore, Indiil
'Drputment of Orgilnl( Chemistry. Indi.an Innitule of Sc:ienc:c,
Biln,.Io,e.
IncH~
signa.ls in the nuclear magnetic;. resonance spectra. The p igment was then eluted with chloroform.
The purity of the 8reen pigment was further confirmed
by high performance liquid chromatography . HPLC was
carried out on Waters Model 6000 A, packed with Waters I'
Bondapak C 18 column eQuipped with ulltav io let detector.
The mobile phase was methanol at 1-2 ml/mln . The retent ion time of the green pigment was compared with that of
bililJerdin dimethyl este"r.
2.3 Electronic absorption spectroscopy
Ultraviolet vis.ible absorption spectrum was recorded in
spectroscop ic grade chloroform on a Gilford 250 double
beam recDr ding spec tr ophotometer.
2.4 In frared spectroscopy
Infrared spectrum was recorded on the Perkin Elmer
(Model 700) spectrometer.
2.5 Nilclear magnetiC resonance spectroscopy
Pro ton nuclear magnetic reSonanCe spectrum was obtained by p ulse d and Fourier-uamform nuclear magnetic
spectrometry on a Brucker 270 MHz spectrometer at She
Department of Physics, Indian In!.titute of Science , Banga·
lorc. Samples were disso lved in deuterated chloroform and
tetramethyl,ilano was the internal standard .
2.6 Qualitative rests for rhe green pigment of cow milk
As the concentratio n o f the green pigment was extremely
low, systematic method, of idtntification generally used for
the qualitative analysis of organic compounds ,"ould not be
.employed. Hence it waS tested for the possibility of bile
p igment' react ions base d on the analogy to the green colour
of biliverdin of buffa lo milk. Gmelin', test and Van den
Bergh 's tc>t were carried out as described by OSE R (12).
Jaffe·Schlesinger's lest and Stovik's reaction were carried
o ut as described by WITH {I 6). An etheral extract of the
co lour ing maliN was te sted for brown phase reaction
answered by ch lorophyll, (1). A methanal i, solution of the
non-esterified pigment wa s treated with 2.4-dinitrophenylh ydra.ine to detect the ener group (6).
3. Result5 and discussion
The e,terified green piament of cow milk m'oved as a
single band on TLC with 'llicagel (Rr, 0.76), while biliverdin
dimethyl est<r had a Rr value of 0.30. The retenlion time
of the green pigment on II- Bondapak C 18 rolumn
6 min,
while the retention time of the biliverdin dimethyl ester of
buffalo milk was 2 m in. From the above results , it is clear
that the new green' pigment of cow milk is different from
biliverdin.
.
The ultraviolet·visible· spectrum in ·.chlaroform of the dimeth)'! ester of the green pigment shows a 'ingle absorption
wo,
J
K.umar,
14
pj9m~I?'
of CQW mill"
l a.Q'WI'Is.. ..
l Indi9lno...
1 "'oll'l,"f1'~f;l'Jit~
n2
200
600
Wl.ve~en9th
Fig . I:
"'"
f-nml
Absorptiun '>,pcdr.l of tile ~r~~11 pi,ioIm.:nt e\t~r rrQIil 'n<.iigl!riOUS JndoexQtic
tHc:-t!d~
of cow.;' mm. in (Illomform,
maximum in the vi'5ible region along with a ~mall inflexion
in the region, 265-270 nm (Fig. 1). The speWum of the
green pigment of cow milk differs from that of biliverdin
present in buffalo milk in. lacking the absorption band Jt
380 nm though it closely resembles biliverdin in showing the
absorption maximum at 660 nm. According to BONNETT
and MCDONAGH (3), the dimethyl ester of biliverdin in
<hloroform shows peaks Jt 380 om and 656 664 nm. The
green pigment of cow milk J.tSD exhibits. J. b.athochronlic
shift in dill! tcd Hel solt.: lion leotding to ..! '5ubstJ,ntidl Sohift of
the 660 nm peak to longer wavelengths - 700 nm (Fig. 2).
This change was. accompanied by an increase in the intensity
of absorption to twofold. In the presence of a strong base,
the pigment undergoes a change to lime-green and a marked
shift of the 660 nm peak toward, the near infrared (Fig. 2).
The green pigment of -cow mil k. re5emble:s bili\'crdin with
regard to the above mentioned property, .a"i ii simil;lr phenomenon was rec::orded with biHverdin diP1ethyl e~ter by
MCDONAGH (111. The green pigment of cow milk, how·
ever, does flot belong to porphyrin pigments, due to the lack
of the characteristic absorption.
The infrared spectrum of the esterified green pigment in
nujol has absorption bands at 2950 om-I due to the --CH
group and at 1740 cm- I due to the carbonyl group (Fig. 3).
The s.pectrum lac;ks. the c;:har~u;::teri .. tic -NH vibration .of
porphyrin ·pigments whkh is obser;,.ed with biliI,Perdin Jt
3200 to 3500 em - I (10).
The nLJclear magnetic resonance spectrum recorded at
270 MH, show,.n aromatic peak at 7.5 6 (Fig. 4). The est.r
linkage in the green pigment -shows up at 4.5 8. The peaks
at 1.5 6 could be due to methyl and methylene groups.
NO·-NH protons Jre resolved indi-cating the ab'Scnce of
-NH group'.
Because of certain similarities- between the green colour
of cow milk pigment and bili'\lerdrn~ it Wil5 tested for colour
reactions of bile pigm~n ts_ The green pigment of Cow milk
doe5 not answer any of the colour rea-.::tions of biladcine.
bilatriene and pentdyopent pigml;!nu_ The pigment of fresh
-cow milk was- also tested for chlorophylls .and the result was
negatiye_ There are only a few reports about the occurrence
of chlorophyll in the separator slime. GRIMMER and
SCHWA RZ (7) attributed the presence of chlorophyll to
the contamination of milk from feed-s during milking.
PANETSOS et 01. (13) however, identified a water soluble
pigment in 50ft .r.:;hees.e as. chlorophyll III. In recent studies
with farm produced cow and buffalo milk, DANIEL (5)
wa.s able to identify chlorophyll in the SEparator slime. The
green pigment of cow milk. however answered pos.itively
j
for the pres.ence of the ester group.
W.a....I-IInglh Inml
Fig. 2:
Absorption sper;;tr.Ol of the green pigment ester in acid (----) and base
(--_._-) compared with the spectra in chloroform.
Mi!cllwj~st':nsch<1ft
42 0) 1937
Kum(Jf, Pigment of cow milk
60
40
20
4000
Fig. 3:
3000
1600
2000
1200
BOO
400
Infrirtd .!pe.::trum of t he green pigment of cows' rr e!oh mil~ .
r
1/
'"
IV
I A temllhc peak It 1.56
II £511'1' "nboe~' " .!lll
III Mrlhyl.nd mrlhvl~ne ~oton, 11.1.5
Iv Sall,lr.ted il1iprwl,c prcrl(lrn'[ 0.9 ()
G
"
I
I ~
-
-
-1
--.---J v
a
,
7
I
,-
,
4
'"
•
PPM I!l
jJ
!
2
'V
I
1
J
,....L-
0
t'i,lo;. 4 : ' H NMR ~ P(,(lqJlT'lof~ll·cn pigm(,l1t CXIr.J<.Icd Iro m co ....· miJk in Cd Cl\
From the abolle results., it is clear that the new green pig~
ment of cow mill(is neither biliverdin nor chforophyll . ft is
quite distinct from the green pigment) ,biliverdin, identified
in buffalo milk. The presence of a new green pigment in cow
milk is reported for the first time. Though the green pigment
of cow milk has not becn fully identified, the results have
shown the presence of on ~romatic rinK as evidenced by the
aronutic; signals in the: nucTear m,)gnet~G resonance ~ctr.a.
The pigment is highly polar owing to the presence of a
arbonyllVoup. 't has an ester linkage as evidenud by the
peaks in the nucl.... magnetic reS<)nance spectra and further
confirmed by the qualitative test. Carotenoids are present
in cow milk, but absent in buffalo milk and the revers. is
true for ~jfj~rdin.
MilchwisstMchafl '1 (1) 198i
There is a passing reference to the pre~ce of urobilinogen
in cow milk in the studies of PAULSMEYER (14). His
Hudies were limited to the tests based on the positive benz
aldehyde reaction. This reaction is also answered by other
substances like tryptophan, ind·o le and i, not specific for
urobilinogen •. BAUMGARTEL (2) subsequently demon"
strated that the po.itive benzaldehyde reaction observed by
PAULSMEYER (14) from the acetic acid preeipilated whey
was due to the presenCe of ingested phylloerythrinogen.
Porphyrins arc synthesized in skim milk by Propionibacterium shermanll (8), but the gr... n pigmeniocctWs ~ a normal
constituent in fresh cow milk. To the several distinguishing
characteristics in the milk of buffalo and cow,anew property
of each type of milk having a characteristic green pigment
has come to )igh!.
r
Kumar, Pigm.nI of ,,,..
16
5. S u m m a r y . I
A ckl10wledgement
The financial .support by the Indlijln Cou"r:.H of Medkal R-esearch
[s. acknowledged.
4. References
(1) AOAC: Officl .1 Methods of Anal ysi,. A.sacl.,;on of Offld,l
Analytic.al Chernis15, Washlngton, D.C •• W. Horwitz (ed.). pp.
580 (1970)
(2) BAUMGARTEL, T.: Physlolog le und P.,holog;. des Bilirubin'
stoffwechsels. ills GrundlOlgm def IkterusforSChul'lg. Th;errlt.
Stuttg.art, pp. 271. 'n Bile pi.ment5, T.K. With (td.), Academic
Pr ... lnc. (1968)
\3) BONNETT, R., MCDONAGH, A.F.: , . Cnem. So c. D 23 6
(1970)
.
(4) CHAN DRAVA DANA, M.V., DASTU R, N. N.: Mlichwi... nsch.ft
31 26 (1976)
(5) DANIEL, E.V.~ Studies on bioc.hemic.al d),angIC5 ll1 milk constituent! during the prt~n. ~tlon of ghec (butterf.n). Ph .O.
The.i" Paniab UniY''';ty (Inda) (1976)
(6) FIEGLE, F.: In: Spot Te!llin Organ ic.: Ana~'Y5i5 . 61h Englis h cdr. ,
pp. 249-253. (bevl", Amncrd.m (1960)
(7) GRIMMER, W"SCHWARZ,G.: Milohwirtsch. FD"ch . 2163;
cit.d In Chom. Abstr. 19 2988 {l9B}
(8) ILiEVA, M., POPAVA, Y.. ATANASOYA, T. : Prom i,hleoDst
17 (3) (1970, Pobl. 1972). Cited in Oairy Sci. Abstr. 3513)
Abstr. No. 930 (1911)
(9) KUMAR, M.A., DASTUR, N.N., BHATT, M.Y.: Milch",i, .. n·
sch.ft 39 (J) 29 (1984)
(101 MANITTO, P. , MONTI, D.: GiUZ. Chim. ltal. 104 513 (197 4)
Ill) MCDONAGH, A.F .: In: Th. Porp hyrins, Dolphin, O. I<d.) 6,
pp. 293, A"domic P,.", London (1979)
(12) OSER , B.L: H ..... '. Phyolologiu l Ch.mistry, pp. 488 - 509 .
Tata Mac:Grilw-Hili Pl1bJishlngCompany Ltd., New DC"fhi, India
(1979)
(13) PANETSOS, A., KlliKIDES, S., KALOXYLOS, P. : Hom .
HrQn . )6 147 (1971) j ci1ed in O.. iry Sci. Abstr. 34 n 1 , Abstr.
No. 895 (1972)
. (14) PAUlSMEYER, F.: Quant il.il; ...e Bes;tirnmung der Urobj{ i..,·
F arbstuf1e jn der Milch von Kuhen
fur dtn Nachweh des
Drs~ru.Lion
~
un d ihr
d~ainosl ischer
Wrrt
der KUoC' . Ini.ugural ·
(Vet.-Med_L Tler:irllJ. Ho <:h,c hulf H.ln ....over. pp.
Fris(hmjkhc"d~jn~
25 (1937)
115) ROOIGER, W., O'CARRA : Europ. ) . Biochem . 7 S09 (1969)
(16) WnH, T. K. : Bllt pigmenl5. Chemlcolf, 8,010gi ,a l and C lin~'cal
Aspecu. , An.demk Prr$S, london (1968)
0"
KUMAR, M.A., OASTUR, N.N . , BHATT, M,V.: Studies
IJj
new ,reen pl,ment of cow milk. I. o.~racteriz.aUon . MUdtwi
schaft42 (I) 13- 16 (1987).
34 Milk compo.ltion (pigments)
Roml nant milk contains f,at soluble oInd water soluble pig me
.md pigmenr.ation in mflk '1i1rie5 from species to spedes. Studies fro
our labo ratory have. brought to light the pre$Cnce of bile pigmenu
blliverdlr1 and bilirubin as a norm.al constituent of fresh b",ffdo , ~
ilnd butterf.ilL Fresh cow milk conuins.a dormant irccn pigm~nt .111
'sodated with thie a~l" fraction which un becxtracted with acetone
Th e uftra'Vio!el·visiblc , Infrared and nucle.ar magnetic: rtSOniln~
spectra of the ~urificd green pIgment have betll retarded. From
result!> of the quafiutlvr. tuts iind the ~petlral data, it has beeJl (.Oft
eluded that the new green pli,incnt of cow mJFk b diurncl from bili·
verdin - the blue-green pig me nt rdentiffcd in fresh buffillo mHk .
t':J
1(UMAR, M.A., DASTUR , N.N . , BftATT, M.V.: UnlOnuchunl"
liber d~s neUe Gri.illpl,ment Von Kuhmll ch. I. Ch ..rilk •• risleru~,"
Milc~wi"ensch>fI42 (1)13 - 16 (1987).
MilchlUSimmenwuung tPigmenle)
Die Milch von Wieder.kaucrn cnChlLlt f-eIt- und wuser'01ljcf:!e Pie·
mente. und die Pigmcntteruna der Mi"l ,h v~,ilerl von Specits l\rSpec;ies. Laboralorlum:iilJntersuc;nu,ngen hiilben gl!leigt. dag Gailenpi,.
m,ente - Biliverdin und Bilirub in - normilre BestiindteUe von frisGhcr
Buffelmilct) und Mn,hfctt sind. FriSGhe Kuhmilch enthjtt tin·liten·
tcS, mil der Caseinfuklion ii1sSOziierlCs Griinpjgment. d.iS mit Aceton
34
c .... trahierl wl"rdt!n kann . Die UV·s..ichlb~rc-n . IR- un d Nuklearm.3·g(iel'
res-onan 7spcktrrn des gc-reinigten Griinpigmcnu wurden au(gl!zfl,.h·
net. AU:!i Ctno Er8~bnissc-n der qualitativc:n Tests ul1d der 5pektul.
daten w urdc grschlossen, daB. d~ ncue GrUnpijmrent der Kuhmllch.
,,;e h von Bllivesdin, dem blilugriinen PiK~nt in trjsc:bes Biiffelml.Jch,
unlerscheidct.
KUMAR, M.A., DASrUR, N.N. , BHATT, M.V.: Rocherd.... ur
1(" pigment Io'crt nouveau du hlit bovin. I. Car.JCterisarjon. Mlfch·.~~5,.n,chaft 42 (1)13-16 (19B7).
34
Compo~hio"
du
I~it
(pigments)
KUMAR , M.A., DAST UR, N.N., BHATT, M.V.: Investi.acionn
)Obrc: e1 nue\lo "llme-nlo verde de Itche bo\'ln~. I. CiiTilltle:riz,uion.
Milchwi.sen><h.ft 4211) 13-16{l987).
34 CQmposicion de leche- (pi&mcnl.()!.)