(("p,tiol# from jViimal . of Food Scimc< and Techrwlogy, Vol. . 16, Nov.- Dec. 1979, pp. 242-24;7 .. .. , . .

,;' 'Grain Formation in Ghee (Butter Fa~) as Related to Structure of T riglycerides .

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C. ARUMUGHAN AND K. M, NARAYANAN National Dairy Research Institute, Karnal-132001, India

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M()lfusc"ripJ received 16 March 79; re~j.s.ed 8 lIme 1979

0;';'.18Hoo '01" buft'alo .rid "".. gb.. (butter fat) as Inll.meed by storage period d ~lyCerld. ,tructure .... IIndled. ,1i~o ubee prvducc.(g;.;;io, of larger .ilre and were b!&ber i. quantity. The solid (grains) fractlon of. gbee dlll'< ...... r.:..m ~,:;''''''Id fracti900 .... fro. wbole ghee In pbyS!co-1y~ori.des and fatty acids.

MntaJs aod Methods .i!oo~ed samples of milk were collected separately f '~Qm :.,bliffaloes and cows of the Institute', berd . Gbee \~~Pt'~pared by the cream'buttermethod:

tlriillUlalioJl:Bufl'~lo and cow ghee samples were Illillted' at 80 ' C and 100 ml samples of each were kept

iI~ 19~C; for 1-'7 iIlIYs. Granulation was compelle on tM '9f!td day, and, tbis temperature-time combination WId i~ in aU ~ubsequent studies. The size of 100 )1J:i.\IIi\!es was measured with a microscope with the help vf ~n optical micrometer calibrated with a stage micro-Illeter;' . Grain$ were separated by filtration through

cheese cloth and the quantity of l,quid fraction. 'Vl\S recorded . .

Physico-chemical 'collstants: Ghee samples and their fractions were analysed for ' their physico-chemical characteristics like melting point, and Reichert, Poienske, Iodine and Saponifkation values acCording to Indian Standards Institution Methodss.

Polyunsaturated fatty acids (PUPA) contenl$ were measured by the alkali isomorisatfon method described by the AOCS9.

Paltern of lipase hydrolysi.: Susceptibility of whole gllee and of solid (grains) and liquid fractions to pan-creatic lipa.se hydrolysis was determined by the method of Jack el al lo with tbe exception ' that NH4CI-NH.OH buffer Was replaced by I M NaCI as suggested by Free man el a/" . in order tbat the progress of the' renction could ' be followed by titration: with 0.1 l'( NilOH ID maintain a constant pH of 8.0 . . A pH mctet was used to monitor tbe pH changes during lipase action. :Hy-drolysis was carried out for a period of 20 min.

Intramolecular distribution of fatly ad'" in trigly cerides: Buffalo and cow milk fa ts were extracted with chloroform-methanol (2: 1) and iriglycerides were isgl~t~ ed by preparative TLC described by Kuksis and Breakel'\-ridge12, The triglycerides thus obmined,oiere hydrolysed

. for 3 min with pancreatic lipase (Sigma.) by the method' of Brochkerhoff 13, The glycerides and fatty acids wete then extracted with ethyl ether-he:muc ( I :1) from the reaction mixture. Monoglycerides obtained by lipase action were isolated by preparative TLC on 20 X 20 em plates coated with a thin layer (0.5 mm) of silica gel 0 1

Gas liquid chromatography: Methyl estel'S of fatty acids prepared ' following deMan lS were analys~ ina gas chromatograph (F & M Model 6(9) equipped With

'JQUIlNAL OF FOOD SCIENCE AND TECHNOLOGY, VOL. 16, NOV.-DEC. 1979

'Sto.'.,.,riod ,Buffalo Co .. daYs)

Grain size Liquid Grain size Liquid" (I"m) (%) (I'm) ( %)

I 359 69 95 91 . 2 413 65 105 85

.,,' J 420 59 108 80

'4 417 59 100 80 '" 388 59 96 79

' ,6 357 60 102 80 ,7 334 59 99 80 'A~", or 4 .ampl .. ,

.: .. ~":~

" 1I4me ionisation detector. Column was pacle.ed with Gns,9!rom-P (1OO(120) coated with 10 percent EGSS-X. AJJJ.ijiiis was carried out at temperatures programmed (fOtit})O to lSOC. The injection and detector remper-rati4:#, \vere ~SO'C and 2SO'C respectively. Nitrogen :It '9J:t:'lnljmin w~ used as carrier gas, Peak areas were

P1~~ted by triangulation, and results expressed as n,iril~' ' percentli:~es. ' Triglyceride types wero calculated tlx!Ill, P, a,ncreatic,' lipase hydrolysis data following the

1: ... " . ltil!ll\od of Vander Wallo.

. >::;' ., .

'Resi!''- and Discussion ,ii1f~CI of srora;:e period on granulation: Results of storlo:iQ of ghe.: samples at 29'C, as sbown in, Table I, liJd,~te totli:1 granillaiion in both buffalo and cow ghee ,saqipJ,CB by the third day, on wbich the minimum

jlcr~tages of 'liquid fractions (59 per 'cent for buffalo ~illt.~O per cent for cow) and maximum grain size (420 ifii!tfor, bum,fo ghoe and ,108 f'm for cow ghee) were ~rd,ed. The stain ,size was observed to incre'dse from tb,e ' firSt to th~, ihirdday in both buffalo and cow gboe ;

tbereafter grain size decreased gradually in buffalo but not in cow ghee. Similar observations were recorded by Joshi and Vyas7 in commercial, billfal()' ghee on storage.

Physico-citemicai characteristics: Buffalo 'and, cow gbee samples and their solid (grains) and liquid fractions obtained after storage for three days at 29'C were analysed for various physico chemical characteristics (Table 2). The average size of grain was 410 I'm, ,and 98 I'm for buffalo and cow ghee respectively. Average percentage of the liquid fraction was 62 for buffalo sboo and 83 for cow ghee. Buffalo shee grains occurred in clusters witb irregular margins. Cow shee grains, on the contrary, were discrete independent spherulites composed of fine divergent monocrystals rad\aiing, from tbe centre. Inftuence of chemical composition and glyceride structure on tbe kinetics of crystli:lIiti!.tiQ.u, has been studied by various worlcers 1l 17,18. Grains larger in size and number, as in buffalo ghoo, may' be: attributed to the differential make up of the triglycerides and fatty acid composition of buffalo mille. fat (fables 4, 5 and 6). Crystal size and structure are determined by the number of nuclei fonned, temperature" super-saturation, chemical composition etc. I It has, been reported that high melting triglycerides increase the crystli:l size and numberI7- 1R Buffalo milk rat is lepOrted to contain a greater amount of higb melting triglycerides (8.7 per cent) as compared to cow milk fat (4.9 per oonOl9 and tbis may explain why buffalo ghee grains are latger in size and higher in quantity. Higber melting 'point (33.") and lower iodine value (27.1) for buffalo ghee as compared to cow', {31.5" and 32.2", respectively} also correlate with greater content of saturated fatty acids and lower amount of unsaturated acids In buffalo gbee (Table 4). A marked differen:~ in:lhe physico-cbemical characteristics between the solid ' (grainS) and liquid fractions of buffalo and cow ghoo was also obser'\'-

TA:li,~: ~, PHVSICO' GHIMICAL CHARACTERISTlCS OF WHOLe OI-leJ! AND ITS FRACTI01>."S OO'TAINEO AfTER 3 DAYS OJ! ORANULATION AT 29Q G )':':':'. '.,~ . .

)'by"~h.mical cMracteri,tics . '

:\ ':'

Mej!~l' poinl(OC} '~,c;liIitl va lue - .\: ; .. , 1'l'ilf!!,lt;e vlliu. 'WQ!i.iptiOQ ya""", Iodi;,;.$luo ,Si~ .br'irain qtr:n)

' :toi.;:~~ liquid rr4ctioD (:Yo)

Whole ghoe

33,7 33.5 1.3

227 27.1 410

62

Butr.lo

Liquid Solid Whole )Ill ... fraction fraction

2S.~ 37.2 31.5 36.9 28,9 28.0

1.4 1.3 1.5 229 219 221 29.6 24.1 J2,2

9!1 83

~II ,values are. aVefale of 2 samples except the vol. of liquid fraction which is the av, value of 4 samples.

Cow

Liquid Solid (raction fraeiiOfi.

24.S 35.3; 29.6 213

1.6 1.$ 224 2,6

33.9, 27.6

ARMUOAM AND NARAYANAW GRAIN FORMATION IN GHEE AND STRUCTURE OF TR)GLYCBIlIDI!S : .~ ;".

"i ,. TABLa. 3. POLYUNSATUR.A'fII.O FATTY ACJI>S 0) WHOLE GHEE AND OF LNlUID AND SOLID FRACl'IONS OBTAINED

" AFTER 3 DA.VS Of GRANUL .... nON ,o\T 29C

Conjugaled ( %)

Buffalo Cow Dl-.!lIl.o;e

.Whoi.mercially feasible methods for sepa-r.,fWg milk fa t 'by crystallization into higb melting and luW melting rr:i.Ctions with a wide range or physico-~h.T1li~r properties. have been developed by many wojkcri1&,22. Such fraCtionation proomes provide a '''' il,tinIlOUs economiCal method of modifying the physical prQPeit.iC$ of milk fat to make it more sUltable for vanOilS . purposes.

P~/~aturated .fallY acids (PUFA): All the PUFA fi:

4, ..... ; , ' JOURNAL OF FOOD SClBNCI! AND. TECHNOLOGY, VOL. 16, NOV.-DEC. 1979 ,~ .....

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.,- " .. TABU 4, .I'A"ITY . ~CID COMPOStrlON (MOLl! Yo) OF w:ElOLE GHEE ANO OF LIQUID AND SOLID FltAC'tJONS (FR.) ,',', . .

OBTAINED AFTI!:R 3 DAYS Of GRA...'lULATION AT 29C Buffalo Cow

Fatty acids " . Whole shee . Liquid fro Solid fro Whole gbee Liquid fro Sojid .tr.

4:0 1l.8 lU 10.2 9.4 11.6 1.4 ., . . 6:0 5.2 5.8 4.2 5.0 6.1 404 ..

8:0 1.6 25 1.0 2.4 2.6 2,4 10:0 2.4 3.7 2.2 3.6 3.7 3.Q 10:\ 0.2 0.3 0.2 0.4 0 .7 .. 0.6, n:O 3.0 3.0 3.0 3.6 H 3.~ '14:0 12.5 10.8 14.2 11 .9 11.0 14 . 2 14:1 0.8 0.9 0.7 1.4 1.6 1;2 is:.O 1.3 0.6 1.2 1.7 l.l 1.& 16:0 lO.l 27.7 3M 27.8 25.0 33,5 16.;1 1.9 J.5 1.2 I.l 1.6 LI lS:O 10.2 6.2 11.2 9.2 7.2 12.0 1&.;1 16.0 20.5 12.7 20.3 22.1 15.7 '18:2' 0.9 0.9 0.8 1.2 ' 1.2 1.0

:.:- . 18:3 0.7 0 .7 0.6 0,7 .'0:8 . 0.6

Il.ruJ cow ghee wa~ observed as compared to the corres-.ponding whole ghee and the liquid fraction. LipMe

~~dfolysis. that tak~ place at an oilfwater interface is : ~ff,tcted by unsatUrated ratty acids, type of triglycerides,

physical stale of Ihes ubslrale, eic2.-2~. The lower. rate of hydrolysis of the solid fracti6'o mal' be due to the higher level of long-cbain ~turilted . fatty .acids (Table 4).

TAPl.;E 5. FA'ITY ACID COMPOiSI.TION (MOLE %) OF WHOLE FAT A.ND MONOGLYCERlDBS OBTAINED FROM PANCREATIC LIPASE HYOJlOLYSlS OF TRlG.L-YCIiEUbES

Buffa10 Cow Fatly -acid

Whole fat Monogly- Proportion of Whole fat Monogly- . Proportion of ceride~ Iatty acids in cerides fatly acids .in

z,.position (%) 2-posjtion (%) 4:0 12.8 2,5 6 9.4 15 6' 6:0 5.2 1.5 7 5.0 2.7 18

-;- 8:0 1.6 1.0 20 2.4 1.5 ~ 10:0 2.4 4.6 57 3.6 6.0 60 10,:1 0.2 0 .4 60 0.4 I.l 66 12;0 3.0 6.3 66 3.6 7.8 70 N:O 12.9 22.8 66 11.9 20.0 S8 14.:1 0.8 1.2 55 1.4 3.0 6$' B:O 1.3 1.6 40 1.7 J.6

.' 35

., -," 16:0 30.3 36.3 40 27.8 18.9 . ). : 34 16:1 1.9 3.3 57 1.1 U 41 1.8,0 10.2 6.6 22 9.2 S.~' 2~ 18:1 16.0 11 .0 22 20.3 )7.2 26' 18:2 0.9 0.7 27 J.Z '0.6 19

1 ~:3 . 0.7 0.5 30 0.7 0.4 20

Percentage of fatly add eSterified in the 2po,ition of trigiyoerides. Calculak

ARMUGAM AND NARAYA~AN: GRAIN FORMATION IN GH!iI; AND ST\l.UCTURE OF TRIGI.YCERIDBS

I;(i (f)l .ncid cOlnpo.sitiQll.' Table 5 shows tbe fatty acid "omjIDsition of \luffaIo and cow gboe and of solid I ~, rliniO .and liquid fractions. Higher contents of C4, ( '" ,. ,a:Ud':Ci8 fatty acids and a lower level of CIS: I fatty ... ,' Itl '$'eerti ,[0 be major points of difference beTWeen the btl:;, llcidcompQsit,on of buffalo and cow ghee. The p,.,q;I\tages: of IQng-chain saturated falty acids, viz. t ' , . eli! and CIS were higher in the solid fraction. I '1",10 blrlited and short chain fatty acids were concentrat cd itt the liquid fraction. The fatty acid compositions " I wbolQ. sheo and thoir fractions showed a direct

!C klt'O~P: with their physico,chemical constants, (T:.ble2,')! ::Greale1 concentrations of C 14 , CI6 and C'8 hltv ,acids' in buffalo ghee can be correlated with grains 1:1(;~~.ln siz< and ql!llntity as these fatty ",dds have been I",..,iliv-ely. correhiled with high-melting triglyceride ,:ont(:nt2S.

Mtramolecular dis)ribu(ioli of fatty acids and trig/y-r;,(~ $ti'licture: lluffalo and cow milk fat triglycerides

"eF" $\lbJected to pancreatic lipase hydrolysis to study I hc!tribution of fatty acids between primary and ' ,,",udal)' hydrox.Yl gtoUps of triglycerides (Table 5). ~, hor" cb'aui fatty acids (C4, C6 and Ca) showed a specifi-c;I'; Fot the extemal. positions, whereas medium chain !,\I:lyacids. (eun e12 and C 14) were preferentially attach ,,01 lO. th internal p()'Sition of triglycerides. Among the 1,,,,(,' .e.h~l!i fatty acids, Ct6 did not seem to show any prC'"kteritiJ but C'8 fatty acids were found 10 concentrate ... !hil' eil,ternal positions, The pattern of distribution J Iltly "aCids between external and internal positions , j tnglycerides seem!)d to be similar ill both buffalo and

,-,,,,,mllk .fat triglycerides (fable 5). The difference in !i ",' j!i1YiIi~ behaviOur of buffalo and cow milk fats "'I:'.' '\lI\!ii' not be due to the pattern of distribution of "ft)' acids in lbe triglyceride molecules, However, i ic'Afl.olle and Savary2 demonstrated how physical

,i:,~r"c.tai$~tics of natural fats are governed by the "n; !h~tG;r.llistribution of fatty acids, in the Ught of " " t obtained by pancreatic lipase hydrolysis of the

hiSIYccride types calculated as described by Vander W" l i~ !lothe basi$of pancreatic lipase hydrolysis data " 'C' presented in Table 6. A greater content of trisaturat-,, ' Rt)lcerides (SSS) in buffalo milk fal (49.25 per cent) ,I' ,~,imPared to cow milk fat was found to be a major .J lilccllllce in the make up of the triglycerides of those ,\", species, Ai l ,and Vander WallQ and Coleman21

l~ '.itl1[i\~ triglYceride types of various uatural fats ",c1u\lill,f cow m.lIk fat and discussed the influence "i' lrj~iyCCltide strii~ture on properties offal. It is tempt "':

10URNAL OF FOOD SCIENCE AND TECHNOLOGY, VOL. 16, NOV.-DEC. 1979 ~'C;-:: , . .,. _ -

6

nt : G)'~bchenko. A, D.and IvantsoVtl, L S . XIXJlIlerflot. Dairy 23 . . ~'Ct>IW., NeW Delhi,' India, 1974, lB, 653.

Et-Sadek, G . Rifa.t. L D., Abd E~SaI.m. M. H. am! EI-Bagary, E., Indian I. Dairy Sci .. 1972, 25,167. 1 9~ ; ~ Murlby, M. K~ and Narayanan, K. M., XIX I.ternol. Z4. Desnuelle, P., In AdWIIICea i. Enzymology, edited by Nord,

F. P., Interscience, New Yorle, 196J, Vol. 23,129. /': .~y Congr., New Delhi, India, 1974, IE. 208. IQ, 'iliiJi,y, R. M., XlIJ111nlerlUll. lJqi,y GoV' .. Sydney. Australia

; c:'1;70, IE, 24i. "11,_ ':~n, R.S.", X ,VIlI lltlernat. Daily Congr., Sydney, Austra.lia,

)(- (970 IE '240 , ".:. ,' ' ~ .

,Z, .llo;>ltis, R, Gray, I. K.,McDowell, A.K.R. and Dolby, R. M . '':i?.J . :, Dairy Rea., 1971, 38, 17~.

25. ShertJon,1. W. and Dolby, R. M., J, Voiry Sci 1971 . 5 '2. 26, Ast. H . 1. and Vander Wal, R. J" I. Am. 011 Ch"",. Soc.,

1961 , 38,.67. 27. Coleman, M. H .. In Adv. lipjd Res