Triglyceride Composition of Milk Fat: II. Separation of Triglycerides by Argentation-TLC and GLC

Triglyceride Composition of Milk Fat: II. Separation of Triglyceride. by Argentation-TLC and GLC A. Adel Y. Shehata and John 1\1. deMan Department of Food Science and

J. Craig Alexander Department of Nutrition University of Guelph Guelph, Ontario

Abstract Fractions of milk fat separated by column chromatography on silicic acid were further analyzed by thin-layer chromatography on silver nitrate impregnated silica gel and gas chromatography of triglycerides and their component fatty acids. The triglyceride types in 8 bands on the thin-layer chromatograms were characterized and their composition determined.

Resume Des fractions de graisse de lait sepan§es sur colonnes chromatographiques d'acide silicique ont ete analysees sur couche mince de gel de silice impregne de nitrate d'argent et aussi par chromatographie en phase gazeuse des triglycerides et de leurs acides gras. La caracterisation et la composition ont eM faites sur les types de triglycerides de 8 bandes de chromatogrammes de couche mince.

Introduction Milk fat contains as many as 142 different fatty acids, (Jensen et at., 1967) and is one of the most complex natural fats. The study of its triglyceride composition and structure, therefore, is a difficult task. In the early stage of milk fat triglyceride analysis studies, the methods employed were mainly fractional crystallization (Greenbank, 1953) and countercurrent distribution (Haab et at., 1959). Lipase hydrolysis has been applied to the whole milk fat (e.g. Jensen and Gander, 1960; Jensen et at., 1961; 1964; Ast and Van del' Wal, 1961; Jack et at., 1963; Freeman et at., 1965); or applied to fractions previously separated by fractional crystallization and countercurrent distribution (Smith et a7., 1965) or silicic acid column chromatography (Clement et aT., 1963). The presence of short chain fatty acids in milk fat has greatly limited the value of fractional crystallization as a separation technique (Chen and deMan, 1966) and also made some of the results obtained from lipolysis studies rather questionable due to the preferential cleavage of short chain fatty acids by the enzyme (Clement et aT., 1963; Jensen et at.) 1964; Boudreau and deMan, 1965). However, the difficulties in the early stage of milk fat triglyceride studies resulted mainly from the inadequacy of the separation techniques used. In recent years, several powerful chromatographic techniques have been developed and this helped in resolving the complex mixture of milk fat triglycerides into much simpler fractions. These methods were the subject of a recent review (Shehata and deMan, 1971). Kuksis et at., (1963) separated and quantified the triglyceride molecular species* of bovine milk fat. This was found to cover the range CS4-C Sb• They concluded that butyric and caproic acids are found exclusively in combination with medium and long chain fatty acids. A similar conclusion wa' ':' Molecular species: A group of triglycerides which may have different fatty acid compositions but having the same acyl carbon number.

13

drawn by Dimick and Patton (1965) after they used combination of silicic acid column chromatograp~ and low temperature crystallization from acetone WIj GLC of the intact triglycerides for the study of hyd genated goat and cow milk fat triglycerides. Bla and Privett (1964) separated milk fat triglycerid by silicic acid chromatography into long and sho chain fractions. The triglyceride types (of differe number of double bonds) in each fraction were detd mined by two combinations of methods, namely: SilVj' ion TLC-pancreatic lipase hydrolysis and ozonolysi 'l'LC. Thirty-five different groups of triglycerides we estimated and it was concluded that the distributio of fatty acids in milk fat triglycerides is non-rando~ and that there is a preferential association of long (oi medium) and short chain fatty acids. Nutter an~ Privett (1967) studied the short chain triglycerid fraction of bovine milk serum. Their findings co firmed the previously drawn conclusion that sho chain fatty acids were widely distributed among thl triglycerides. 1\1ore recently, the triglycerides of bovin. and human milk fats were separated by a combinatiOj of silicic acid TLC-silver ion TLC and GLC of intac triglycerides. A further analysis of some fraction with a stereospecific analysis technique was also per formed and several individual triglycerides and tri glyceride types were identified (Breckenridge an Kuksis, 1968; 1969; Breckenridge et at., 1969). In a previous report (Shehata et at., 1971) silici~ acid column chromatography was used for the preparative separation of milk fat triglycerides into 17 major groups with gradually increasing polarity and the distribution of both the fatty acids and triglyce·, rides in these fractions was determined. The first four fractions A, B, C and D, accounted for more than 40% of the total triglycerides. In the present paper, further study of these fractions is reported. A combination of silver nitrate impregnated TLC and GLC of both the intact triglycerides and their fatty acids was em' ployed. The first two fractions, A and B, were com' bined before the analysis and coded as (A,B) and similarly, C and D were combined and coded as (C,D).

Experimental The method of preparation of the methyl esters of milk fat and its fractions, and the gas chromatographic conditions have been reported previously (Shehata et al., 1970) and also the GLC method for analysis of intact triglycerides (Shehata et at., 1971.) For silver nitrate impregnanted TLC, 56 g of silica gel G was slurried with 120 ml of 10% AgN0 3 solution by shaking for 40 - 60 seconds. The slurry was spread Can, lnst. Food Science and Technol. J. Vol. 5, No. I, 1972

er 5 plates of 20 x 20 cm size and 0.5 mm thickness oV'ing an anodized aluminum spreader (Fisher Scien:UiC Co. Limited). Plates were activated for one hour t 110°6 and placed in a desiccator for 1 - 2 hours :efore use. In the preparation and handling of the iJnpregnanted plates, light was excluded as much as possible. Samples were applied as a streak in 4% chloI roform solution with a Desaga Sample Applicator TNO.Delft System lVlodel 12 09 50. Plates were developed twice, the first development in 0.2% ethanol in chloroform, the second in 0.4% methanol in chloroform. The double developing technique permitted increase of the maximum load of the plate and also improved resolution. Developing time was about 30 minutes for each run and was limited to 12 cm from the base line which was set at approximately 2 cm from the edge of the plate. Plates were left to dry for 10 . 15 minutes after the first development. The concentration of methanol in the second solvent can be changed to suit the type of separati,on desired. The reproducibility of the separation will also depend on the activity of the adsorbent which in turn will depend on exposure to atmospheric humidity if other factors remain constant. The triglyceride bands separated were made visible under ultra-violet light aher spraying with 0.05% 2,7-dichlorofluorescein in ethanol. Bands were marked with a needle and later scraped off onto a piece of aluminum foil and transferred to a 125 ml glass-stoppered flask and extracted six times by shaking with 50 ml portions of diethyl ether containing 5% methanol. The combined extracts obtained by filtration through glass wool were decolorized to remove 2,7-dichlorofluorescein by passing under suction through a small column of silica gel. This column was made up of a medium porosity sintered glass funnel (cap. 15 ml) which was filled to about half its height with a slurry of silica gel in petroleum ether. The silica gel was activated at 110°C for 35 minutes. The 2,7-dichlorofluorescein remained adsorbed in the silica gel and the fat solution ran through clear. The column was washed with petroleum ether to remove any trace of fat. The decolorized extracts were washed four times in a separatory funnel with 50 ml portions of water to eliminate methanol and residual silver ion, dried with anhydrous sodium sulfate and filtered through a sintered glass funnel. The filtrate was transferred to a rotary evaporator and the volume reduced to 3 - 4 ml. This solution was transferred in portions to a small vial and evaporation completed under nitrogen. The vials were flushed with nitrogen and kept in the freezer until used for analysis. Quantification of the silver nitrate impregnanted TLC plates was done by scraping each band into a glassstoppered 125 ml flask and adding internal standard solution (50 p.g tricaprylin in 1 ml of chloroform). The extraction and purification procedure described above was then used with this material. The total am,ount in mg of triglyceride in each band was computed according to Kuksis (1967) as follows: Total area of all triglycerides Weight of within the band x the internal Area of the internal standard standard (mg) J. lnst. Can. Science et Techno!. Ailment. Vo!. 5, No l, 1972

Results and Discussion The silver nitrate impregnanted TLC separations for (A,B) and (C,D) are shown in Figures 1 and 2 respectively. The weights of the fractions are reported in Table 1 and the fatty acid composition data in Tables 2 and 3. Both (A,B) and (C,D) were separated each into at least nine bands (Figures 1 and 2). The last two bands (8 and 9) were c'ombined for further analysis. The amount (wt %) of each band was determined using tricaprylin as internal standard for GLC of the intact triglycerides. Table 1 shows the results obtained for each of a total of eight bands, reported in decreasing order of R f values, and for both (A,B) and (C,D). The tentative identification of some of these bands was done by applying known triglyceride mixtures of simple composition as a reference spot on the plate. Cocoa butter has the symmetrical monounsaturated triglyceride type SlVlS as a dominant component and lard has the asymmetrical monounsaturated triglyceride type lVlSS in appreciable amount, and these were used as reference compounds. The confirmation of this tentative identification as well as the identification of the rest of the bands was generally based on the fatty acid composition as S2lVl should contain 33.3 mole % of lVl and SlVl2, 66.6 mole % (Tables 2 and 3). It was found that for (A,B) and (C,D), the major triglyceride types of band number 1 was trisaturated (SS8), band number 2 was symmetrical monounsaturated (SlVlS) , band number 3 was asymmetrical monounsaturated (SSlVl), bands 4, 5 and 6 were diunsaturated of different types ('M 2S and S2D), band number 7 was a mixture of triunsaturated type lVllVllVl and diunsaturated of the type S2D and band number 8 (bands 8 and 9 in the original separations of Figures 1 and 2) as several triglyceride types in addition to a small amount of the polyunsaturated types as will be discussed later. Since the symmetrical diunsaturated triglyceride type lVlSlVl interacts more strongly with the adsorbent, it has a smaller R f value than the asymmetrical diunsaturated triglyceride type SlVlM, and since the majority of the diunTable 1.

Separation of milk fat fractions on silver nitrate impregnated TLC.

Band No. Major1 triglyceride type

-------

1

2 3

43

5 6

7 85 1

2 3 4 5

___ ~eight %2 (A,B)

-------

SSS SMS SSM SMM MSM S2D+MSM S2D+M34 Others

(C,D)

------

16.7 7.9 30.4 9.0 19.5 6.3 6.5 3.6

19.2 7.4 33.0 9.0 16.7 4.4 6.0 4.2

=

S = saturated acyl group, M = monoenoyl and D Dienoyl. Average of two independent determinations each from two plates or more. This type migrates on the TLC plate farther than MSM in clear separation. In case of (C,D) this band has also MSM. Possible types are S2Tr, SMD, SMTr and S2T (where Tr = Trienoyl and T = Tetraenoyl.

14

Figure 1.

Table 2.

Band No.

Silver nitrate impregnated TLC separat:on of the milk fat combined fractions (A,B). 1. SSS, 2. SMS, 3. SSM, 4. SMM, 5. MSM, 6. M2S and DS 2, 7. MMM and S2D, 8 and 9 other types. The fatty acid composition l of (A,B) -

Major l Triglyceride type

Figure 2.

Silver nitrate impregnated TLC separation of the 1l11~ fat combined fractions (C,D). 1. SSS, 2. SMS, 3. MS 4. MMS, 5. MSM, 6. M2S and DS2, 7. M2S, MM and S2D, 8 and 9 other types.

tryglyceride types separated by silver nitrate impregnated TLC.

Fatty acid mole % 4:0 6:0

8:0 10:010:1 12:0 12:1 14:0 14:1 15:0 16:0 16:1 18:0 18:1 18:2 18:3

-

27.96 1.33

1

SSS

0.57 1.07 4.45

tr

5.33

17.24

tr

2.51

?

SMS

1.15 0.88 2.45

tr

3.38

11.73

tr

2.04 30.09 1.92 19.86 26.51

3

SSM

0.16 0.601.87

tr

2.88

10.20

tr 1.50 30.26 1.68 18.13 32.68

4

SMM

0.85 0.55 1.24 0.07

5

MSM

0.63

6

HSM

1.45

S~D "-

tr

39.51

-

1.80 0.72

6.82 1.45 1.29 18.26 4.34

9.11 53.46

1.05

2...1J. 0.68 0.70 .1.7.&. 3.63

~

2.19

6.48 1.71 0.92 15.09 5.47

6.20 53.43 7.02

-

4.16 56.29 14.23

7

MMM SZD

1.28

4.47

1.67

8

Others4

1.45

5.55

0.640.4415.69 1.81

11.23 6.64

!il..!B

20:4 Other]

1. 22

---- -

8.81 ~ ~ ~ 4.59

0.76

; Four major fatty acids in each band are underlined. S = saturated acyl group, M = Monoenoyl and D = Dienoyl, 3 Includes some of the C20 fatty acids. 4 Possible types are S2Tr, SMD, SMTr and S2T (where Tr = Trienoyl and T = Tetraenoyl).

Rntnr'nled tl'iglycerides in milk fat are known to be of

th0 t.y pc MS~I (Blank and Privett, 19(4), the diunH
);11'~(,Ht dllllH;atUl'ated Imnd in the

0TOUP

and therefore

il( •1Ollg- t 0 type :\ISM. The b " separation between 1I1t·" two 1)·\ .1. ' . .' 'lIuS IS a clear one (see FIgures 1 and 2).

11111'"

.

;~; (j ;~:.(.\IIl~.I> all lJands after band 5 of ~he diunsatul'at;-, .\« 1)(le t,ype M2 S must also be of the type .M:S1II.

l~

This applies with the exception of S2D which can be calculated independently. A small problem with over; lapping of triglyceride types was encountered. This posed no major difficulty since a correction based 011 the fatty acid composition could easily be applied. The corrected percentage of each of the triglyceride types is given in Table 4 for (A,B) and (O,D) an d • I I compared wIh th e resu ts of Blank and t Privet: (1964) and the recent work of Breckenridge ana Kuksis (1969). (O,D) contained more of tbe i

Can. Inst. Food Science and Techno!. J. Vo!. 5. No. I, 19~

Table 3.

The fatty acid composition 1 of (C,D) -

--

1'1ajor 2

Band No.

triglyceride type

triglyceride types separated by silver nitrate impregnated TLC. Fatty acid mole

4: 0

6: 0

8: 0

10: 0 10:1 12: 0 12:1 14: 0 14:1

-

2.21

1.75 6.75

-

6.43

1.65 3.74

-

1

SSS

2

SMS

4.47 2.86

3

SSl:I

0.96

4

SMM

3.04 1.16 2.38

1.26 3.19 0.13

3.06

-

4.28 0.95

11.82

-

1.24 29.19 1.72 15.08 22.95

-

3.98 0.95

12.47 0.60

1.59 28.68 2.19 12.71 .R.2.!:

3.31 2.39

10.26 1.40

1.17 19.843.37

7.36 43.15

-

2.18 0.68

7.65 1.04

0.86 17.86 3.99

6.65 56.73

1.26

0.69 12.58 3.97

4.19 36.20

12..&i

2...12 !:il...Sfl

0.96 1.17

-

1.42 1.00

24.35 2.17

7

MSM

0.63 1.27

-

1.31 1.05

6.11 1.58

8

others

~

18:3

2.65 38.37

1.08

6

18:2

-

MSM S2 D

-

18: 0 18:1

18.22

-

-

MSM

%

15: 0 16: 0 16:1

tr

6.04

tr

0.93

-

20.53

3.61

J..6-£l:i 2.79

20:4 Others J

8.05

-

3.23

~

-

1,.22

8.61 ~ ~ ~ 9.79 2.78 ..

_.~

-

--- - - - - - - -

Four major fatty acids are underlined. 2 S = saturated acyl, M = Monoenoyl, D = DienoyI. 3 Includes some of the C zo fatty acids. 4 Possible types are SzTr, SMD, SMTr and S2T (where Tr = Trienoyl and T = Tetraenoyl). 1

saturated trigl.yceride types (8S8, 8M8 and 88M) but less of 8MM and much less ,of '~h The contents of other triglyceride types were comparable. ~With the exception of band 8, all of the triglyceride types and contents of (A,B) were in agreement with the results of Breckenridge and Kuksis (1961:1) on long chain milk fat triglycerides. The triglyceride types of (O,D) a ppeared to be more saturated. There was general agreement between the present results and those for long chain milk fat triglycerides of Blank and Privett (1964) for both 888, 8M8 and S~Bf, there were appreciable differences in case of the rest of the triglyceride types which could be due to differences between the milk fat samples used. However, results for fractions (O,D) were closer to those obtained by Blank and Privett (1964) than (A,B). The major fatty acids in both (A,B) and (O,D) were comparable (Tables 2 and 3). The S88 types had palmitic, stearic and myristic as the major fatty acrds. The major fatty acids of the unsaturated classes 8 2M, 8M2 and M3 included oleic and palmitoleic acids in addition to the saturated fatty acids. D8 z also had linoleic acid as the major fatty acid as was expected. In general, a lesser contribution of relatively shorter chain fatty acids, which are largely saturated, isobserved in going from band number 1 to band number 8. It is also interesting to note that linolenic acid was restricted to the last band (Band 8 in Tables 2 and 3). However, as mentioned before, the so·called band 8 l'epOl'ted in all the tables is actually the mixture of the last two bands 8 and 9 in the original separations (see Figures 1 and 2). These two bands, which were C~ll1bined before the analysis, were the compounds ~vlth the lowest R I value, and one of them (number 9 In the ol~iginal separation) did not mo\'e from the stal·ting line. 'l'herefore, this mixture (now called band 8) should contain triglycerides with higher degree of un saturation (or strongest interaction with the adsorbent) than the rest of the triglyceride bands J, lust. Can, Science et Techno!. Aliment. Vo!. 5. No 1, 1972

with higher R I values. The fatty acid composition of band 8 in both (A,B) or (O,D) (Tables 2 and 3 respectively) showed about 31 mole % saturated acids (8), 34 -39 mole % monoenes (M) and about 30 - 32 mole % of a mixture of diene (D), triene (Tr) and tetraene (T) fatty acids. This compares with 8 (40.1 mole %), lH (36.7 mole %), D, Tr and T (23.2 mole %) reported by Breckenridge and Kuksis (1969) fOl' their polyunsaturated band 'of the long chain milk fat triglycerides. It appears that the majority of triglycerides in this band contain either two 8 (largely 16 :0, 18:0 or 14 :0) and one Tr to form the triglyceride type 8 2Tr which may interact with the adsorbent more strongly than either lHlHM or 8 zD of the preceding band (7), or they may contain one 8, one M (largely oleic acid) and one D (largely linoleic acid) or Tr (largely linolenic acid) to form the triglyceride types SMD or 8lVl'l'r respectively. Arachidonic acid will most likely be associated with saturated acids and/or monoenoic acids. The likelihood of the presence of triglycerides containing more than one ·of D, Tr or '1' in significant proportions is not very high since their presence in significant amounts would make supply of unsaturated acids insufficient to form the majority of triglycerides with interacting ability with the adsorbent stronger than the preceding triglyceride band with higher R I value (band number 7 which contains lVlMM and D8 z). However, it is to be expected tllat there will be overlapping of triglyceride types containing appreciable amounts of trienoic and tetraenoic acids, during silver ion TLC separation (Litchfield, 1968). On this basis, the only polyene triglyceride types (more than three double bonds pel' molecull') in these fractions (A,B) and (O,D) of milk fat are essentially those containing arachidonic acid. These trigl;ycerides represent a small proportion of band 8 in addition to those belonging to the type 8M'l'r, which contain four double bonds per molecule. Blank and Privett (H)64) found trace amounts of pol~'elle

'16

AA .....02

AA

SMS

SSM

llANO 3

~ ...;L ......,. ~

SM/oI

AA WCl5

MSM

....

Figure 3.

The molecular species of the triglyceride types of the milk fat combined fractions (A,B), bands from 1 to 9. 1. SSS, 2. SMS, 3. SSM, 4. SMM, 5. MSM, 6. M2S and DS2, 7. MMM and S2D, 8 and 9 other types.

t.1·igJj'("eI"ideH (with more than 3 double bonds per in the long chain fractions of milk fat. i\'nttPI' Hud 1'1'ivett (19G7) reported the presence of [he (riglyeeriuc types S2Tr, (2.2%) and SMD, (1.9%) jilt hI' short elm in triglycerides of milk serum fat. HI'(,l"kl'lll'idge allll Kuksis (19G9) reported the presence of ti.:~'lo pol.YllIlsaturatcd triglyceride types (with more thall :: <10111111' 11011111,; per molecule) in long chain milk rat tJ'iglyeel'ides ('rable 4). 11101('("1111')

'rhe triglyeeride types of band 8 contain all of the linolenic aeid of milk fat (12.72 mole %) in case of (A,B) an~ 11.82 mole % in case of (C,D), in addition to appreCIable amounts of arachidonic acid (4.59 mole % in (A,B) and ~.79 mole % in (C,D)). These triglycerides must play an important role as initiators of milk fat auto-oxi~ation. The triglyceride composition for ea~h of the _eIght bands of (A,B) and (C,D) is given III Tables" and 6 and typical molecular species chroma~ogr~ms for the different triglyceride types are shown III FIgures 3 and 4 for (A,B) and (C,D) respectively. T~e mol~cular species found (Tables 5 and 6) in tbe tr.Iglyceride types of (A,B) and (C,D) were essentially III ~he range between C 56 and C34 , though some triglycerIde tY~es contained a small proportion of the molecular speCIes smaller than C34 . A I. the same time, band number 8 (in both A,B and C,D) did not 17

Table 4.

Triglyceride types 1 of milk fat fractions, (A,B) and (C,D) compared with those of o~her investigato.rs (Blank and Privett, 1964; Brekenndge and KukslS, (1969). Weight %

Triglyceride type 2 (A,B')

(C,D)

Blank and Privett 1964

16.4 7.2 31.3 8.1 22.3 4.4 6.1 4.7

18.0 8.6 35.3 6.8 22.2 4.5 1.3 4.2

19.6 8.9 49.4 8.6 13.5 N.R.4 tr 4.4

- -

SSS SMS SSM SMM MSM DS2 3 MMM Others 5

Breckenridge and Kuksis 1969 16.5 36.7 27.7 12.9 6.3

Calculated from the data in Table 10 to correct for the overlapping of some types. 2 S saturated acyl group, M = Monoenoyl and D = DienoyJ. 3 Overlaps the band of (MMM) but calculated on basis of the mole percentage of D in the original band. 4 Was not reported. 5 possible types are S2Tr, SMD, SMTr and S2T (where Tr ::: Trienoyl and T = Tetraenoyl). 1

=

contain any molecular species smaller than C44 . The molecular species C42, C.. and C46 were present in maximum proportion in bands 1 (SSS), e.g. about 39 mole % in (A,B) and 35 mole % in (C,D). There was a general trend for these molecular species to decrease in amount in going from band 1 to band 8 (with the exception of band 6) resulting in minimum values of 0.80 and 3.87 mole % for band 7 in (A,B) Can. lnst. Food Science and Techno!. J. Vo!. 5, No. I, 1972

co cD

eN'll1 555

CD

6AND3

BAND2 SUS

SSU

.,

!

1\ J~l

~_~p}·~V ~"\J~~

~,~JJJJv~ CD CD WClS MSM

BAND' 5MM

u

lAND •

""",s,o

i\

~

Figure 4.

.. .... . .

J

The molecular species of the triglyceride types of the milk fat combined fractions (C,D), bands from 1 to 8. 1. SSS, 2. SMS, 3. MSS, 4. MMS, 5. MSM, 6. M2S and DS 2, 7. MMM, and DS 2 , 8. others.

and (O,D) respectively. The distribution of the four molecular species 0 54 , 0 52, 0 50 and 04B in each of the eight bands for both (A,B) and (O,D) is also shown in the form of histograms in Figures 5 and 6. In both (A,B) and (O,D), 04B was the dominant molecular species in the SSS bands and 0 54 was the lowest. In the bands number 2 (essentially SSM) and 3 (SMS), 0 50 was the dominant species and 0 54 was the lowest. In bands number 4 and 5 (essentially M2S), the dominant molecular species shifted to 0 52 and the species present in lowest amount changed from 0 54 to 04B' In the last three bands, 6 (essentially M2S and DS 2), 7 (essentially M3 and DS 2) and 8 (polyunsaturated and other types), the dominant molecular species again shifted to 0 54 and those in lowest amount were 04B. The trend in both (A,B) and (O,D) reflected the increasing contributIOn of OIB unsaturated acids (especially 18 :1) in the triglycerides in going from band 1 to band 8. The maximum proportion of 0 54 was found in bands 7 (MMM and S2D) and 8 (polyenes and others) in both (A,B) (52.4 mole % and 43.3 mole % respectively) and (O,D) (35.0 mole % and 38.1 mole % respectively)". The maximum proportion of 04B was present in band number 1 (SSS) for (A,B), (20.8 mole %) and in band number 3 (SSM) for (O,D), (22.0 mole %). J. Inst.

Can. Science et Techno!. Ailment. Va!. 5. No 1. 1972

I' II

'.J\.A

A

wJ '\",-_

--YJVJV

The major molecular species for each of the eight bands were largely similar for both (A,B) and (O,D) though varied in amounts. Major molecular species in type SSS were 0 50 , 04B and 0 46 in (A,B) and 0 50 , 04B and 0 52 in (O,D), listed in order of decreasing quantity. The major molecular species in S2M (bands 2 and 3 in both A, Band 0, D) were 0 50 and 0 52 or 04B. The dominant molecular species in M2S (bands 4 and 5 in both A,B and O,D), 0 54 , 0 52 and 0 50 were the dominant molecular species. This is in agreement with the recent work of Breckenridge and Kuksis (1969) on long chain milk fat triglycerides. It should also be mentioned here that molecular species greater than 0 54 (essentially 0 56 ) were present in appreciable proportions in band 7 (3.3 mole %) and band 8 (10.1 mole %) of (A,B) and also in bands 7 (4.9 mole %) and 8 (12.1 mole %) of (O,D). These molecular species are assembled with contributions of 0 20 and possibly 0 22 fatty acids which were not determined fully in the present work. It was not expected from the consecutive separation system (silicic acid column - silver nitrate impregnated TLO - GLO of intact triglycerides) to resolve the individual triglycerides of milk fat. However, close examination of Tables 2 and 3 on fatty acid composition and Tables 5 and 6 on triglyceride composition of the different triglyceride types of (A,B) and (O,D) will reveal a good deal of information about major individual triglycerides in these triglyceride types provided that only major molecular species and 18

Table 5.

The molecular species 1 of (A,B) Band No.

56

54

52

50

4g

46

% 3g

40

42

44

3.4g LOO

0.27

34

32

0.06

-

3.01

lLg4

20.64 20.76 17.64 12.g9

2

SMS

7.69

22.53

24.31 14.49

6.02

3.92 3.56

5.1g

6.5g

3.g4 L39

3

SSM

g.OO

26.86

30.g4 19.09

7.7g

3.51

L65

L30

L13

0.61

0.22

4

SMM

22.11

42.1g 19.52

4.90 3.79

2.75

2.02

2.66

0.90

0.15

-

5

MSM

2L02

42.19

19.14

5.75

2.S7

L75

LOg

L60

2.19

L5g

0.62

6

MSM DS 2

26.47

17.79

13.42

g.22

5.14 3.75

3.73

5.72

6.63

4.S7

2.45

}1MM

l..ll

DS 2

10.11

Others3

g.34

36

SSS

8

3

Triglyceride mole

1

7

1 2

Major triglyceride type 2

triglyceride types separated by silver nitrate impregnated TLC --._- - - - - - - - - - - - - - - - - - - - - -

30

0.45

0.17

-

L15 0.64

2b3.Q 30.1g 10.g2 2.52 O.gO ll..lQ

~

12.00

5.50

2.80

LOO

Four major molecular species are underlined. 5 = saturated acyl, 1\1 = Monoenoyl, D = Dienoyi. Possible types are 5MD, 5MTr and 5 2T (where Tr = Trienoyl and T = Tetraenoyl).

fatty acids are considered (see Table 7). For example, the trisaturated band (band number 1) in fraction (A,B) contains the fatty acids, 16 :0, 18 :0, 14:0 and 12:0 as the major fatty acids and 0 48 , 0 50 , 0 46 and 0 44 as the major molecular species, both listed in order of decreasing quantity. Therefore, the major individual triglycerides in the trisaturated band of (A,B) have to be 16, Hi, 16 and 18,16,14 (the order of arrangement of the fatty acids having no structural significance) as the major 0 48 triglycerides (20.8 mole %). Similarly, 0 50 would essentially be 18, 18, 14 and 18, 16, 16. As 0 48 and 0 50 are the dominant molecular species in this band (20.8 mole % and 20.6 mole % respectively), the l'l'(~\'iously mentioned single triglycerides must be the Table 6.

The molecular species 1 of (C,D) -

dominant ones in the trisaturated band. Molecular species, 0 46 (17.6 mole %) will be formed of 18, 16, 12; 18, 14, 14 and 16, 16, 14, and 0 44 (12.9 mole %) will be formed of 18, 14, 12; 16, 16, 12 and 14, 14, 16. Using the same argument for SMS (essentially band 2) a monoenoic acid has to be in position 2 for all the molecular species of this type. Since 18:1 is the major monoenoic acid, the molecular species 0 50 would in· clude triglycerides 16, 18 :1, 16 and 18, 18 :1, 14; 0 52 would be the single triglyceride 18, 18 :1, 16, and the last three triglycerides must be the major single trio glycerides among all others of this type since 0 50 (24.3 mole %) and 0 52 (22.5 mole %) are the major molecular species of this triglyceride type. The single

triglyceride types separated by silver nitrate impregnated TLC. --~---

Band

:"0.

Major triglyceride type 2

-------------._._--_._..

56

54

52 7.74

4g

50

46

42

44

40

3g

36

34

32

30

9.M

5.61

2.44

L02

0.61

4.5g

O.gl

1

SSS

3.35

2

SMS

5.94 16.17

2L7316.17

6.46

5.02

5.60 7.95

-'

~

SSM

4.23 19.17

29.01 2L9g 12.10 7.61

4.25

l.62

4

SMM

15.17 35.54 19.05

6.75

3.65

2.73

4.26

6.94

5.g5

-

5

MSM

17.03 42.31

g.15

3.91

1.72 l.07

O.gl

0.g4

-

to

DS2 MSM HS!1 DS 2 M3

7

Others 3

L55

2.2...3.0

12.07

lL44 12.21 12.35 lL75 lL30 10.39

24.13

9.55

1.2...1l 12.79 g.g5 7.27 5.74 14.42 4.54 0.51 0.34 ~

~

6.00 3.51

1.44

3g.12 25.66

~

6.62 3.08

0.79

4.90 J.5.....Ql

~ ~·(~I~r~~-;-0~~lecularspecies

are underlined . 3 I; satj"jrated acyl group, 11 = Monoenoyl and D Dienoyl. ()SSI ) e types are 52Tr, 5MD, 5MTr and 52T (where Tr

1H

_-

Triglyceride mole %

=

L07

1.66

4.00

3.9g

4g

Lg4

2.37

-------

=

Tricnoyl and T =

Tetraenoyl).

Can. lnst. Food Science and Techno!. J. Vo!. 5, No.1, 1972

60

A,B

50

60

50

60

CD

50

50

54 52

~

52

40

i

AO

40

~ 30

•• :i

20

575;9

5/il"

.1

1:1.8' i=O

1" : 0

• •

504.8

i ••

54

1

II :!

Ii:!. ;:

"••

:~.

•

BAND

Figure 5.

50

• 10 •• ••-

50

• ••

l

i 48 =•• i:! I:~

5

· ·••

.48

·48

-

~i

6

8

7

Main indiviuual triglycerides 1 of the triglyceride types of fractions (A,B) and (C,D).

--_._.-.---.---.

Triglyceride type

Hajor molecular species

SMS

SSM

SMM

MSM

C 54 C52 C50 C48

Nole

(A,B)

%2

(C,D)

l.Q....L

~

ll....Q.

!.L.A

17.6

1b...1

12.9

11. 7

l4.....3.

lL.1.

.ll....5. 14.5 7.7 6.0

l.Q.....d 16.2 5.9 9.5

..J.Q...d

ll...Q

l.2....9.

19.2

18.1 8.0 7.8

ll...Q

.4b.f. ll.c!

.:J.L..i

4.2 12.1 15.2

18.5 4.9

1H .7

.4b.f. bL..!

Q...l 17.0

19.0 5.7

1.L.!

~

~

~

~

10.8 2.5

14.0 6.0

8.1

0

~

Main individua1 3 triglycerides

16, 18, 18, 18, 18, 18, 16, 18, 16, 16,

16, 16, 18, 16, 16, 14, 16, 14, 16, 14,

16 14 14 16 12 14 14 12 12 14

16, 18, 18, 16, 18, 14,

18: 1, 18:1, 18:1, 18:1, 18:1, 18:1,

16 14 16 14 18 14

18: 1, 18:1, 18:1, 18:1, 18:1, 18:1,

18, 16, 18, 16, 18, 14,

18:1, 18:1, 18:1, 18:1,

18: 1, 18:1, 18:1, 18:1,

18:1, 18:1, 18:1, 18:1,

16, 18, 14, 12,

18:1, 18:1, 18:1, 16:1,

18:1, 18:1, 16:1, 16:1,

14 16 16 14 18 14 16 18 14 12

18:1 18:1 18:1 18:1 18:1 16:1 16:1 16:1

Many other individual triglycerides could be existing in appreciable amounts and bands number 6 and 8 were not 2 considenid. The two dominant molecular species of each triglyceride type 3 are underlined. lhe arrangement of fatty acids within individual triglycerides as no structural significance except when positional isomers Were originally separated by silver ion TLC. 1

J. lnst. Can. Science et Techno!. Aliment. Vo!. 5. No 1, 1972

I I.

54

.. i

52

.i

• i

i i : • -. 5;; 4:8 i" ! i 10 52:! I:! i i 54= • • 545.. i i:i lUi 5~i: ii i • i---l.ll.L.l1J 52 :48

:

:

54

I I·

;: 0 30::;; 52

.50

48

50

123

NUMBER

The distribution of tour molecular species C 54 , C 52, C 50 and C 48 in the eight banus from (A,B) separated on silver nitrate impregnated TLC.

triglyceride 16, 18 :1, 14 is the main triglyceride in the molecular species C48 (14.5 mole %). Molecular species CS4 (7.7 mole %) of the same type would contain the triglyceride 18, 18 :1, 18. In 881\1, (band 3), all of the molecular species have to have a monoenoic acid at an external position. Again, the monoenoic acid is largely 18:1. Therefore, Table 7.

50

20

5.

5 •• 4

2

~ ~30

20

..., !~

n B! •• : ! I:i U! i: 4! : i :••i I.:i 1:- 1:1' 0.-. I.! 11-

~

~

54 :."

: •

:i

10

30

I

54

.- ...- 1....- If. .. . .In ii =•

54

:. 51 :

I: :. :

48

i.

: !• :

52

5. = .48 i· .

!" •

: :

p!

:

4

~ 5

40

54

52

;:

i

50

1 8

1.

"

!" • =1 48 p.

5• i H. Ii: i 6

7

~

r

....

20

150

5• "

10

1:4R

:

:

5 ••

:

:

8

BAND NUMBER

Figure 6.

The distribution of four molecular species C 54 , C 52, C 50 and C 4g in the eight bands from (C,D) separated on silver nitrate impregnated TLC.

molecular species C so would contain 18 :1, 18, 14 and 18 :1, 1G, 16; C S2 would contain 18 :1, 18, 16 only, and these triglycerides would be the major single triglycerides of this type since C so (30.8 mole %) and C S2 (26.9 mole %) are the major molecular species for this triglyceride type. .Molecular species C 48 (18.1 mole %) would contain 18 :1, 16, 14 and CS4 (8 mole %) 18:1, 18,18. In band number 4 (essentially 8MM) two mOlloenoic acids (largely 18:1) have to be in the a and (3 positions. ~lVIolecular species C S2 (42.2 mole %, major molecular species) would contain 18 :1, 18 :1, 16 and this has to be the major individual triglyceride among all others of the type SMM. Molecular species C S4 (22.1 mole %) would contain 18:1, 18:1, 18; Cso (18.5 mole %) would contain 18 :1, 18 :1, 14 and C48 (4.9 mole %) 18 :1, 18 :1, 12. In band number 5 (essentially M8.M), molecular species Csz (42.2 mole %) would contain 18 :1, 16, 18:1 and this will be the major single triglyceride in this band. Molecular species C S4 (21 mole %) would essentially contain 18 :1, 18, 18:1. Molecular species C so (19.1 mole %) would be 18 :1, 14, 18:1 and molecular species C48 (5.7 mole %) would contain 18 :1, 12, 18:1. Band number G ClVI8:~1 and 8 zD) is similar to band number 5 in its major molecular species and fatty acids, so it will have some triglycerides similar to those of Land 5. Band number 7 contains a mixture of l\1.MM and 8 zD. It can be expected that molecular species CS4 (52.4 mole %, major molecular species) of the type MMM contains 18 :1, 18 :1, 18:1; molecular species C S2 (30.2 mole %) to contain 18 :1, 18 :1, 16:1; molecular species Cso (10.8 mole %) to contain 18 :1, 16 :1, 1G:1 and molecular species C48 (2.5 mole %) to contain IG :1, 16 :1, 1G:1. Triglyceride type SzD must be largely assembled of 18:2 and long chain saturated acids. The previous discussion on the fatty acid composition of band 8 indicated the possibilty of the presence of several triglyceride types within this band, ego 8 zTr, 8 zT, ("Tr" is a triene or linolenic acid and "T" is a tetraene or arachidonic acid), 8MTr and 20

probably others. These triglyceride types, however, have to be confined essentially to the major molecular species OS6, OS4, OS2 and Oso. The triglyceride types of (O,D) have more or less the same major molecular species and fatty acids though in different proportions. Using arguments similar to those used with (A,B), the main individual triglycerides of the different triglyceride types of (O,D) were determined and compared with those -of (A,B) in Table 7.

References Ast, H. J. and R. J. Vander Wal. 1961. The structural components of milk triglycerides. J. Amer. Oil Chemists' Soc. 38: 97. Blank, M. L. and O. S. Privett. 1964. Structure of milk fat triglycerides. J. Dairy ScI. 47: 481. Boudreau, A. and J. M. deMan. 1965. The composition of mllk fat diglycerides and partial glycerides obtained by pancreatic llpase hydrolysis technique. Biochim. Biophys. Acta. 98: 47. Breckenridge, W. C. and A. Kuksis. 1968. Structure of bovine milk fat triglycerides. I. Short and medium chain lengths. Lipids 3: 291. Breckenridge, W. C. and A. Kuksis. 1969. Structure of bovine milk tat triglycerides. II. Long chain lengths. Lipids 4: 197. Breckenridge, W. C.• L. Marai and A. Kuksis. 1969. Triglyceride structure of human mllk fat. Can. J. Biochem. 47: 761. Chen, P. C. and J. M, deMan, 1966. Composition of mllk fat fractions obtained by fractional crystallization from acetone. J. Dairy ScI. 49:612. Clement, G., J. Clement, J. Bezard. G. DiCostanzo and R. Paris. 1963. Hydrolysis of the triglycerides of butter by pancreatic llpase. Location of butyric acid. J. Dairy ScI. 46: 1423. Dimick, P. S. and S. Patton. 1965. Structure and synthesis of milk fat. VII. Distribution of fatty acids in mllk fat triglycerides with special reference to butyrate. J. Dairy ScI. 48: 444. Freeman, C. P., E. L. Jack and L. M. Smith. 1965. Intramolecular fatty acid distribution in the milk fat triglycerides of several species. J. Dairy ScI. 48: 853.

G;'eenb,mk, G. R. 1953. The fractionation and properties of ~ glyceride fractions of butter fat. Proc. XIIIth Intern. D Congr. 3: 1269. Haab, W., L. M. Smith and E. L. Jack. 1959. Countercurrent dlstrl bution of mllk fat triglycerides. J. Dairy Sci. 42: 454. Jack, E. L., C. P. Freeman, L. M. Smith and J. B. Mickle, 1963. ~ creatic lipase hydrolysis of cow mllk fat. J. Dairy ScI. 46:284. Jensen, R. G. and Gander, G. W., 1960. Fatty acid compositio~ the monoglycerldes from llpolyzed milk fat. J. Dairy Sci. 43: 17 Jensen R. G., J. Sampugna and G. W. Gander. 1961. Fatty composition of the diglycerides from lipolyzed mllk fat. J. Da Sci. 44: 1983. Jensen, R. G., J. Sampugna and R. L. Pereira. 1964. IntermolecUl!l specificity of pancreatic lipase and the structural analysis It milk triglycerides. J. Dairy ScI. 47: 727. Jensen, R. G., J. G. Quinn, D. L. Carpenter and J. Sampugna. 1gei1 Gas-llquid chromatographic analysis of mllk fatty acids: .. review. J. Dairy ScI. 50:119. Kuksis, A. 1967. Gas chromatography of neutral glycerides. (Lipid chromatographic analysis. G. V. Marinetti, editor, Vo!. pp. 239-337. Marcel Dekker, Inc., New York). . Kuksis, A., M. J. McCarthy and J. M. Beveridge. 1963. Quantitathl gas-llquid chromatographic analysis of butterfat triglycerideQ J. Amer. all Chemists' Soc. 40:530. Litchfield, C. 1968. Trigiyceride analysis by consecutive llqUldl llquid partition and gas-liquid chromatography. Ephedra nevadl ensis seed fat. Lipids 3: 170. Nutter, L. J. and O. S. Privett. 1967. Structures of triglycerides lC bovine milk serum. Short chain triglycerides. J. Dairy ~ 50: 1194. Shehata, A. A. Y., and J. M. deMan, 1971. Chromatographic metholij for. separation and quantitative analysis of triglycerides. review. Can. Inst. Food Technol. J. 4: 38. . Shehata, A. A. Y., J. M. deMan, and J. C. Alexander. 1970. A Sim~l' and rapid method for the preparation of methyl esters of fa in mllligram amounts for gas chromatography. Can. Inst. F Technol. J. 3: 85. Shehata, A. A. Y., J. M. deMan, and J. C. Alexander. 1971. TriglYi ceride composition of mllk fat. I. Separation of triglycerides column and gas-liquid chromatography. Can. Inst. Food Tech~ nol. J. 4: 61. Smith, L. M., C. P. Freeman and E. L. Jack. 1965. Distribution o(j fatty acids in mllk fat fractions. J. Dairy ScI. 48: 531.

1

1

b'

Received Nov. 20, 1970.

21 Can. Inst. Food Science and Technol. J. Vol. 5, No.1, 1972