J . Sci. Fd Agric. 1975,26,507-521

Selective Extraction and Quantitative Analysis of Non-Starch and Starch Lipids from Wheat Flour William R. Morrison, David L. Mann; Wong Soon and Anne M. Coventry Department of Food Science and Nutrition, University of Strathclyde, Glasgow GI ISD (Manuscript received 24 September 1974 and accepted 6 November 1974)

Wheat flour non-starch lipids (lipids not bound to starch) were quantitatively extracted with water-saturated n-butanol (WSB), benzene-ethanol-water (10 :lo :1, by vol.) or ethanol-diethyl ether-water (2: 2: 1, by vol.) in 10min at 20 "C. Starch lipids (lipids bound to starch) were subsequently extracted with WSB at 90-100 "C. Carotenoid pigments were quantitatively extracted with the non-starch lipids. There was no significant hydrolysis of esterified fatty acids and no detectable autoxidation of unsaturated acids in the hot solvent extracts. Non-starch and starch lipids from a high grade spring wheat flour and three grades of winter wheat flour were separated by thin-layer chromatography and quantified as fatty acid methyl esters (FAME) by gas-liquid chromatography (g.1.c.) using heptadecanoic acid (or its methyl ester) as internal standard. Total flour and starch lipids after acid hydrolysis were also converted to FAME for quantitation by g.1.c. Non-starch lipids consisted of 59-63 % non-polar (neutral) lipids, 22-27 % polar glycolipids and 13-16 % phospholipids. Steryl esters, triglycerides, and all the diacyl galactosylglyceridesand phosphoglycerideswere present only in non-starch lipids. Starch lipids consisted of 6-9 % non-polar (neutral) lipids (mostly free fatty acids), 3-5 % polar glycolipids and 86-91 % phospholipids (mostly lysophosphatidyl choline). Starch lipids were almost exclusively monoacyl lipids. Factors are given for converting weight of FAME into weight of original lipid for all individual lipid classes in wheat which contain 0-acyl groups. Factors for total lipids are: total starch lipids = FAME x 1.70, total non-starch lipids = FAME x 1.20, and total flour (non-starch starch) lipids = FAME x 1.32. Similar factors could be used to convert weight of lipids obtained by conventional acid hydrolysis methods into weight of unhydrolysed lipids. Phospholipid contents are given by: total starch phospholipids = P x 16.51, total non-starch phospholipids = P x 23.90, total flour phospholipids = P x 17.91.

+

1. Introduction Numerous attempts have been made to find solvents which will extract wheat flour lipids quantitatively and without damage,l but in our opinion none has been entirely successful. In many cases incomplete extraction was not evident because insensitive methods were used to quantify residual unextracted lipid, or because values obtained a

Present address: Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford MK44

1LQ. 507

508

W. R. Morrison et al.

for unhydrolysed extracted lipid were wrongly compared with values obtained for total lipid after acid hydrolysis. Since we wished to compare the extraction efficiency of solvents on a small scale, we first had to develop a more sensitive method for quantifying total, extracted and unextracted lipids. The method, described in this paper, is based on acid hydrolysis in the presence of an internal standard, followed by quantitation of an aliquot of lipid as fatty acid methyl esters (FAME) by gas-liquid chromatography (g.1.c.). The weight of original lipid is obtained from the weight of FAME multiplied by an appropriate conversion factor. The best solvents for extracting wheat lipids are considered to be ethanol-diethyl ether-water (EEW, 2:2:1, by V O ~ . benzene-ethanol-water ),~ (BEW, lO:lO:l, by ~ 0 1 . ) ~ and water-saturated n-butanol (WSB).4-6 Flour lipids extracted with WSB contain much more lysophosphatidyl choline (LPC) than those extracted with EEW6 and since LPC is the principal lipid in wheat ~ t a r c h we ~ - thought ~ that EEW (and possibly BEW) might be used to extract flour non-starch lipids (i.e. lipids other than those in the starch fraction), and WSB could then be used to extract the starch lipids. The rates of diffusion of lipids into solvents have received scant attention, most workers being content to extract for several hours or overnight, with several changes of solvent until "exhaustive" extraction has been achieved. We therefore decided to study the rates of extraction of flour and starch lipids by EEW, BEW and WSB. The results described in this paper show that non-starch lipids can be quantitatively extracted from flour in 10 min with EEW, BEW or WSB at 20 "C,and starch lipids can then be extracted in about 3 h with WSB at 90-100 "C.The non-starch and starch lipids each have characteristic compositions.

2. Experimental 2.1. Materials Unbleached, untreated commercial flours were used. High-grade spring (HGS) flour (73 % extraction rate, 12.9 % protein) was milled from a grist consisting of 60 % No. 2 Manitoban, 20% Australian FAQ, and 20% US Hard Red Winter wheats. Winter flours were milled from grists of 70 % English and French wheats and 30 % Australian FAQ wheat. Three grades of flour were used-high grade, top divide (HGW, 50% extraction rate, 9.0 % protein), straight run (SRW, 76 % extraction rate, 9.5 % protein), and a low grade, bottom divide (LGW, 74 % extraction rate, 9.0 % protein). Flours were stored aerobically at -20 "C. Starches were washed from flour-water doughs, the wash-water filtered through a cloth and centrifuged, and the light coloured starch sediment recovered. After repeated washing the starches were air dried. For small amounts of starch it was more convenient to make a thin flour-water dough (I :1, by wt) ;this was centrifuged for 2 h at 75 000 x g, the starch layer recovered, resuspended and recentrifuged twice. The nitrogen contents of the starches were 0.03-0.1 % (dry basis). The lipid nitrogen contents calculated from the lipid analyses (below) were 0.025 so that the washed starches were effectively protein-free.

x,

Non-starch and starch lipids in wheat flour

509

All solvents were reagent grade and, except for butanol, were redistilled before use. Methyl heptadecanoate (17 :O), of 98 % purity by g.l.c., was purchased from Sigma London Chemical Co Ltd, Kingston-upon-Thames. 2.2. Methods 2.2.1. Determination of lipids as fatty acid methyl esters Flour or starch (0.5-0.75 g, correct to 0.1 mg) was weighed into a round-bottom tube (16 x 100 mm, Sovirel, 92 Levallois-Perret, France) fitted with a screw cap and a PTFE-faced liner. Methanol (2 ml) containing an appropriate amount of heptadecanoic acid or methyl heptadecanoate (17:O) was added under nitrogen and the sample dispersed by mixing. Concentrated hydrochloric acid (1.8 ml, 12 M) was added under nitrogen to the tube which was then quickly sealed, shaken vigorously, and heated (shaking at regular intervals) in a boiling water bath for 45 min. After cooling, hydrolysate lipid was extracted by adding 2 ml chloroform, shaking vigorously and centrifuging. The hydrolysate lipid in the chloroform layer consisted of steryl esters, triglycerides, free fatty acids and FAME. An aliquot of the chloroform layer was transferred to a similar tube with a Pasteur pipette, the solvent was removed with a stream of nitrogen, and 2 ml of methanolic boron fluoride (14 % w/v)-methanol-benzene (35 :35 :30, by vol.)l0 was added under nitrogen. The tube was closed and heated in a boiling water bath for 45 min. FAME were extracted with pentane and purified by chromatography on silica gel G thin-layer chromatography (t.1.c.) plates developed with benzene. lo The esters were visualised with 2',7'-dichlorofluorescein under ultraviolet light and eluted from the silica gel with diethyl ether. FAME were analysed by g.1.c. on instruments fitted with flame ionisation detectors and 1.8 m x 3.3 mm diameter columns packed with 15% EGSS-X on 100120 mesh Gas-Chrom P (Applied Science Laboratories Inc.), operated at 185-195°C with an argon carrier gas flow rate of 10 ml min-l. Solvent-extracted lipids were quantified in a similar manner. Internal standard (17 :0) was added to an aliquot of extract, and the solvents removed under nitrogen over a boiling water bath or by rotary evaporation. The lipids were then treated with boron fluoride methanolysis reagent and the FAME analysed by g.1.c. as described above. Individual lipid classes were separated by t.1.c. and converted to FAME'O (in the presence of silica gel and 17:O internal standard) for quantitation by g.1.c. Identifications of lipids were based on previous studies of flour and starch l i p i d ~ , ~ -using ~,~l reference lipids prepared in this laboratory. Phospholipids were also quantified by phosphorus content." Solutions of lipids extracted with hot WSB tended to form white precipitates which were removed by centrifugation without affecting the composition of the lipids. The t.1.c. solvents for separating neutral lipids and phospholipids were described previously." Glycolipids, free from neutral lipids, were obtained by developing plates to 12 cm height with chloroform-acetone-acetic acid-water (10:90:2: 3, by vol.), followed by air drying and developing in the same direction to 18 cm height with diethyl ether-acetic acid (99:1, by vol.). Digalactosyl monoglyceride (DGMG) and N-acyl phosphatidyl ethanolamine (PEA) overlapped in this system, but the amount of DGMG could be calculated, knowing the amount of PEA from phosphorus determinations.

510

W. R.Morrison ef al.

2.2.2. Cold solvent extraction offlour and starch Flour or starch (0.75 g correct to 0.1 mg) was weighed into a round-bottom tube (16 x 100 mm) fitted with a screw cap and a PTFE-faced liner, and 6 ml solvent added under nitrogen. The tube was capped and shaken immediately. For prolonged extraction the tube was mechanically inverted three or four times each minute. After extraction at 20 "C the tube was centrifuged and a 4 ml aliquot of solvent removed for analysis. Flour or starch were re-extracted three or four times before commencing hot extractions with WSB. 2.2.3. Hot extraction ofjour or starch Cold solvent-extracted flour or starch was extracted under nitrogen with WSB as described in section 2.2.2, except that the tube was kept in a boiling water bath and was shaken intermittently by hand. The tube was cooled to 20 "C before removing aliquots of extract. 2.2.4. Determination of wheat carotenoids Flour was extracted with WSB at 20 "C and the absorption spectrum scanned from 550 to 380 nm using 10 mm path length cells and 0-0.1 or 0-0.5 absorbance range. The reference cell contained a similar extract of a freeze-dried aerobically mixed dough made from CIO,-bleached flour, and the instrument was balanced at 550 nm. Carotenoid absorbance was then measured in WSB at 447 and 494 nm without further correction for background absorption from substances other than carotenoids. In practice the ratio of A447/A494was almost constant, indicating that adequate background correction had been achieved. 2.2.5. Moisture determination Moisture was determined by the AOAC method13 of oven drying in air for 1 h at 130 k 3". All results are expressed on a dry matter basis. 3. Results 3.1. Determination of lipids as FAME The yield of total FAME was the same when the acid hydrolysis step was carried out at 60 or 100 "C(Table 1). Hydrolysis at 100 "C resulted in very dark chloroform extracts, but the FAME were colourless after purification by t.1.c. Hydrolysis at 100 "C was Table 1. Yields of fatty acid methyl esters from HGW flour and starch (mg/100 g, dry basis) after acid hydrolysis at 60 or 100 "C,followed by methanolysis. Results are given as mean k standard error (8 determinations)

Fatty acid methyl ester Sample Flour, 60 "Chydrolysis Flour, 100 "C hydrolysis Flour, 0.05% BHT, 100 "C Starch, 100 "C hydrolysis

16:O

18:O

409-i.7 414 _+ 5 387 5 13 204 k 6

21k 1 21 +_ 2 17 k 1 9k 1

18:l 187_+2 189 5 4 174 f 4 39 k 1

18:2

18:3

Total

1102f23 1080 & 78 1003 f 27 231 8

87f4 84 k 5 76 k 6 19 k 2

1806+36 1788 ? 28 1657 k 35 502 k 10

511

Non-starch and starch lipids in wheat flour

preferred since there was less chance of incomplete hydrolysis and there was no difficulty from emulsificationat the chloroform extraction stage. Addition of antioxidant (0.05 % or 0.5 Zjbutylated hydroxy toluene) did not improve the yields of linoleate or linolenate (Table l), but there was a very slight improvement when half-strength acid was used. Losses of poly-unsaturated FAME during acid-catalysed methanolysis are reported to be negIigible,1° but in the present study there were occasionally significant losses during the initial acid hydrolysis step which could not be prevented. Almost all lipids in wheat contain fatty acids, mainly as 0-acyl esters.6 Lipids separated by t.1.c. can therefore be quantified conveniently and accurately as FAME by g.1.c. after methanolysis in the presence of silica gel and an internal standard. There may be substantial autoxidative losses of linoleate and linolenate if lipids are extracted from the silica gel before methanolysis, and methanolysis in the presence of silica gel is therefore strongly recommended. The weight of original lipid can be obtained from the weight of FAME multiplied by a conversion factor (see section 3.7).

3.2. Cold solvent extraction of flour and starch At the beginning of this study it was thought likely that there would be some loss of polyunsaturatedfatty acids during the more rigorous extraction and analysisprocedures. Since the principal lipids in wheat all contain palmitic acid6*14*15 it was decided to follow the extraction of palmitate only (Figures 1 and 2), thereby eliminatingthe autoxidation problem. In fact with some experienceit was found that all fatty acids in extracted lipids could be quantified satisfactorily. 300 r B L

0 3 -

c

h

4

I5O

t Extroclion lime (min)

(log scale)

Figure 1. Extraction of lipid (palmitate) from HGW flour and washed starch with water-saturated n-butanol (WSB), ethanol-diethyl ether-water (EEW, 2:2:1, by vol.), or benzene-ethanol-water (BEW, lO:lO:l, by vol.) for various times at 20 "C. Washed starch was also extracted with WSB at 100 "C.0, Flour; B,washed starch.

512

W. R. Morrison et al.

When HGW flour was mixed with EEW, BEW or WSB at 20 "Cthere was very rapid extraction of lipid (Figure 1). The yield remained constant up to 30 min, and then increased slowly, particularly with WSB extraction. It was thought that non-starch lipids were extracted almost instantaneously, and that starch lipids were extracted very slowly by WSB. WSB (20°C)

/e-*

c

/

/

m 01

I I

WSB(2O"C)

'a, . ' '

I EEW 120 "C)

I

I

3

10

32

100

316

1000

E x t r a c t i o n time (min)

( l o g scale)

Figure 2. Extraction of lipid (palmitate) from HGS flour and washed starch with water-saturated n-butanol (WSB), ethanol-diethyl ether-water (EEW, 2: 2:1, by vol.), or benzene%thanol-water (BEW, lO:lO:l, by vol.) for various times at 20 "C. Washed starch was also extracted with WSB at 100 "C. a, flour; H washed starch.

The extraction of lipids from washed starch was therefore examined (Figure 1). EEW and BEW did not extract significant amounts of lipids, but WSB extracted starch lipids very slowly at 20 "C. The extraction of washed starch should not be compared too closely with the extraction of starch in flour. since some amylose (which probably contains lipid) would have been leached out from starch during washing, and the washed sedimented starch granules cannot be considered completely representative of the range of starch granules in the flour. Similar results were obtained with HGS flour and washed starch (Figure 2), but there were indications that starch lipid was more readily extracted. Since HGS flour was milled from harder wheats it would contain slightly more damaged starch, and this may account for the greater extractability of the starch lipids. The yield of lipids (Figures 1 and 2), which were calculated from analyses of solvent aliquots, could have been subject to significant volumetric errors. It was calculated that sorption of water by flour from WSB5 would result in a volumetric error of less than 3 %, and it was assumed that there would be similar sorption of water from EEW

Non-starch and starch lipids in wheat flour

513

and BEW. In practice, successiveextractions of flour with EEW and WSB gave theoretical yields on non-starch lipids in the second and third aliquots, and the total lipid recovered with three extractions was 97% of the amount calculated from the first aliquot.

3.3. Hot solvent extraction of washed starch Since Acker and Schmitz' obtained best yields of wheat starch lipids with hot butanolwater (65 :35, v/v), it was decided to use WSB at 100 "C to extract lipids from washed starch in the present study. After 300 min the yields of palmitate (Figures 1 and 2) were -67 % of the acid hydrolysis values. Since there was little increase in extracted lipids after 100 min, it was decided to extract starch three times for periods of 100 min each. The yields of lipids (based on palmitate) were successively 55,28 and 14% of the total, giving a recovery of 97 %. Unlike the cold WSB extraction of flour, the yields of lipid in the second and third hot extracts were larger than predicted from the first aliquot, showing that complete equilibration of solvent with lipid in the starch had not occurred during the first extraction. 3.4. Degradation of lipids during solvent extraction Since comparatively high temperatures were used in the extraction of starch and in the evaporation of solvents, some degradation of lipids was considered possible, even although Acker and Schmitz' had reported that phosphatidyl choline was stable for 2 h in hot butanol-water. Non-starch lipids were extracted from flour with WSB at 20 "C, heptadecanoic acid internal standard added, and aliquots heated at 100 "C under nitrogen for 3 h. The lipids in the heated extracts were then examined for changes due to transesterification,hydrolysis and autoxidation. Lipids in control and heated lipid extracts were separated by t.1.c. on silica gel G plates developed with diethyl ether-hexane (2 :98, v/v), and the lipids detected by charring with 50% sulphuric acid. Synthetic butyl palmitate was used as a standard. No traces of fatty acid butyl esters formed by transesterificationwere detected in any of the lipid extracts. The free fatty acids (FFA) of unheated and heated extracts were isolated by preparative t.1.c. (solvent: diethyl ether-hexane-acetic acid, 20 :80 :2, by vol.), converted to FAME and quantified by g.1.c. There were no increases in the levels of FFA after heating at 100 "C for 3 h (Table 2). Table 2. Fatty acid composition of WSB extracts of flour lipids before and after heating under nitrogen for 3 h at 100 "C. Results are given as means standard error of means (n= 6)

+

-

~

~~~

Fatty acid content (pg/ml) Sample

16:O

18:O

18:l

18:2

18:3

Total

Total lipid, unheated Total lipid, heated FFA, unheated FFA, heated

3 8 6 k 10 388k5 38k1 40+1

305 1 29k1 3kO

363 2 4 364+7 25+1 25+2

1738k 13 1740t17 152+_3 152t4

136+2 13956 12+1 12t-1

2653+ 10 2660526 23024 232+5

18

3+0

514

W. R. Morrison e# al.

Heating the WSB extracts under nitrogen did not affect the fatty acid compositions of the total lipids (Table 2). The FFA, which are particularly susceptibleto autoxidation, were likewise unaffected. It has already been shownio that there is very little loss of polyunsaturated fatty acids through autoxidation or artifact formation during methanolysis under nitrogen. Autoxidation in hot solutions may be prevented by the greatly reduced solubility of oxygen in hot organic solvents coupled with its low partial pressure after flushing out the tubes with nitrogen.

3.5. Extraction of carotenoids Wheat carotenoids (principally xanthophylls) are relatively non-polar lipids, and they do not occur in starch granules. Flour carotenoids were completely extracted by WSB at 20 "Cin 5 min together with the non-starch lipids. Extraction with EEW or BEW was a little slower. 3.6. Composition of non-starch lipids and starch lipids Our interpretation of the extraction curves in Figures 1 and 2 was that flour non-starch lipids were extracted quantitativelywith the cold solvents with very little contamination from starch lipids in the flour. This could be confirmed if certain lipids were present exclusively in one or other fraction, or if the residual lipids in flour after extraction at 20 "Cwere quantitatively the same as those in an equivalent amount of washed starch. The flour and washed starch lipids were therefore quantified and compared. Flours (100 g batches) were extracted with EEW or WSB at 20 "Cfor 10 min and the solvent extracts used for lipid analysis. The flourswere re-extracted four times to remove interstitial lipid solution and these extracts discarded. The residual lipids in the cold solvent-extracted flours were then extracted three times with hot WSB (the solvent temperature only reached 83-93 "Cin bulk extractions), and the combined hot extracts were taken for analysis. Washed starches were extracted once with EEW or WSB at 20 "Cto remove any superficialnon-starch lipid, the extracts discarded, and the starches then extracted three times with hot WSB. The compositions of the non-starch and starch lipids in the various extracts are given in Tables 3 and 4. The non-polar (neutral) lipids separated by t.1.c. contained small amounts of glycolipids (6-0-acyl monogalactosyl diglyceride = EMGDG, esterified steryl glucoside = ESG) which for convenience were quantified along with the neutral lipids steryl ester (SE), triglyceride (TG), diglyceride (DG), monoglyceride (MG) and free fatty acid (FFA). The polar glycolipid fraction consisted of mono- and digalactosyl diglycerides (MGDG, DGDG) and the corresponding monoglycerides (MGMG, DGMG). The phospholipids were N-acyl phosphatidyl ethanolamine (PEA), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl inositol (PI), and the corresponding monoacyl or lysophospholipidsLPEA, LPE, LPC, LPS and LPI. There were two minor unidentified phospholipids( x and y in reference 15), which moved close to PE and LPE respectively on t.l.c., and these were tentatively identified as phosphatidyl glycerol (PG) and lysophosphatidyl glycerol (LPG).l6 The non-starch lipids extracted with EEW and WSB from HGS flour were very similar (Table 3), and the small differences are consistent with WSB being a slightly more efficient solvent than EEW. The data in Figure 2 indicate that there should be very

Non-starch and starch lipids in wheat flour

515

little starch lipid, especially in the EEW-extracted lipids. This is supported by the low levels of LPC which constitute 1.44%(EEW) and 2.1 1 % (WSB) of the non-starch lipids compared with 73.3 % of the washed-starch lipids. The non-starch lipids are rich in TG and other non-polar lipids, and contain almost all the diacyl galactosyl- and phosphoglycerides. PEA and LPEA are exclusively nonstarch lipids. Since the flour was stored at -20 "C from the time it was milled there Table 3. Composition of non-starch lipids (first extraction with EEW or WSB at 20 "C) and starch lipids (subsequentextractionwith WSB at -90 "C)of HGS flour and HGS washed starch (mglipidjl00 g dry flour or starch) HGS flour Lipid

Non-starch (EEW)

SE 67 TG 657 DG 93 FFA 101 EMGDG, MG 67 ESG 66 MGDG 90 MGMG 23 DGDG 219 DGMG 56 PEA 71 LPEA 33 PE PG" 9 LPE LPG" 11 PC 69 LPC 24 PS, PI, LPS, LPI 12 Total non-polar 1051 (63) (neutral) lipid (%) Total polar 388 (23) glycolipid (%) Total phospholipid 229 (14)

+ +

Starch

HGS flour Total

Non-starch (WSB) Starch

87 72 719 674 86 99 125 110 75 66 72 71 94 87 34 23 230 214 58 81 72 71 34 33 2 11 13 55 66 I0 32 101 66 683 707 36 28 40 11 126 (13) 1177 (44) 1079 (63) 20 62 6 24 8 6 4 11 11 25

439 (17)

382 (23)

800 (82) 1029 (39)

242 (14)

51 (5)

Total

18 35 6 19 7 6 6 7 12 25

HGS Washed Starch

90 709 92 129 73 77 93 30 226 83 72 34 6 19 59 69 38 104 657 693 24 35 91 (10) 1170 (45)

95 (9)

432 (16)

51 ( 5 )

50 ( 5 )

784 (85) 1026 (39)

3 2 2 66 18 4 2 15 2 32

8 77 36 807 27

955 (86)

( %)

Total lipid (100%) 1668 (100) 977 (100) 2645 (100) 1703 (100) 925 (100) 2628 (100) 1101 (100) % P (calc.) 0.57 4.91 2.17 0.60 5.07 2.17 5.21 Tentative identification.

would be negligible Iipolysis after milling. The partially acylated glycerides and the FFA were therefore present in the wheat before milling, and may be residues of intermediate stages in lipid b i o ~ y n t h e s i s .'~ ~ , ~ The washed starch lipids contained very little SE, TG or diacylglycerides(Table 3), but there were significant amounts of SE and TG in the residual starch lipids in flour after cold solvent extraction. This is attributed to inefficiency in the bulk solvent extractions.The principal starch lipidswere LPC (73.3 %), LPE (7.0 %) and FFA (6.0 %), with lesser amounts of other monoacyl glycerides (MG, MGMG, DGMG, LPG,

SE

10 10 3 15 3 3 2 5 3 9

43 909 67

525 (19)

19 (2)

753 (92) 1046 (38)

39 1198 (43)

644

44 ( 5 )

615 30

-

35 73

53 919 70 79 56 21 117 22 325 61 95 33 19 40 80 96

Total

24 (3) 796 (93)

483 (27) 240 (1 3)

49 (5)

183 26 62 (6)

936 (89)

-

26 84

-

-

4 7 2 20 3 3 2 7 3 12

Starch

654 32 39 (4)

5

42 805 79 64 58 19 101 16 321 39 76 39 14 3

Non-starch

SRW flour

70 23 10 1067 (60)

-

23 104

-

-

7 27 8 3 4 10 11 24

15

2

HGW Washed Starch

84 (8) 57 ( 5 ) 968 (87)

1036 (39)

796 35

-

33 104

-

-

-

507 (19)

3 16 9 34 16 6 4 15 11 27

46 812 81 84 61 22 103 23 330 51 76 39 14 29 89 70 677 42 1106 (42)

Total

285 (16)

407 (22)

35 892 60 61 54 15 92 21 283 11 85 42 20 4 9 84 33 8 1117 (62)

757 (89)

31 (4)

601 38 59 (7)

-

29 89

-

-

-

4 3 7 7 14

4

3 20

20

8

84

85 42 20 33 98

1

17

1042 (39)

438 (17)

-

973 (91)

35 (3)

806 31 59 (6)

36 100

-

31 9 3 2 9

5

25

3 8

LGW Washed Starch

43 912 63 81 58 19 95 28 290

Total

634 46 1176 (44)

SRW LG W flour Washed Starch Non-starch Starch

1953 (100) 816 (100) 2769 (100) 1047 (100) 1790 (100) 859 (100) 2649 (100) 1109 (100) 1809 (100) 847 (100) 2656 (100) 1067 (100) 5.47 0.55 0.62 5.64 2.09 5.67 2.21 5.33 0.66 5.48 2.20 5.58

'Tentative identification.

Total lipid (100%) % P (calc.)

( %)

+

-

Starch

Non-starch

DG FFA 64 EMGDG, MG 53 ESG 18 MGDG 115 MGMG 17 DGDG 322 DGMG 52 PEA 95 LPEA 33 PE PG" 19 LPG" 5 LPE 7 PC 96 LPC 29 PS, PI, LPS, LPI 9 Total non-polar 1154 (59) (neutral) lipid (%) 506 (26) Total polar glycolipid (%) Total phospholipid 293 (15)

TG

Lipid

HGW flour

Table 4. Composition of non-starch lipids (first extraction with WSB at 20 "C)and starch lipids (subsequent extraction with WSB at 90 "C) of HGW, SRW and LGW flours and their washed starches (mg lipid/100 g dry flour or starch)

Steryl ester Triglyceride Diglyceride Monoglyceridc Free fatty acid Estd. monogalactosyl diglyceride Estd. steryl glycoside Monogalactosyl diglyceride Monogalactosyl monoglyceride Digalactosyl diglyceride Digalactosyl monoglyceride

Lipid

667 848 596 344 270 1010 829 758 506 920 668

Mean mol. wt

2.35 0.995 1.05 1.21 0.95 1.18 2.93 1.32 1.76 1.60 2.32

FAME factor N-acyl phosphatidyl ethanolamine N-acyl lysophosphatidyl ethanolamine Phosphatidylethanolamine Lysophosphatidyl ethanolamine Phosphatidyl glycerol Lysophosphatidyl glycerol Phosphatidyl choline Lysophosphatidyl choline Phosphatidyl serine Lysophosphatidyl serine Phosphatidyl inositol Lysophosphatidyl inositol

Lipid

762 510 764 512 839 587

498

971 719 719 467 750

Mean mol. wt

1.14 1.26 1.26 1.64 1.31 1.74 1.33 1.79 1.34 1.79 1.48 2.05

factor

FAME

Table 5. Factors to convert weight of fatty acid methyl ester or phosphorus into weight of individual wheat flour lipids

24.22 16.08 24.60 16.47 24.67 16.53 27.09 18.95

15.08

31.35 23.22 23.22

P factor

W.R. Morrison et al.

518

LPS, LPI). This is in good agreement with the results of Acker et UI.'-~ and Wren et ~ 1 The lipids extracted by Rogols et al.I9 were obviously extensively hydrolysed during their acid hydrolysis extraction procedures. Analysis of the non-starch lipids (cold WSB extraction only) and starch lipids of HGW, SRW and LGW flours (Table 4) confirmed these findings. The separation of non-starch lipids and starch lipids from the winter flours was slightly better, judging by the small amounts of LPC in the former and the traces of SE and T G in the latter. HGW and SRW flours were taken from different mill-streams at the same time and are strictly comparable, but LGW flour was taken from a similar grist about two weeks later. There are minor differences in lipid compositions between the three flours which are probably of little significance. An increase in neutral lipids such as TG (from bran and germ oil) might have been expected with lowering of flour grade, but this was not observed. I t is possible that recent changes in milling technique and higher flour extraction rates have resulted in a more uniform lipid distribution between various grades of flour.

3.7. Factors to convert weight FAME into weight lipid The fatty acids of individual lipid classes determined during the present and previous studies6,14*15' l7 were fairly constant in composition, and lipid and FAME mean molecular weights could therefore be calculated. Factors were derived from these data to convert weight FAME (determined by g.l.c., section 2.2.1) into weight original lipids (Table 5). Similar factors for lipids of different fatty acid composition have been published by Christie et aLzo Using the factors in Table 5 and the lipid compositions in Tables 3 and 4, factors were then calculated to convert weight FAME in whole lipid fractions into weight of original lipids (Table 6). These factors will vary, depending on Table 6. Factors"for calculating weights of wheat lipids from weights of FAME or phosphorus

Conversion FAME to neutral lipid FAME to glycolipid FAME to phospholipid FAME to total lipid P to phospholipid

Flour, Starch and non-starch Flour, starch lipids lipids 1.06 1.59 1.32 1.20 23.90

1.15 1.92 1.76 1.70 16.51

Total flour lipids

1.32 17.91

a Factors for converting weight FAME vary by +3 %, and factors for converting weight P vary by +1% for flours studied (Tables 3 and 4).

the composition of the lipids, but they should not differ by more than +3 % for flours or starches similar to these used in the present study. Factors for non-polar (neutral) lipids and total lipids will be significantlylower if there are large amounts of TG present (e.g. in wholemeal flour, bran, germ and whole wheat). Factors were also calculated for converting weight of phosphorus into weight of phospholipid (Tables 5 and 6).

.

~

~

Non-starch and starch lipids in wheat flour

519

4. Discussion

4.1. Quantitation of lipids

No direct comparison was made between quantitation of lipids as FAME by g.1.c. (section 2.2.1) and conventional acid hydrolysis gravimetric method^.^^-^^ The hydrolysate lipid from 1 g flour, usually about 12-18 mg, can be weighed with an error of about k0.3 mg (51.7-2.5 %). With the new hydrolysis-methanolysis-g.1.c. method it is possible to determine as little as 100 pg FAME with an error of 25 pg (55 %). With larger amounts of lipid the standard error of determinations is +2 %. The new hydrolysis method is therefore equally precise, but it is much more sensitive than gravimetric methods, non-lipid artifact^^^*^^ do not cause trouble, and the additional information which can be derived from the composition of the FAME is frequently valuable. Since the yield of FAME in the new method is generally similar to the weight of purified acid hydrolysate lipid, conversion factors similar to those in Table 6 could be applied to results obtained by acid hydrolysis methods, especially when the lipids contain a lot of steryl esters, glycolipids or phospholipids. Phospholipids are often determined from their phosphorus contents using factors such as P x 24, but for many phospholipids this could lead to significant errors (Table 5). Average factors for total wheat phospholipids (Table 6 ) are : non-starch phospholipids = P x 23.90, starch phospholipids = P x 16.51, and total flour phospholipids = P x 17.91. It is likely that similar factors should be used with phospholipids from other cereal starches since they have similar compositions to wheat ~ t a r c h . ~ 4.2. Solvent extraction efficiency

There are conflicting reports in the literature on the relative efficiencies of solvents for extraction of wheat flour lipids. In some cases the extraction techniques were obviously suspect, but in other cases they were sound and adequate proportions of solvent :flour were used. Wootton2 dried flours to < I % moisture and then extracted 97 % of the flour lipids (quantified as hydrolysate lipid) with EEW, compared with only 64 % using WSB. On the basis of this result he claimed that EEW is the best solvent for extracting wheat lipids, although this was not apparent when he extracted other samples with these solvents. MacMurray and Morrison6found that WSB extracted more flour lipids than EEW, and the increase was mainly in LPC. The results in this paper explain MacMurray and Morrison’s result, but do not support Wootton’s conclusions. Graveland3 claimed that BEW extracted all flour lipids, since no lipids could be detected in the extracted flour residue after acid hydrolysis, but quantitative data were not given. It is possible that residual starch lipid could not be detected by this method in the small samples he used. The compositions of the lipids he extracted with EEW and BEW are very similar to the non-starch lipids shown in Tables 3 and 4, and since he found very little LPC it seems likely that he did not extract starch lipids with EEW or BEW. Comparison of Graveland’s data3 with Tables 3 and 4 indicate that non-starch lipids are almost completely extracted with ethanol-diethyl ether (2: 1, v/v) or chloroform-methanol (2 :1 , v/v).

520

W. R. Morrison ef al.

The Bligh and Dyer solvent system (chloroform-methanol-water, 10: 20: 8, by vol.) advocated by Tsen and H l ~ n k was a ~ not ~ studied, but there seems to be no reason why this solvent (or other binary solvent-water m i ~ t u r e s should ~ ~ ~ ~extract ~ ) more lipids than the solvents discussed above. The lipid phosphorus content of 0.68 :!reported by Tsen and H l ~ n k indicates a ~ ~ that very little of the starch lipid in flour was extracted with chloroform-methanol-water. There was nothing in the present study to indicate that non-starch lipids remained unextracted or unquantified (by t.1.c.) because of lipoprotein or phospholipid-metalprotein c ~ m p l e x e (only s ~ ~ ~minor ~ ~ acidic phospholipids would be involved in the latter case). All phosphorus-positive material in crude lipid extracts migrated as identifiable phospholipids on t.1.c. plates. There was also good agreement between the total lipids recovered in cold + hot solvent extracts of flour and the total amounts determined by acid hydrolysis. EEW, BEW and WSB are all convenient solvents for cold extraction of lipids, but WSB has advantages when wet dough is macerated in solvent on account of its lower volatility and higher flash point. So far starch lipids have only been quantitatively extracted with butanol-water (65 :35, v/v) or WSB at elevated temperatures. EEW and BEW would be less convenient for hot extraction, and it is unlikely that they would be as effective as WSB. The particular efficiency of WSB may be associated with the ability of butanol to enter the amylose helix, possibly replacing lipid with an amylosebutanol complex. Butanol has been criticised on account of its high boilingpoint and odour, but neither criticism is serious. WSB can be removed by rotary vacuum evaporation without damaging lipids, and the solvent can be readily trapped so that vacuum pump oil does not become contaminated. Odour is not a problem if work is carried out in a ventilated space, which should be standard practice with all lipid solvents in any case, since they must all be considered as proven or potential health hazards. 4.3. Comparison with previous quantitative analyses of wheat lipid classes In previous studies1,6~14~15 flours were extracted with cold WSB for considerable times, and the extracted lipids would therefore consist of non-starch lipids and unknown proportions of starch lipids. Fortunately reproducible extraction procedures leave constant amounts of unextracted starch lipids,15 so that comparisons within a single study are valid. Lin et aLZ9extracted flours for 30 min with cold WSB, and their data may be compared with the non-starch lipid analyses in Tables 3 and 4. Lin et al. found comparatively high levels of FFA, EMGMG, MGMG, and DGMG in Chris HRS flour which can be attributed to lipolysis during storing theflour for 2 years at ambient temperatures before analysis. Lipolysis in stored flours thus occurs in the non-starch lipid fraction which is involved in gluten and dough properties. We do not know whether changes occur in starch lipids during storage, but such changes would be unlikely to affect dough although they could affect starch gelatinisation during baking.

References 1. Mecham, D. K. Wheat, Chemistry and Technology 1971, 2nd ed., p. 393 (Pomeranz, Y. Ed.), St. Paul, Minn., Amer. Assocn. Cereal Chemists Inc. 2. Wootton, M. J. Sci. Fd Agvic. 1966,17,297.

Non-starch and starch lipids in wheat flour 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

521

Graveland, A. J. Am. Oil Chem. Soc. 1968,45,834. Mecham, D. K.; Mohammad, A. Cereal Chem. 1955,32,405. Bloksma, A. H. Cereal Chem. 1966,43,602. MacMurray, T. A.; Morrison, W. R.J. Sci. Fd Agric. 1970,21, 520. Ackcr, L.; Schmitz, H. J. Starke 1967,19,233. Ackcr, L.; Schmitz, H. J. Starke 1967,19,275. Acker, L.; Becker, G. Starke 1971,23,419. Morrison, W. R.;Smith, L. M. J. Lipid Res. 1964,5,600. Clayton, T.A.; MacMurray, T. A.; Morrison, W. R. J. Chromat. 1970,47,277. Morrison, W. R.Analyt. Biochem. 1964,7,218. AOAC Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D.C., 1970,llth ed., method 14.004. Clayton, T. A.; Morrison, W. R.J. Sci. Fd Agric. 1972,23,721. Mann, D. L.; Morrison, W. R.J. Sci. Fd Agric. 1975,26,493. Hirayama, 0.;Matsuda, H. Agric. biol. Chem. (Tokyo) 1972,36,2593. Arunga, R. 0.;Morrison, W. R.Lipids 1971,6, 768. Wren, J. J. ; Merryfield, D. S. J. Sci. Fd Agric. 1970, 21,254. Rogols, S.; Green, J. E.; Hilt, M. A. Cereal Chem. 1969,46, 181, Christie, W. W. ; Noble, R.C. ;Moore, J. H. Analyst 1970,95,940. AOAC Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D.C., 1970, 11th ed., method 13.019. Wren, J. J.; Wojtczak, P. P. Analyst 1964,89, 122. Morgan, R.H.; Rawlings, H. W. Analyst 1951,76, 161. Tsen, C. C.; Hlynka, I. Cereal Chem. 1962,39,195. Schmid, P. Physiol. Chem. Phys. 1973,5,141. Schmid, P. ; Calvert, J.; Steiner, R.Physiol. Chem. Phys. 1973, 5, 157. Fullington, J. G. J. Lbid Res. 1967, 8, 609. Fullington, J. G. Bakers’ Digest 1969,43, (6), 34. Lin, M. J. Y. L.; Youngs, V. L.; D’Appolonia, B. L. Cereal Chem. 1974,51, 17.