lipid-protein interactions during dough development

J. Sci. Food Agric. 1981, 32, 811-897

Lipid-Protein Interactions During Dough Development

Peter J. Frazier, Norman W. R. Daniels and Peter W. Russell Eggitt

Dalgety Spillers Limited, Research and Technology Centre, Station Road, Cambridge CBI 2JN

(Manuscript received 30 October 1980)

Radionuclide-labelled glycerol triolein has been used to follow the fate of triglyceride lipids during the mixing of wheat flour doughs. After removal of residual free lipid by petrol extraction, initial fractionation of freeze-dried dough identified the acetic acid-soluble protein as being the only component involved significantly in work- induced lipid binding during dough development under nitrogen, and also in lipid release on admission of air. Very little labelled lipid was found in either the water- soluble proteins or the starch residue. Sub-fractionation of the acetic acid-soluble protein by ammonium sulphate precipitation from acetic acid-urea-cetyltrimethylammonium bromide (AUC) solvent showed bound lipid to be distributed almost entirely between high-molecular-weight glutenin and protein which remained soluble in the AUC supernatant even in the presence of 20% ammonium sulphate. Precipitated gliadins contained very little labelled lipid. However, significant levels were found in classical ethanol-extracted gliadin, and were traced predominantly to the same supernatant-protein owing to its solubility in aqueous ethanol. Examination of this AUC-supernatant fraction showed it to contain a hitherto unreported protein which had a molecular weight of about 9000, was strongly complexed with triglyceride lipid on a 1 : 1 molar basis and showed a tendency to aggregate in solution. Its amino acid frequency was found to differ significantly from both glutenin and purothionin, the latter containing in particular very much more cysteine than the supernatant-protein. Representing 10% or more of the total gluten, this small, highly interactive protein is responsible for a significant, if not the major, part of lipid binding activity in dough and may well have a fundamental role in the formation of an insoluble glutenin structure through both -SH and hydrophobic interaction. Accordingly, the name ‘Ligolin’ is proposed, from the Latin ligare: to bind, to tie.

1. Introduction

Wheat lipids are a minor (1.5-2.0% of the flour, depending on extraction rate) but complex192 fraction of all wheat flours. Controversial early literature reported variously3 on the consequences for bread quality of removal and recombination of flour lipids and the addition of shortening fats,4,5 on the part played by phosphatides and other polar lipid fractions6 and on possible oxidative pathways involving polyunsaturated fatty acids in the dough.’ However, it was generally agreed that the proportion of lipid easily extracted by non-polar solvents (‘free’ lipid) decreased substantially as flour was hydrated and mixed into a dough.8 The consequential increase in ‘bound’ lipid was attributed to the formation of lipoprotein complexes in the dough,g,lO but direct evidence regarding the identity and structure of these lipoproteins remained scanty.

An early suggestion that gluten may resemble an immature myelin, with protein layers held together by lipids11 was supported by independent work based on X-ray scattering at small angles from stretched freeze-dried gluten and led to the now well known Grosskreutz model.12 Later models involved interactions between glycolipids, proteins, starch and also a specific complex between gliadin, glycolipid and g1~tenin.l~

0022-5142/81/090&0877 $02.00 0 1981 Society of Chemical Industry 877

878 P. J. Frazier el al.

In spite of these models, the only flour lipoprotein that has been isolated and studied is purothionin, present in light petroleum extracts of wheat flour and yielding a crystalline globulin-like protein after disruption of the liproprotein complex.14~ 15 Phosphatidyl choline, glycolipids and steroI ester have been identified as associated lipids but mono-, di- and triglycerides appeared to be absent.16 An analogue of purothionin, hordothionin, has been isolated from barley flour,'? leading to the conclusion that these lipoproteins are unlikely to be significantly involved in the unique properties of wheat dough structures. Being preformed in flour, purothionin has not been implicated in lipid binding during dough mixing.

The replacement of traditional long fermentation breadmaking methods by new systems relying on high speed mechanical dough development heightened interest in lipid interactions. Shortening fats previously regarded as optional additives were found to be essential for bread quality in systems such as the Chorleywood Bread Process.l* Studies which followed showed that increasing the rate of work input to the dough also increased the level of lipid binding, mainly involving the added shortening triglyceride.19 Lipid binding was affected by the atmosphere in the mixer chamber, reaching a maximum in anaerobic doughs mixed rapidly to high work levels.20 Mixing in air, on the other hand, released bound lipid through a coupled linoleate oxidation mechanism, particularly in the presence of active soya lipoxygenase.21922 It was suggested that the coupled oxidation acted on the binding site protein, an action later shown to strengthen the dough rheologically, improve loaf volume and retard staling.23-27

It was also observed that the presence of high-melting-point fat appeared to raise free lipid levels in mechanically developed doughs,23 retarding the onset of gas release during the early stages of baking28 and aiding oven spring through a physical, rather than oxidative, mechanism.29 Possibly also involving gas retention, the improving effects of emulsifiers in bread doughs have been related to an increase in free lipid resulting from a modification of binding sites by these surfactants.30, 31 Deteriorative effects of flour storage on bread quality, which are dependent on the type of breadmaking procedure, have also implicated lipid^.^^-^^

Notwithstanding the now generally accepted importance of lipid-protein interaction during dough development, little or no evidence exists regarding the nature and identity of the lipid binding sites in mechanically developed doughs. Objectives in the present study have been, therefore, to examine critically the components of bread doughs for their involvement in lipid binding during high energy mixing, to identify protein fractions involved specifically in lipoprotein formation and to search for a minimum molecular weight unit existing in dough that retains characteristic binding site properties, associated at the molecular level with lipid function in the breadmaking process.

2. Experimental 2.1. Materials 2.1.1. Flour An untreated, unbleached strong bakers' flour, commercially milled from 100 % US Northern Springs grist, was used for this investigation, having a moisture content of 13.7% and containing (on a 14% moisture basis):13.3% protein (Nx 5.7), 1.1 % oil (by Soxhlet) and 0.59% ash. Water absorption was 63.9 % (500 Brabender units, 14 % moisture basis).

Soya flour was a commercial full-fat, enzyme-active material (Diasoy). Added to doughs at a level of 0.72 % (flour weight basis) this provided approximately 5-10 units of lipoxygenase activity (as defined by Surrey35) 8-1 of wheat flour.

2.1.2. Genera[ reagents All usual reagents were AR grade except for general purpose light petroleum (b.p. 4&60°C) which was fractionally distilled before use. Sodium dodecyl sulphate (SDS) was specially purified for biological work (BDH Chemicals). Olive oil was BP grade. Acetic acid-urea-cetyltrimethylammonium bromide (AUC) solvent36 was prepared from acetic acid ( O . ~ M ) , urea (3111) and hexa- decyltrimethylammonium bromide (Cetrimide, Sigma Chemical Co.) (0.01~).

Lipid-protein interactions during dough development 879

2.1.3. Radiotracer reagents Radionuclide-labelled compounds were obtained from the Radiochemical Centre, Amersham as follows: glycerol tri(1-W)oleate, (14C-GTO), specific activity (depending on batch) between 55 and 60 mCi mmol-1, radiochemical purity > 97 % by thin layer chromatography, supplied in vials containing 50 pCi dissolved in dry, deaerated benzene and sealed under nitrogen; standardised n-(l-14C)hexadecane, specific activity 0.503 pCi g-l, radiochemical purity > 98 %, for internal standardisation. Two scintillation cocktails were used : phase combining solution (PCS) supplied by the Radiochemical Centre, Amersham, for aqueous systems and a solution of 4 g PPO (2,5-di- phenyloxazole) and 0.05 g POPOP [1,4-di-(2-(5-phenyloxazolyl)) benzene] litre-1 toluene, for non- aqueous systems.

2.1.4. Gels Precast polyacrylamide gels for electrophoresis were obtained from Pharmacia with a concentration gradient from 4 to 30%. Single strength gels were made up as required from mixtures of 10% acrylamide (BDH specially purified for electrophoresis) and 0.5 % N-N’methylenebisacrylamide (Sigma) using ammonium persulphate and N,N,N’,N’-tetramethylethylenediamine (TEMED, Sigma) as catalyst. Column chromatography was carried out using Sephadex G50 (superfine) supplied by Pharmacia.

2.1.5. Molecular weight markers The following protein molecular weight standards were employed for both gel electrophoresis and column chromatography: (a) insulin, mol. wt 5700 (Sigma); (b) cytochrome C, mol. wt 12 500 (Sigma); (c) chymotrypsinogen, mol. wt 25 000 (Koch-Light); (d) chicken egg albumin, mol. wt 45 000 (Koch-Light); (e) bovine serum albumin, mol. wt 67000 (Sigma); and (f) bovine thyroglobulin, mol. wt 680 000 (Sigma).

2.2, Methods 2.2.1. Preparation of 14C-GTO-labelled olive oil

The majority of this investigation was carried out using a level of 14C-GTO-activity of 5 pCi per dough mix added in olive oil. Sufficient 14C-GTO-labelled olive oil was prepared in one batch for at least ten doughs, employing the entire contents of one or more vials of 14C-GTO. Typically, 3.6 g olive oil were weighed into a 150 ml round bottom flask and the contents of one vial of 14C- GTO in benzene were washed into the flask using light petroleum. The solvents were then removed on a Buchi rotary evaporator at 60°C with a nitrogen bleed, and the remaining 14C-GTO-labelled olive oil was stored at 5°C.

When lipid recoveries were required from dough fractions ultimately separated by column chromatography, up to ten times greater levels of 14C-GT0 were employed in preparing the labelled olive oil before dough mixing.

2.2.2. Dough mixing Doughs of constant weight (80 g) containing 2 % salt (flour weight basis) and distilled water equal to Farinograph absorption at 500 BU less 9% were mixed in a stainless steel-clad junibr Farino- graph bowl attached to the Spillers Compudomixer.37 The reduced amount of water was employed to facilitate handling of doughs after mixing to high work level~.~4 Soya flour and 14C-GTO- labelled olive oil were both included at a level of 0.72% (flour weight basis), the latter being care- fully weighed on to the wheat flour so as not to adhere to the container. The atmosphere above the doughs was controlled by continuously metering humidified air or oxygen-free nitrogen into the mixing bowl at a rate of 4 litre min-1. The dry ingredients were first blended at 20 rev min-l with the gas stream bubbling through a flask containing the required amount of water before entering the bowl. After 1 min the flask was inverted, allowing gas pressure to drive the water into the bowl.

880 P. J. Frazier et al.

(2 X 75 rnl, Silverson for 2 min)

I

When mixing under nitrogen, this method ensured that air was largely eliminated from the system and its re-entry prevented. The wetted ingredients were premixed for a further 1 min at 20 rev min-l. Mechanical development was then carried out at a constant rate of work input of 20 kJ kg-1 min-l for times from 0.5 to 15.0 min, i.e. a range of work levels from 10 to 300 kJ kg-1. Developed doughs were immediately cut into small pieces, frozen by immersion in liquid nitrogen and freeze-dried. On completion, the freeze-drier vacuum was broken with dry, oxygen-free nitrogen.

-X 3 - Water solubles Count (3 X 1 ml in PCS) ( to 500 ml)

2.2.3. Fractionation of whole dough

The fractionation scheme is shown in Figure 1. All procedures were carried out to provide quantitative recovery of lipid and protein. Freeze-dried dough (20 g) was ground to a fine powder using a pestle and mortar, transferred to a Soxhlet thimble and extracted for 7 h with light petroleum.

(2 X 7 5 ml, Silverson for 2 min)

I

Flour Salt Water Soya flour 1 4 c - ~ ~ 0 ( 5 pci) in olive oil

-X 3 - Acetic acid solubles Count (3 X 1 ml in PCS) ( to 500 ml)

Figure 1. Preparation and fractionation of whole dough.


The extract was concentrated by rotary evaporation at 60°C and transferred to a 100 in1 graduated flask with light petroleum. Three 1 ml aliquots were taken into 20-ml scintillation vials and the solvent evaporated off in an air oven at 50°C. After cooling, 15 ml PPO-POPOP-toluene scintillant were added and the samples were counted to 105 counts in an ICN Corumatic 200. Counting efficiencies, by channels ratio, averaged 78 %.

The contents of the Soxhlet thimble were desolventised in an air oven not exceeding 50°C and transferred into two 250 ml centrifuge bottles. Distilled water (75 ml) was added to each bottle and mixed using a Silverson homogeniser for 2 min at maximum speed. After centrifuging the bottles at 1200g for 20 min, the supernatants were decanted into a 500 ml graduated flask. This extraction procedure was repeated twice and the bulked extracts were made up to 500 ml with water. Three 1 ml aliquots were counted to lo4 counts in 15 ml PCS both before and after spiking with 10 pl n-(l-I4C) hexadecane as internal standard. Counting efficiencies averaged 69 %. The remaining extract was freeze-dried and the recovered mass and Kjeldahl nitrogen content were determined.

The centrifuge bottle residues were extracted again by the same procedure using 0 . 0 5 ~ acetic acid instead of water. Counting efficiencies averaged 71 %. Recovered mass and Kjeldahl nitrogen were determined as before. The residue after extraction with acetic acid was examined for bound lipid using a modified mixed solvent procedure.lg Methanol (50 ml), water (20 ml) and chloroform (25 ml) were added to each centrifuge bottle and the residue was Silverson homogenised for 2 min at maximum speed. A further 25 ml of chloroform was added and the homogenisation procedure repeated for a further 30 s. The centrifuge bottles were then cooled for 15 min in a cabinet at - 18°C and centrifuged at 12OOg for 20 min. The lower layer was drawn-off by pipette into a 50 ml graduated cylinder and the aliquot volume immediately noted. This extract was concentrated by rotary evaporation at 40"C, transferred to a scintillation vial with chloroform and the solvent removed in an air oven at 50°C. After cooling, 15 ml PPO-POPOP-toluene scintillant were added and the samples were counted to lo4 counts. Efficiencies, by channels ratio, averaged 77 %. Finally, after decanting the methanol-water layer, the residue was freeze-dried and the mass and Kjeldahl nitrogen were determined as before.

2.2.4. Aqueous ethanol extraction (see Figure 2) Freeze-dried, acetic acid-soluble protein, prepared as described in section 2.2.3, was extracted in 70% ethanol (1 g protein 100 ml-l ethanol) for 12 h with gentle magnetic stirring. The supernatant was separated by centrifugation at 1200g for 20 min and the residue re-extracted with aqueous ethanol for a further 2 h. Centrifugation and re-extraction were continued until the supernatant showed negligible absorbance at 280 nm. Ethanol was removed from the pooled supernatants in a rotary evaporator under vacuuni below 40°C. The ethanol-soluble protein, Fz (gliadin) and the ethanol-insoluble residue, F1 (glutenin) were freeze-dried. Samples were redissolved in acetic acid ( 0 . 0 5 ~ ) and counted in PCS to lo4 counts with an average efficiency of 60%.

2.2.5. Ammonium sulphate precipitation The method of Wasik and Bushuk3g was applied to acetic acid-soluble protein as shown in Figure 2. The freeze-dried protein (prepared as described in section 2.2.3) was redissolved in AUC solvent ( 1 g protein 170 m1V1 AUC), cooled to 4°C and then ammonium sulphate (15 g) added slowly with stirring. After standing overnight, the voluminous precipitate that formed (Pi) was separated by centrifugation at 20 OOOg for 15 min. A second portion of ammonium sulphate ( 1 5 g) was added to the supernatant and precipitate Pz was allowed to settle. Following centrifugation as before, a further 15 g of ammonium sulphate was added to the supernatant and precipitate P3 was obtained. The final supernatant, after centrifugation, was retained for counting together with precipitates PI, PZ and P3, which were redissolved in AUC before counting in PCS to lo4 counts with an average efficiency of 60 %.

Samples of PI, PZ and supernatant quantitatively prepared on a larger scale, starting with about 9 g acetic acid-soluble protein, were exhaustively dialysed in Visking tubing against running tap water, to remove urea and ammonium sulphate, freeze-dried and analysed for protein by Kjeldahl.

59

882

Extract in ethanol-water (for 2 h with gentle stirring)

P. J. Frazier ef ai.

Remove kthanol (rotary evaporator)

I Freeze dry

I Freeze dry

Ethanol-insoluble Ethanol-soluble protein, F , protein, F, (glutenin) (gliadin)

I a Dissolve in AUC

(1 g 170ml-l)

I

I I

Add ammonium sulphate (1 5 g, allow to stand at

4°C for 12 h)

Centrifuge ____t Precipitate P, (20 O O O g for 15 min)

Add ammonium sulphate

(glutenin)

(gliadin)

IISg)

r5 g,

Centrifuge - Precipitate P, ( zoooog for 15 min)

Add ammonium sulphate I

t Precipitate P, (gliadin)

Centrifuge ( 2 O O O O g for 15 min)

Supernatant S

'0.1 \ I Acetic acid f 3 . 0 ~ urea + 0.01\1 cetyltrimethylammonium bromide.

Figure 2. Fractionation of acetic acid-soluble protein.

2.2.6. Gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was carried out on vertical slab gels (82 x 82 x 3 mm) using a Pharmacia GE-4 apparatus and either gradient (430%) or single strength (10%) gels (see section 2.1.4).

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at pH 8.9 using a tris-borate-SDS buffer (0.125~, 0.1 % SDS) as described by Bietz and Wall.39 Protein samples were dissolved in running buffer, with the addition of extra SDS to two to four times the concentration of protein, and heated for 5 min at 95°C. ,B-Mercapto-ethanol was added to the solvent (10 ml litre-1) when reduction of the protein was required. After electrophoresis at 20-30 V cm-l for 1.5-2.5 h the gels were fixed with aqueous trichloroacetic acid (TCA) (120 g litre-l) and stained overnight in a freshly prepared mixture of Kenacid Blue R (BDH Chemicals) (0.5 g litre-1) in acetic acid-methanol-water [l : 5: 5 (viv)]. The gels were destained by immersion in methanol-acetic acid-water [lo: 15: 175 (v/v)] as described by Weber and Osborn40 and stored in aqueous acetic acid (70 ml litre-1).

Gradient-PAGE was carried out in sodium lactate-lactic acid buffer (0.0042~, pH 3.1) following the method of Wrigley and M ~ C a u s l a n d . ~ ~ Protein samples were dissolved in running buffer and the electrophoresis was performed at 25 V cm-1 for up to 3.5 h. The gels were then fixed in TCA as described and stained overnight in a freshly prepared mixture of Kenacid Blue R (0.125 g litre-l)


in the TCA fixative. After destaining in TCA (120 g litre-l) the gels were stored in aqueous acetic acid (70 ml litre-l).

2.2.7. Gel chromatography Gel filtration chromatography was carried out on a 1 m x 25 mm column of Sephadex G-50 (Super- fine) packed in ammonium hydrogen carbonate-ammonium hydroxide buffer (0.01 M, pH 8.1). Protein and peptide solutions were applied to the column via a three-way tap and eluted in the buffer at a flow rate of approximately 12 ml h-1 controlled by an LKB Vaiioperpex peristaltic pump. Detection was by ultraviolet absorbance at 280 nm using an Isco Type 6 Detector and UA5 Monitor. Fractions (10-12 ml) were collected for 14C-GT0 detection, 3-ml aliquots being counted in 15 ml PCS with an efficiency of 55-60%. Appropriate fractions were then pooled and freeze- dried for subsequent amino acid analysis.

2.2.8. Amino acid analysis Amino acid analysis was carried out on a JEOL JLC-6AH automatic amino acid analyser. Protein fractions were hydrolysed in 6~ hydrochloric acid by autoclaving at 108.5 kPa for 7.5 h. Serine and threonine recoveries were corrected for approximately 10 % loss on hydrolysis. Cystine and cysteine were determined together as cysteic acid in a separate run following oxidised hydrolysis using 0 . 2 5 ~ dimethylsulphoxide in 6~ HCI. Amino acid standards were chromatographed before each hydrolysate sample analysis to ensure accurate calibration.

3. Results and discussion

3.1. Fractionation of whole dough 3.1.1. Doughs mixed in nitrogen Dry solids, protein and lipid distributions in fractions obtained by successive extraction of freeze- dried dough with light petroleum, water and 0.05~ acetic acid are shown in Table 1. Overall protein recoveries averaged just over 90%. Little change occurred across work levels, with extracts per gram of dough averaging 15 mg for water-soluble protein (20% of recovered protein), 57 mg for acetic acid-soluble protein (75 %) and 3.4 mg for residue protein (4 %). Thus, the efficient system of protein dispersion used at each stage avoided proportionality problems resulting from any increase or decrease in protein solubility with dough work input.42, 43

Overall labelled lipid recoveries averaged 97 %. The majority of the labelled lipid appeared as ‘free’ lipid in the petrol extract, decreasing from 55 to 40 nCi 8-l dough as work input increased from 10 to 300 kJ kg-l. Of the ‘bound’ lipid, most occurred in the acetic acid-soluble fraction, increasing three-fold from 6 nCi g-l dough at 10 kJ kg-1 to 18 nCi g-1 dough at 300 kJ kg-1. Lipid recovered from the water-soluble protein was an order of magnitude lower, averaging 0.4 nCi g-l dough and not changing with dough work input. Residual solids, mainly starch and accounting for almost 70% of the dough dry mass, attracted the lowest proportion of lipid (under 0.4 nCi g-l dough at 10 kJ kg-1) although this showed some increase at high work levels.

Plotted as percentage total 14C-GT0 recovered, Figure 3 illustrates the change in lipid composition of dough fractions with work input, and clearly shows the acetic acid-soluble material to be almost totally responsible for the binding of lipid during dough development under nitrogen.

3.1.2. Doughs mixed in air

Dry solids, protein and lipid distributions in fractions from air-mixed doughs obtained by successive extraction with light petroleum, water and 0.05~ acetic acid are shown in Table 2. Overall protein recoveries averaged 94 %. Again, little change occurred across work levels, extracts per gram of dough averaging 15 mg for water-soluble protein (19% of recovered protein), 60 mg for acetic acid-soluble protein (77%) and 3.3 mg for residue protein (4%).


Table 1. Dry solids, protein and lipid distribution in fractions of whole dough mixed under nitrogen

Work level (kJ kg-l)

Determination 10 40 100 150 200 300

Dry solids (mg g-1 dough) Fraction

Light petroleumQ Water Acetic acid Residue

Total Recovery (:<)

Protein (mg g-1 dough) Fraction

Light petroleum Water Acetic acid Residue Total Recovery (;<)

Lipid ('4C-GTO) (nCi g-ldough) Fraction

Light petroleum Water Acetic acid Residue Total Recovery ( %)

(13.2) (12.2) (11.2) 63.5 63.9 74.2 75.2 70.7 75.9

352.5 313.5 350.3

504.4 460.3 513.4 89.5 81.7 91.1

ND N D ND 13.9 14.2 15.0 58.8 55.8 60.0 3.8 3.1 3.0

76.5 73.1 78.0 91.5 87.4 93.3

(10.9) 72.1 75.5

360.9

519.4 92.2

ND 15.0 59.5 3.6

78.1 93.4

(10.0) 12.2 75.6

335.5

493.3 87.5

ND 15.0 54.8 3.2

73.0 87.3

(9.8) 87.6 70.8

358.9 527. I

93.5

ND 18.9 52.7

3.4 75.0 89.7

55.2 52.9 44.3 47.0 36.1 39.9 0.48 0.34 0.30 0.36 0.30 0.55 5.90 11.0 13.7 15.5 15.6 17.8 0.39 0.35 0.54 0.87 1.33 1.93

62.0 64.6 58.8 63.7 53.3 60.2 99.1 103.4 94.1 101.9 85.3 96.3

a Calculated assuming total lipid distributed as labelled lipid. N D = Not determined. Total solids content of dough = 563.6 mg g-I dough; total protein content of dough =83 .6 mg g-l dough; total

lipid content of dough = 14.8 mg g-1 dough; and lipid (14C-GTO) added to dough =62.5 nCi g-1 dough.

Overall labelled lipid recoveries averaged 99%. Most of the lipid was extracted in the light petroleum 'free' fraction and, unlike the nitrogen-mixed doughs, remained almost constant with work level (average 54 nCi g-' dough). Most of the 'bound' lipid occurred in the acetic acid-soluble fraction and again unlike the nitrogen-mixed doughs, remained almost constant with work input (average 6.5 nCi 8-1 dough). Lipid recoveries from the water-soluble fraction and the residue fraction were both very low (average 0.7 and 0.3 nCi g-l dough, respectively) and were unchanged by work input.

Figure 3 compares the lipid distribution in fractions from air- and nitrogen-mixed doughs, as a percentage of the total labelled lipid recovered, and confirms earlier gravimetric findings20 that dough development in unrestricted air in the presence of active lipoxygenase results in little or no lipid binding.

3.1.3. Doughs mixed in nitrogen followed by air

Table 3 shows the dry solids, protein and lipid distributions in fractions from doughs mixed first to 150 kJ kg-1 under nitrogen and then developed further in air. Overall protein recoveries were poorer than before (average 86%) but again showed little change across work levels. Water- soluble protein averaged 15 mg g-l dough (21 % of recovered protein), acetic acid-soluble protein averaged 54 mg g-l dough (75 %) and the residue contained an average of 3.2 mg 8-1 dough (4.5 %).


W o r k input ( k J k g - l ) Work input (kJ kg-l)

Figure 3. Distribution of triglyceride lipid (14C-GTO) in fractions of doughs mixed in nitrogen and in air. LP, light petroleum-soluble; W, water-soluble; A, acetic acid-soluble; R, insoluble residue; x , acetic acid-soluble after admission of air to a nitrogen mix at position shown by arrow.

Figure 4. Distribution of triglyceride lipid (f4C-GTO) in fractions of doughs mixed under nitrogen, showing sub-fractionation of acetic acid-soluble protein by aqueous ethanol. FI, ethanol-insoluble (classical glutenin); Fz, ethanol-soluble (classical gliadin); other abbreviations as described in legend to Figure 3.

Labelled lipid recoveries averaged 92%. After mixing to 150 kJ kg-1 under nitrogen and a further 10 kJ kg-1 in air the 'free' lipid fraction contained 39 nCi g-' dough, falling between the values at 150 and 200 kJ kg-1 in Table 1 . Further mixing in air resulted in an increase in free lipid to almost 50 nCi 8-1 dough after a total work input of 350 k J kg-1. The acetic acid solubles, relatively high in 'bound' lipid after 150 kJ kg-1 under nitrogen, rapidly released lipid during the further mixing in air, 14C-GT0 falling from 15.7 to 6.3 nCi g-l dough. Lipid levels in the water-soluble and residue fractions were low and relatively constant (averaging 0.5 and 0.6 nCi g-' dough, respectively).

The effect of air admission on the 14C-GT0 content of the acetic acid-soluble protein, expressed as a percentage of the total lipid recovered, is shown in Figure 3. Rapid release of bound lipid can be clearly seen.

These results, using more precise radio-tracer techniques, provide an elegant confirmation of earlier gravimetric observations20J2p 24 and, in addition, clearly identify the acetic acid-soluble protein as that fraction of dough intimately involved in oxidative interactions with the dough lipids.

3.2. Fractionation of acetic acid-soluble protein 3.2.1. Aqueous ethanol extraction The lipid distribution in gliadin and glutenin fractions obtained by classical aqueous ethanolic extraction of freeze-dried, acetic acid-soluble gluten is shown in Table 4. Recoveries of 14C-GT0 averaged 96%. At low work levels under nitrogen about three-quarters of the bound lipid was recovered in the glutenin residue (F1). As dough work level increased, accompanied by greater lipid binding in the acetic acid-soluble protein, labelled lipid increased in both gliadin and glutenin

886 P. J. Frazier ef a/.

Table 2. Dry solids, protein and lipid distribution in fractions of whole dough mixed in air

Determination

Dry solids (mg 8 - 1 dough) Fraction

Light petroleurna Water Acetic acid Residue Total Recovery ( 70)

Protein (mg g-l dough) Fraction

Light petroleum Water Acetic acid Residue Total Recovery (%)

Lipid (I4C-CTO) (nCi g-1 dough) Fraction


Work level (kJ kg-1)

10 I00 200 3 00

(13.5) 65.7 75.8

384.7 539.7

95.8

N D 14.3 60 .6

5 . 4 80.3 96.1

56.1 0 .73 4 .73 0.33

61.9 99.0

(12.7) 69 .0 15.7

376.9 534.3

94.8

N D 15.5 60.1 2 . 6

78.2 9 3 . 5

52 .6 0 .65 7 .70 0 . 2 9

61.3 98.1

(12.8) 70.9 71.7

371 .O

526.4 93.4

N D 15.2 5 8 . 3 2 . 5

76.0 90.9

53 .3 0.71 7.29 0 .30

61.6 98.5

(13.0) 12.9 71.1

376.5 539.5

95.1

N D 1 5 . 1 61.1 2 . 5

78.7 94.1

55.2 0.88 6 .40 0.32

62.8 100.5

For details see footnotes to Table 1 .

fractions. However, the overall increase with work input was greatest in the ethanol-soluble gliadin fraction (F2), with the result that, at high work levels, bound lipid was distributed almost equally between the two fractions. Expressing lipid distribution as a percentage of the total dough lipid recovered, Figure 4 shows clearly the contribution of the gliadin and glutenin fractions. From this, F1 glutenin appeared to be the fraction of primary importance in determining the pattern of lipid binding during dough development under nitrogen, but with a significant contribution from the ethanol-soluble material, particularly at high work levels.

3.2.2. Ammonium sulphate precipitation Using a more discriminating fractionation method, the lipid distribution was determined in protein fractions produced by ammonium sulphate precipitation from AUC solutions of acetic acid- soluble gluten. The results are shown in Table 5. Recoveries of *4C-GT0 averaged 99%. It can be seen that the highest proportion of labelled lipid (between 55 and 72% of the total recovered from solution) was found in the P1 precipitate. This fraction, when prepared directly from Durum flour by Wasik and Bushuk38 was characterised by PAGE as mainly glutenin.

The P1 fraction prepared from acetic acid-soluble protein isolated from a dough mixed to 150 kJ kg-1 under nitrogen was therefore examined by PAGE. Unreduced Pi (not illustrated) remained mostly at the origin except for a small amount of material that entered the gel but was not present in the region of the main gliadin bands. These lower molecular weight components of glutenin have been observed even after extensive purification steps to remove g l i a d i ~ ~ . ~ ~ The reduced PI


Table 3. Dry solids, protein and lipid distribution in fractions of whole dough mixed in nitrogen followed by air

Work level (kJ kg-l)

Nitrogen

150 150 150 150 Air

Determination 10 50 100 200

Dry solids (mg g-1 dough) Fraction


Protein (mg g-1 dough) Fraction


Lipid (14C-GTO) (nCi g-l dough) Fraction


(10.3) 68.6 68.6

355.5

503.0 89.3

ND 14.4 53.1 3 .0

70.5 84.3

39.3

15.7 0.60

1 .oo 56.5 90.5

(12.5) 70.2 71.9

369.3

523.9 93.0

ND 16.2 57.3 3.2

76.7 91.8

47.7 0.37 7.92 0.39

56.4 90.2

(12.5) 69.3 71 . O

348.3 501. I

88.9

N D 13.8 52.6 3.0

69.4 83 .O

51.4 0.34 8.70 0.53

61 .O 97.6

(12.9) 72.2 68.8

351.6

505.5 89.7

ND 14.9 51.7 3.5

70.1 83.9

49.5 0.82 6.34 0.38

57.1 91.3

For details see footnotes to Table 1

Table 4. Lipid distribution in aqueous ethanol fractionated acetic acid-soluble protein from doughs mixed under nitrogen

Work level (kJ kg-1)

Determination 10 40 100 200 300

Lipid (14C-GTO) (nCi g-1 dry acetic acid-soluble protein) Stock acetic acid-soluble protein 52.3 176.6 189.2 225.2 257.2

Fraction Aqueous ethanol Fz (gliadin) 11.2 74.8 59.0 91.4 127.0 Residue F1 (glutenin) 34.9 103.6 122.1 135.1 119.8

Total Recovery

46.1 178.4 181. I 226.5 246.8 88.1 101.1 95.7 100.6 96.0

P. J. Frazier ef al. 888

Table 5. Lipid distribution in ammonium sulphate fractionated acetic acid-soluble protein from doughs mixed under nitrogen

Work level (kJ kg

Determination 10 40 100 150 200 300

Lipid ( 'T-GTO) (nCi g- dry acetic acid-soluhle proteirrf Stock AUC solution 9 5 . 5 186.7 192.1 222.7 197.6 251.3 Fraction

Precipitate PI 5 6 . 4 99 .3 126.8 156.1 127.4 164.5 Precipitate Pz 3 .07 6 . 2 2 6 . 2 4 7 . 7 2 7.38 6 . 3 2 Precipitate P3 0 .75 1.24 I .44 0 . 9 5 1.05 1.29

Total 103.6 178.8 196.3 215.6 186.7 247.9 Recovery (%) 108.5 95 .8 102.2 9 6 . 8 9 4 . 5 9 8 . 6

Supernatant S 43.4 72 .0 61 .8 50 .8 50 .8 75.7

(Figure 5 ) showed the characteristic sub-unit banding of glutenin. Thus, as found for Durum flour extracts,38 the PI fraction prepared from a developed bread flour dough also appears to be reason- ably pure glutenin. Figure 6 shows that the 14C-GT0 content of the PI precipitate, expressed as a percentage of the total labelled lipid recovered from dough, increased markedly with work input.

The gliadin fraction by ammonium sulphate precipitation from AUC solutions is reported to occur mainly in the PZ precipitate with some in the P3 precipitate.38 A similar distribution was

Figure 5. Tris-borate-SDS-PAGE (PH 8.9) of /3-mercaptoethanol-reduced, ammonium sulphate-precipitated fractions of acetic acid-soluble gluten. 1 , Standards (molecular weight key see section 2.1 3 ) ; 2, acetic acid-soluble protein stock; 3, PI-precipitated protein; 4, Pz-precipitated protein; 5, Ps-precipitated protein; and 6, supernatant protein.


.LP - U E >

Figure 6. Distribution of triglyceride lipid (14C-GTO) in fractions of doughs mixed under nitrogen, showing sub-fractionation of acetic acid-soluble protein by ammonium sulphate-precipitation from AUC. PI, glutenin precipitate; PZ and P3, gliadin precipitates; S, supernatant; other abbreviations as described in legend to Figure 3. I \ I I\ 4 p3 , A

0 100 200 300 400

Work input I k J kg-')

obtained in the present work (Figure 5) and confirmation was provided by aluminium lactate gradient PAGE (not illustrated) which showed a typical gliadin pattern for PZ and some gliadin bands in P3. In marked contrast to the ethanol-soluble gliadin described in section 3.2.1, Table 5 shows that very little labelled lipid was associated with the PZ and P3 gliadin fractions. Under 4P: of the total I4CC-GTO in solution precipitated in PZ and less than 1 % precipitated in P3. This appar- ently insignificant contribution of PZ and P3 gliadin fractions to the pattern of lipid binding during dough development is depicted clearly in Figure 6. The low level of label was particularly sur- prising in view of the presence of some high molecular weight gliadin (or low molecular weight glutenin) seen at the origin of the unreduced PZ and P3 electrophoretograms. However, it must be concluded on this evidence that glutenin is the major high molecular weight protein responsible for lipid binding during dough development and that the apparent contribution from the gliadin fraction (see section 3.2.1) resulted probably from the ethanol solubility of a gluten component other than gliadin.

In addition to these observations, it was noted that a significant proportion of the labelled lipid was not accounted for by either the glutenin or gliadin fractions but remained in the supernatant even after the ammonium sulphate precipitation procedure was complete. Table 5 shows that between 24 and 42% of the total 14C-GTO remained in solution. The composition of the supernatant is not described by Wasik and B ~ s h u k , ~ ~ but since gliadin was completely precipitated in Pz and Pa (cf. Figure 5 ) and albumins are normally present in P3,38 it seems likely that only low molecular water-soluble proteins could normally persist in the supernatant. However, in the present work. triple water extraction of the dough had removed the majority of the water- and salt-soluble material before the acetic acid-soluble protein was prepared. Furthermore, the water solubles were found to contain very little labelled lipid (see section 3.1.1). The high proportion of label in the supernatant fraction (second only to P1 glutenin in its contribution to the lipid binding pattern, see Figure 6) was therefore very unexpected. Free lipid that may have been detached from the protein during the fractionation procedure was one possible explanation. However, this was discounted after addition of 10% trichloroacetic acid established that most of the label was associated with

890 P. J. Frazier et a/.

precipitable protein. Both SDS (Figure 5) and sodium lactate gradient gel electrophoresis of the supernatant fraction indicated the presence of a protein band corresponding to a low molecular weight (10 000 or less) which was absent in the Pi and P2 fractions. Examination of a more concentrated supernatant sample by SDS-PAGE (Figure 7) confirmed this result, which suggested that the AUC-ammonium sulphate fractionation procedure was splitting off a fragment from glutenin, of very low molecular weight compared with the glutenin molecule, but which appeared particularly important for lipid binding.

Figure 7. As Figure 5-1, standards (molecular weight key see section 2.1.5); 2, PI-precipitated protein; 3, Pz-precipitated protein; and 4, supernatant protein (concentrated).

To understand the full significance of this supernatant protein compared with other fractions, a quantitative determination of protein in each fraction was required. The preparation procedure, involving both urea and ammonium sulphate, makes this determination particularly difficult. However, after establishing suitable quantitative dialysis procedures, results for a single work input (150 kJ kg-1 under nitrogen) were obtained as shown in Table 6. Over 92 % of the wet material was recovered from the four fractions and, after exhaustive dialysis and freeze-drying, Kjeldahl nitrogen determinations indicated an overall protein recovery of 97 %.

It is interesting to note the high mass of the Pi precipitate compared with the Pa precipitate. Most of the difference was due to water-the Pi fraction being very swollen and voluminous but drying down to a weight not much greater than Pz. Thus the PI glutenin fraction accounted for just over 50 % of the total protein while the P2 gliadin represented around 37 % of the total protein. Emphasis- ing the absence of water-soluble protein from the system, the P3-precipitate contained too little material for dialysis and nitrogen determination. On the other hand, the supernatant solution after dialysis still contained an appreciable amount of material, accounting for almost 13 "/o of the total recovered protein.

When the lipid distribution in nanocuries is expressed in terms of actual mass of triglyceride, either added (as olive oil) or total (including flour triglycerides), and related to the protein content

Lipid-protein interaction during dough development 891

Table 6. Wet mass, protein and lipid distribution in ammonium sulphate fractionated acetic acid-soluble protein from dough mixed under nitrogen to 150 kJ kg-1

Kjeldahl protein Lipid Wet mass - (14C-GTO)a Lipid distribution on protein

dry acetic dry acetic % total dry acetic mg added mg total acid-soluble acid-soluble protein acid-soluble nCi g-1 triglyceride triglyceride

Fraction protein) protein recovered protein) protein 8-1 protein g-1 protein

(g g-' mg g-' (nCi g-1

Precipitate PI 3.40 384 50.2 156.1 406.5 29.3 53.7 Precipitate PZ 0.827 284 37.0 7 .72 27.2 1.96 3.59 Precipitate P3 0.0230 - - - - - 0.95 Supernatant 202.4 98.4 12.8 50.8 516.3 37.2 68 .2

Total 206. 7b 766.4 I00 I0 215.6 281.3 20 .3 37.1 Recovery ( %) 92.4 97.3 - 96.8

solution S

- - -

1 pCi = 72 mg added triglyceride or 132 nig total triglyceride lipid. Total wet m a s = 1 .O g dry acetic acid extract+ 177.8 g AUC+45 g ammonium sulphate=223.8 g.

of each fraction (Table 6), the marked difference in lipid binding ability between gliadin and glutenin is clearly seen. Thus, weight for weight, glutenin protein bound 15 times as much lipid as gliadin protein, in a dough mixed to 150 kJ kg-1 under nitrogen. However, the most striking feature of this distribution is the very high mass of lipid associated with the supernatant proteins-over 25 % greater even than that bound to the P1 glutenin. The significance and composition of this important new protein fraction is examined further in section 3.3.

3.2.3. Aqueous ethanol and ammonium sulphate fractionation In an attempt to resolve the apparent anomaly concerning the extent of lipid binding between gliadin prepared by ethanol extraction (section 3.2.1) and by ammonium sulphate precipitation (section 3.2.2), both fractionation methods were used on a single sample of acetic acid-soluble protein, with the results shown in Table 7. As expected, the ethanol-insoluble glutenin (F1) when refractionated by the AUC-ammonium sulphate procedure, precipitated mainly as PI glutenin,

Table 7. Lipid distribution in ammonium sulphate sub-fractions of aqueous ethanol fractionated acetic acid- soluble protein from dough mixed under nitrogen to 150 kJ kg-I

Fraction

Determination

Stock acetic Ethanol-insoluble Ethanol-soluble acid-soluble residue protein Total

protein (Fi) (F2) Fi + Fn

Lipid (I4C-GTO) (nCi g-l dry acetic acid-soluble protein) Stock AUC solution 270.6 Sub-fraction

Precipitate PI 128.6 Precipitate PZ 8.00 Precipitate P3 2.36 Supernatant S 145.0 Total 284.0

124.4 Recovery (%) 105.0

TCA precipitate of supernatant S

1 1 1 . 3

72.3 1.46 0 .32

22.6 96.7 86.9 24.6

104.1

12.2 5.24 1.46

81.3 100.2 96.3 66.0

215.4

84.5 6.70 1.78

103.9 196.9 91.4 90.6


most of the F1-bound label being associated with this fraction. Very little material or label precipitated in PZ or PS and the activity of the supernatant was also relatively low.

Examination of the ethanol-soluble ‘gliadin’ fraction (Fz) showed that its high 14C-GT0 content arose largely because of the presence of some glutenin (PI precipitate, contributing about 12 nCi g-1) together with a high level of supernatant protein, contributing over 80 nCi 8-1 of the total activity (100 nCi g-l). In fact the PZ and PS gliadin protein precipitates prepared from ethanol- soluble gliadin had low levels of bound lipid similar to those found by direct precipitation from the acetic acid-soluble gluten.

Adding together the ethanol-soluble and insoluble 14C-GT0 recoveries for each ammonium sulphate fraction produced a lipid distribution similar to that obtained directly from the acetic acid-soluble protein. Thus it may be concluded that the two fractionation methods are fundamentally in agreement regarding lipid distribution between glutenin and gliadin. Any confusion that occurs is due largely to the behaviour of the supernatant protein, which is classified separately by the ammonium sulphate precipitation method but which appears together with the gliadin fraction by nature of its ethanol solubility. Table 7 confirms also that most of this supernatant activity was TCA coaguable, and therefore not due to ‘free’ lipid.

3.3. Fractionation of AUC-ammonium sulphate supernatant protein For this part of the work, dough preparation (Figure 1) was modified by omitting soya flour and increasing the level of 14C-GTO activity ten-fold (i.e. approximately 50 pCi 0.36 g-’ olive oil). A single work level of 150 kJ kg-l was used, followed by preparation of acetic acid-solubles and ammonium sulphate-supernatant as before (sections 2.2.3, 2.2.5).

3.3.1. Gel filtration Initial attempts to fractionate the AUC-ammonium sulphate supernatant protein, using dialysed, freeze-dried samples revealed some difficulty in resolubilising the material and also the presence of a large amount of protein at the void volume of the column (> 50 000 mol. wt). This was unexpected in view of the failure of the material to precipitate with ammonium sulphate and the observed presence of low molecular weight material during gel electrophoresis (Figures 5 and 7, section 3.2.2). One possibility was that aggregation was taking place during either dialysis or freeze-drying. Accordingly, fractionation was attempted on the supernatant directly, without dialysis or freeze- drying. Results of a typical column run are presented in Figure 8 and show a large, clearly separated protein peak at an elution volume of 325 ml, corresponding to a molecular weight of approximately 9000. In addition a small peak was present at the column void volume, possibly representing an aggregate of the low molecular weight material.

Calibrated molecular weight (~1000)

50 20 10 5 2 I I I l l I 1 1 1

Figure 8. Fractionation on Sephadex GSOSF of supernatant protein from AUC-ammonium sulphate precipitation of acetic acid-soluble gluten. -, distribution of protein; 0 , distribution of 14C-GT0.

Elution volume (rnl)


Recovery of radioactivity from the column is also shown in Figure 8. Despite the considerable number of fractionation steps from dough mixing, through Soxhlet extraction, triple homogenisation in both water and acetic acid, freeze-drying, AUC-solubilisation, ammonium sulphate precipitation and finally gel filtration, a substantial level of 14C-GT0 was found to coincide with this single protein peak at molecular weight of approximately 9000. Furthermore the continuing strong association of the triglyceride lipid with this protein was confirmed by the co-precipitation of radioactivity with the protein on treatment of this fraction with TCA. Addition of SDS was found to disrupt the complex, l4C-activity then remaining in solution when the protein was precipitated with TCA.

3.3.2. Amino acid analysis Since the supernatant 9000 mol. wt peak appeared well separated from other material and was probably relatively pure, amino acid analysis was clearly justified. Two separate column prepara- tions of this fraction were used and for comparative purposes PI-precipitated glutenin was also analysed using a sample weight similar to that available from a column run. Table 8 shows experimental replicates of the PI glutenin amino acid analysis. Nearest integer values (in mol lo5 g-1 protein) appear close to typical literature values for glutenin. (Residue frequency in mol lo4 8-l protein is also shown in Table 8 for comparison with the supernatant protein analysis.)

Amino acid analysis of the 9000 mol. wt peak from replicate gel filtration runs of supernatant is shown in Table 9. Comparison with Table 8 shows a remarkably different amino acid frequency. Aspartic acid was found to be much higher and glutamic acid much lower than in glutenin so that the asp: glu ratio for the supernatant protein was almost 1 : 1 compared with 1 : 14 in glutenin. Supernatant proline was about half and alanine about twice the frequency in glutenin. Cysteine was higher in the supernatant protein than in glutenin, but nowhere near as high as found in purothionin (15 residues lo4 g-l for cc-purothioninlG). Methionine, however, was absent, in common with purothionin. Lysine and arginine were both considerably higher in the supernatant protein than in glutenin, but only about half the values reported for purothionin.10

Assuming one residue of histidine per molecule and complete amidation, the minimum molecular weight for the 80 residue supernatant protein was calculated as 8583. Its ratio of ionic and polar residues to non-polar residues was just below unity (0.95).

Table 8. Amino acid composition of PI glutenin

Mols amino acid per lo5 g protein Residues

Amino acid replicates integers values45 g protein

-

Experimental Nearest Literature per lo4

ASP Thr Ser Glu Pro G lY Ala CySH Val Met Ileu Leu Tyr Phe His LY s Try Arg

20 .2 23.7 52.1

268.3 114.5 44.2 25.3 13.4 29 .4 10.1 24.7 48 .4 17.4 3 1 . 3 11.8 9 . 8

ND 17.7

22.6 24.1 5 3 . 3

267.2 101.5 47.7 26 .4 13.4 30.8 8 . 0

25 .6 50.8 17.9 33 .O 1 1 . 8 9 . 8

ND 18.4

21 24 53

268 108 46 26 13 30 9

25 50 18 32 12 10

ND 18

23 26 50

278 114 78 34 10 41 12 28 57 25 27 13 1 3 8

20

2 2 5

27 11 5 3 1 3 1 3 5 2 3 1 1

N D 2

N D = Not determined.

894 P. J. Frazier et a/.

Table 9. Amino acid composition of supernatant protein

Mols amino acid per lo4 g protein

Amino acid Experimental replicates Nearest integers

ASP Thr Ser Glu Pro G IY Ala CySH Val Met Ileu Leu Tyr Phe His LYS Try Arg Total

6.66 4.00 5.80

10.78 5.02 7.79 6.80 2.62 5.09 0.00 3.28 6.84 2.20 3.12 1.44 3.28

4.68 N D

6.55 7 4.43 4 5.81 6 9.72 10 4.99 5 8.46 8 7.52 7 2.39 3 5.24 5 0.00 0 3.23 3 6.60 7 2.42 2 3.04 3 1.38 1 3.73 4 ND N D 4.52 5

80

Calculated minimum molecular weight (assuming complete

Ratio : ionic + polar/non-polar = 0.95. N D = N o t determined.

amidation)= 8583.

Finally, Table 10 presents details of the quantities of labelled lipid and protein recovered in those fractions corresponding to the 9000 mol. wt peak from duplicate Sephadex G50 column runs. Based on added triglyceride alone the mole ratio of lipid: protein approached 0.6. However, it is more realistic to assume that the distribution of the label would be in proportion to the total triglyceride content of the original dough (i.e. including the natural flour triglycerides). When this higher ratio of total triglyceride lipid to label was taken into account (the molecular weights of the flour triglycerides being approximated to that of glycerol triolein) the mean mole ratio of lipid to protein was found to be 0.95.

Table 10. Triglyceride lipid content of supernatant protein

Column run 1

Column run 2

l-'C-GTO in fraction (nCi) Protein in fraction (mg) Labelled lipid (nCi g 1 protein) Added triglyceride (mg g-1 protein)

mol:mol protein mean (mol: mol protein)

mol: mol protein mean: (mol:mol protein)

Total triglyceride (mg g-1 protein)

4.75 4.69 0.78 0.52

6090 9020 48.7 72.2

0.58 79.2 117.3

0.95

0.47 0.70

0.76 1.14

1 PCi-8 mg added triglyceride- 13 mg total triglyceride. Molecular weight of glycerol trioleate= 885; molecular weight of supernatant protein

= 8583.


The supernatant protein therefore appears most likely to be a genuine lipoprotein molecule, formed as a result of dough development and probably representing the minimum molecular size for a stable lipid-protein complex. Its overall molecular weight including a single triglyceride molecule is calculated to be 9468 and the mechanism of attachment of the triglyceride molecule to one molecule of protein is assumed to depend on hydrophobic interaction, since the complex is easily disrupted by SDS.

4. Concluding discussion

The observations made during the course of this work combine to lead to the conclusion that lipid binding during dough development may be attributed largely, or even solely, to the presence in wheat flour of a hitherto unreported, low molecular weight, hydrophobic protein with strong aggregative tendencies. It seems likely that the mobile, interactive nature of this protein has con- tributed greatly to its previous elusiveness, while at the same time giving rise, in the present work, to apparent anomalies in the observed lipid distribution between gluten protein fractions.

An early indication that lipid binding could be associated with a protein of unusual properties was the finding that Pz-precipitated gliadin carried almost no labelled lipid whereas classical ethanol- soluble gliadin appeared to contain nearly half the bound lipid label. The appearance of substantial radioactivity in the AUC-ammonium sulphate supernatant, which was TCA precipitable, provided further evidence for the presence of an unusual protein, since all readily soluble material had been removed earlier by triple water extraction.

The distribution of labelled lipid almost entirely between PI-precipitated glutenin and the supernatant protein suggested that lipid was bound to a low molecular weight peptide chain, strongly associated with the glutenin, so as to resist water-extraction, while yielding in part to dissociative solvents such as AUC and 70% ethanol. Accordingly, when ethanol-soluble gliadin was sub- fractionated by ammonium sulphate precipitation from AUC, the precipitated gliadin again carried little or no lipid while most of the label was found to reside in the supernatant fraction.

It was thought at first that the supernatant protein could represent a fragment of glutenin (part of the polypeptide chain of a subunit) split-off by AUC. However, the amino acidanalysis largelyrules out this possibility and it now seems most likely that the supernatant protein exists as a novel protein class in its own right. Since reduction is not required to achieve separation, it cannot be considered as a true sub-unit of glutenin, although it would normally appear in the very low molecular weight bands on SDS-PAGE of reduced glutenin. Significantly, in many publications using the preparation method recommended by Orth and Bushuk,46 involving precipitation of glutenin from ethanolic- AUC, all or most of the low molecular weight components will have been removed. However, one supernatant fraction described recently47 contained a range of protein bands down to 12 OOO mol. wt with an amino acid analysis somewhat similar to that reported here. The supernatant protein will also appear on ethanol-solubilised gliadin electrophoretograms and has probably been previously dismissed in both cases as a water-soluble albumin/globulin contaminant. The region around 10 OOO mol. wt is dificult for gel electrophoresis and in the absence of 14C-GTO activity to draw attention to this low molecular weight protein, its presence has evidently been overlooked.

The nearest reported protein, purothionin, differs considerably in amino acid analysis and would have been removed from the present system in the light petroleum extract prior to protein extraction and fractionation. Furthermore, purothionin has been reported in flour only in amounts very much lower than the level of supernatant protein seen here. In the present experiments 13 % of the acetic acid-soluble protein was found in the AUC supernatant. Assuming PI glutenin to contain further undissociated supernatant protein attached to lipid, then the true proportion of this small protein in gluten could be much higher. However, not all bound lipid could be accounted for in this way on a 1 : 1 molar basis, since the amount of supernatant protein required would be incompatible with the amino acid analysis of glutenin.

It seems more likely, therefore, that the supernatant lipoprotein may be a key functional ele- ment in the formation of glutenin structure, possibly involving both -SH and hydrophobic interactions, and that the bound lipid so incorporated into the gluten structure under appropriate


dough mixing conditions could form a nucleus for further hydrophobic binding of lipid. Such a mechanism would help to explain the dispersibility of glutenin in long chain fatty acid soap solu- t i o n ~ ~ * and the reported dynamic interchange of lipid during dough mixing.49

While many of these suggestions must remain speculative in the absence of the large body of further research required to explore adequately the properties and function of this novel protein, we feel it appropriate that its strongly interactive nature should be reflected in a suitable name. Accordingly we propose the name ‘Ligolin’ (from the Latin ligare; to bind, to tie) to describe the AUC-ammonium sulphate supernatant proteins, found to complex with triglyceride lipid on a 1 : 1 molar basis during dough mixing under nitrogen. By virtue of its interaction with other gluten proteins and with lipid, ligolin may prove to be of fundamental importance in the formation of an optimum dough protein structure during modern breadmaking processes.

Acknowledgements The authors would like to thank the Ministry of Agriculture, Fisheries and Food for financial support, K. M. T. Shearing and J. E. Stirrup for practical assistance and valuable discussion and the Group analysts for the amino acid determinations.

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Frazier, P. J . ; Leigh-Dugmore, F. A.; Daniels, N. W. R.; Russell Eggitt, P. W.; Coppock, J . B. M. The effect of lipoxygenase action on the mechanical development of wheat flour doughs. J. Sci. Food Agric. 1973, 24, 421-436. Frazier, P. J . ; Brimblecombe, F. A.; Daniels, N. W. R. Effect of enzyme-active soya flour on the rheological and baking properties of wheat flour doughs. Proc. I V Znt. Congress Fd Sci. Techno/. (Madrid) 1974, 1 , 127-1 29, Frazier, P. J . ; Brimblecombe, F. A.; Daniels, N. W. R.; Russell Eggitt, P. W. The effect of lipoxygenase action on the mechanical development of doughs from fat-extracted and reconstituted flours. J. Sci. Food Agric. 1977, 26, 247-254. Frazier, P. 3. Lipoxygenase action and lipid binding in breadmaking. Bakers’ Digest 1979, 53 (6), 8-10,

Daniels, D. G . H.; Fisher, N. Release of carbon dioxide from dough during baking. J . Sci. Food Agric. 1976, 27, 351-357. Bell, B. M.; Daniels, D. G. H.; Fisher, N. Physical aspects of the improvement of dough by fat. Fd Chem. 1977, 2, 57-70. Chung, 0. K.; Tsen, C . C. Changes in lipid binding and protein extractability during dough mixing in the presence of surfactants. Cereal Chem. 1975, 52, 549-560. Jacobsberg, F. R.; Worman, S . L.; Daniels, N. W. R. Lipid binding in wheat flour doughs: the effect of DATEM emulsifier. J. Sci. Food Agric. 1976, 27, 1064-1070. Bell, B. M.; Chamberlain, N.; Collins, T. H.; Daniels, D. G . H.; Fisher, N. The composition, rheological properties and breadmaking behaviour of stored flours. J. Sci. Food Agric. 1979, 30, 11 11-1 122. Bell, B. M.; Daniels, D. G. H.; Fisher, N. The effects of pure saturated and unsaturated fatty acids on breadmaking and on lipid binding, using Chorleywood Bread Process doughs containing a ‘model fat’. J. Sci. Food Agric. 1979,30, 1123-1130. Warwick, M. J.; Farrington, W. H. H.; Shearer, G. Changes in total fatty acids and individual lipid classes o n prolonged storage of wheat flour. J . Sci. Food Agric. 1979, 30, 1131-1 138. Surrey, K. Spectrophotometric method for the determination of lipoxidase activity. PI. Physiol. 1964, 39, 65-70. Meredith, 0. B.; Wren, J . J. Determination of molecular weight distribution in wheat flour proteins by extraction and gel filtration in a dissociating medium. Cereal Chem. 1966, 43, 169-186. Frazier, P. J.; Daniels, N. W. R.; Russell Eggitt, P. W. Rheology and the continuous breadmaking process. Cereal Chem. 1975, 52, 1061-130r. Wasik, R. J . ; Bushuk, W. Studies of glutenin. V. Note on additional preparative methods. Cereal Clzem.

Bietz, J. A, ; Wall, J. S. Wheat gluten subunits: Molecular weights determined by sodium dodecyl sulphate- polyacrylamide gel electrophoresis. Cereal Chem. 1972, 49, 416-430. Weber, K.; Osborn, M. The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J . Biol. Chem. 1969, 244, 4406-4412. Wrigley, C. W. ; McCausland, J. Variety identification by laboratory methods : Instruction manual for barley, wheat and other cereals. CSIRO Wheat Research Unit, North Ryde, New South Wales, Australia, 1977, Technical Publication No. 4. Tsen, C. C. Changes in flour proteins during dough mixing. Cereal Chern. 1967, 44, 308-317. Patey, A. L.; Shearer, G.; McWeeny, D. J. A study of gluten extractability from doughs made from fresh and stored wheat flours. J. Sci. Food Agric. 1977, 28, 63-68. Khan, K. ; Bushuk, W. Studies of glutenin. XII. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of unreduced and reduced glutenin from various isolation and precipitation procedures. Cereal Chem. 1979, 56, 63-68. Kasarda, D. D.; Nimmo, C. C.; Kohler, G . 0. Proteins and the amino acid composition of wheat fractions. In Wheat Chemistry and Technology (Pomeranz, Y., Ed.), American Association of Cereal Chemists Inc., St Paul, Minnesota, 1971, pp. 227-299. Orth, R. A.; Bushuk, W. Studies of glutenin. I . Comparison of preparative methods. Cereal Chem. 1973, 50. 106-1 14. Khan, K.; Bushuk, W. Studies of glutenin. XllI. Gel filtration, isoelectric focusing and amino acid composition studies. Cereal Chem. 1979, 56, 505-512. Kobrehel, K.; Bushuk, W. Studies of glutenin. X. Effect of fatty acids and their sodium salts on solubility in water. Cereal Chem. 1977, 54, 833-839. Wood. p. S . : Daniels. N. W. R.; Greenshields, R. N. The use of radiotracers to study lipid binding in wheat

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