Lipid binding in flour, dough and bread

J . Sci. Fd Agric. 1973, 24, 147-155

Lipid Binding in Flour, Dough and Bread

Nathan Fisher, Brenda M. Bell and Christine E. B. Rawlings

Flour Milling and Baking Research Association, Chorleywood, Rickmansworth, Herts WD3 5SH, England

(Manuscript received 26 May 1972 and accepted 31 October 1972)

A study was made of the binding of lipids during dough and breadmaking, using yeasted and unyeasted doughs, and work input levels during doughmaking of 2 to 6 Wh/lb (0.16 to 0.48 h.p. min/lb; 16 to 48 MJ/kg). Incorporation of bakery fat into dough increased the ratio of (total) “free” to “bound” lipids as compared with corresponding doughs without fat and the distribution was not significantly influenced by the time of addition of the fat during doughmaking. There was no significant change in phospholipid distribution as a result of the incorporation of bakery fat into dough. Neither total work input nor rate of work input were found to affect lipid binding significantly in (air-mixed) dough or in bread, within the range of work levels likely to be encountered in commercial practice in the U.K. Differences in the distribution of total lipid cannot account for the essential requirement for fat in mechanical development processes, where work input is usually about 5 Wh/lb, as compared with conventional processes, with work inputs of about 2 Wh/lb, where fat is an optional ingredient.

1. Introduction

The production of commercially acceptable bread requires the use of fat as an essential ingredient when doughs are mechanically developed, but fat may be omitted if doughs are developed by bulk fermentation.’, ’ The effects of fat subsequent to doughmaking are qualitatively similar in each case, however, namely the production of larger loaves, with finer crumb texture than those obtained from doughs lacking fat. The basis for these effects of fat is still incompletely understood, but has long been believed to involve the lipids naturally present in the flour.3, Both physica14-15 and chemical mechar~ismsl~-’~ have been proposed for the action of lipids in dough and bread. The chemical mechanisms usually involve oxidation of lipid and protein in the dough and have been strengthened by recent e v i d e n ~ e . ~ ~ - ~ ~ Earlier evidence suggested that mechanisms involving lipids in oxidation of sulphydryl groups of proteins were probably of minor significance in breadmaking.l5. 26,27 The importance of solid fat has been stressed.*. 14,28 Work from a number of laboratories has tended to implicate lipid “polarity” and binding in the mechanism of improvement by fat.’, ” 8 23, 29-39 Our present findings,” dealing with lipid binding in flour, dough and bread, show that

Obtained in the course (1964 to 1969) of a project “Studies of interactions between lipids and other dough constituents”, sponsored by the U.S. Department of Agriculture.

147

148 N. Fisher, B. M. Bell and C. E. B. Rawlings

differences in the ratio of “free” to “bound” total lipid in dough or bread cannot be invoked to explain the critical requirement for fat in mechanical development processes of dough and breadmaking, as compared with those involving bulk fermentation, if work level (or rate) during dough mixing constitutes the critical difference between these processes; this was also true for the distribution of total phospholipid between the corresponding “free” and “bound“ fractions.

2. Materials and methods

2.1. Flours For the first experiment (A, below) the flour was freshly Buhler-milled to 71.5% extraction from Bison hard red winter wheat of the 1963 crop, grown in Kansas, U.S.A. [flour: protein (N x 5.7) 14.5%, moisture (by drying at 130 “C for 90 min) 12.3%]. All subsequent tests involved untreated and unbleached baker’s grade flour, commercially milled (J. Rank Ltd) from a mixed wheat grist.

2.2. Doughs These were prepared as given in the text and then freeze-dried, ground finely and desiccated in a vacuum. For both tests following the first, the ingredients were mixed in the following proportions: flour, 840 g; yeast, 18 g; salt, 15 g; fat (“Covo”: slip point 43 “C, Craigmillar and British Creameries Ltd), 6 g; ascorbic acid, 0.063 g; and water (495 ml experiment B, 510 ml experiment C), according to the water absorption of the flour. Experiment C employed double the stated quantities per mixing.

2.3. Bread Doughs each weighing 454 g were baked for 25 min at 430 “F. The bread crumb was comminuted, freeze-dried and finally desiccated in a vacuum.

2.4. Extractions These were carried out at room temperature by intermittent agitation of flour, or the dried, finely-ground product from dough or bread, with several portions of solvent (diethyl ether, followed by water-saturated 1-butanol (WSB)). Solvent : solid ratios and extraction times are given with each experiment. Extraction conditions were more exhaustive than those previously’’ shown to give results comparable with those of an acid-hydrolysis method. Combined extracts were evaporated to dryness under reduced pressure (residual nitrogen) and at a temperature of about 40 “C. Lipid products were puriJied by solvent par t i t i~n,~’ desiccated and weighed. Solvents were purified by standard methods. For the purposes of this paper “total extractable lipid” is defined as the sum of the purified yields obtained by successive extraction with ether and WSB, respectively.

2.5. Desiccation Vacuum desiccation was carried out to constant weight over phosphorus pentoxide and sulphuric acid and in residual nitrogen.

Lipid binding in flour, dough and bread

2.6. Freeze drying This was carried out over calcium chloride.

149

2.7. Analytical Method Lipid nitrogen and lipid phosphorus were determined by methods described previously. l2

3. Experimental and results

3.1. Experiment A. “Free” and “bound” lipid in flour and dough The total extractable lipid contents and the relative proportions of “free” (ether- extractable) and “bound” (WSB-extractable) lipids were determined for flour and for the corresponding doughs containing no fat, fat added at the start of mixing and fat added after the dough had formed, respectively. The simplest possible dough recipe was used, i.e. flour, water and fat where indicated. The experiment was carried out three times on separate occasions.

28 g of flour were mixed in a Simon “Minorpin” mixer with distilled water (1 5 ml for the first two experiments, 14 ml in the last) for a total of 4 min. Fat (0.2 g; 0.71 % based on flour weight as used, 0.81 % of the dried dough) was added either at the start of mixing or after 2 min of mixing, as shown in Table 1. A preliminary experiment using

TABLE 1. “Free” and “bound” lipids of flour and dough (%of dried flour or dough; %of total extracted lipid in brackets)

Sample Composition % “Free” % “Bound” Total

Flour 0.67 (60%) 0.45 (40 %) 1.12 Dough Flour/water 0.20 (20%) 0.80 (80%) 1 .oo Dough Flour/water and

fat added at start of mixing 0.65“ (37 %) 1.13b (63%) 1.78

Dough Flour/water and fat added after 2 rnin of mixing 0.68“ (38 %) 1.12b (62%) 1 .so

a S.E.M. + 0.028 (16 d.f.). Means of values from last experiment only: low and variable recoveries in previous two experiments.

dyed fat established that the fat was adequately dispersed in two minutes of mixing. Triplicate mixings were carried out in the order (l), (2), (3); (2), (3), (1); (3), (l), (2); where (1) was flour and water, (2) flour, water; fat added at start of mixing, (3) flour, water; fat added after two min of mixing. The doughs were placed in tared flasks, weighed, freeze-dried and desiccated. The final moisture content of all products was 2.3%. The products were extracted six times with ether [4 ml/g; 48 h (2), 7 h, 16 h, 3 h (2): “free” lipids] followed by three extractions with WSB (4 ml/g; 16 h, 16 h, 3 h) to give the “bound” lipids. Purified lipid yields are given in Table 1.

The corresponding values for “free” and “bound” lipid phosphorus, determined in the last experiment, are given in Table 2.


TABLE 2. “Free” and “bound” lipid phosphorus in flour and dough (mg Pi100 g dry matter; % of total lipid P in brackets)

Sample Composition “Free” “Bound” Total

Flour 2.0 (14%) 12.0 (86%) 14.0 Dough Flour/water 0.1 (0.7%) 15.1 (99.3 %) 15.2 Dough Flour/water and

fat added at start of mixing 0.1 (0.7%) 14.6 (99.3 %) 14.7

Dough Flour/water and fat added after 2 min of mixing 0.1 (0.7 p/o) 15.0 (99.3 %) 15.1

3.2. Experiment B. The effect of rate of work input on lipid binding in dough

An examination was made of the effect of rate of work input on the “free” and “bound” lipids of doughs mixed to the same total work level [ S Wh/lb, optimal for the Chorley- wood Bread Process (CBP)], using a common CBP recipe (see Materials and methods). The two desired rates of work input were achieved by mixing doughs for 3 min in a Morton mixer or for 12 min in the Brabender “Farinograph” mixer, each resulting in a total work input of 5 Wh/lb of dough.

Three doughs were mixed in each machine, freeze-dried and extracted with ether (6 x 4 ml/g) and then WSB (3 x 4 ml/g). Each extraction was carried out for approximately 24 h. Purified lipid yields are shown in Table 3. Analysis of variance and

TABLE 3. Lipid yields from doughs mixed to the same work level (5 Wh/lb) in different times

Yield as % of total lipid extracted Yield as % flour weight

Morton Brabender Morton Brabender (3 min) (12 min)

I ,

“Free” lipid (ether) 1.18 1.23 48.2 49.2 “Bound” lipid (WSB) 1.27 1.27 51.8 50.8

covariance was carried out (by Dr J. B. Hutchinson) : a pooled error term based on 7 degrees of freedom was derived, and the standard error of means of three calculated to be h0.046, of the difference between means of three k0.065. For means of six (e.g. Morton us Brabender) the standard error of the means was h0.032 or =t0.046 for the difference between means. None of the differences of Table 3 was found to be significant,

3.3. Experiment C. The effect of total work input on lipid binding in dough and bread Previous work both by ourselves and other workers had been complicated by the use of different mixers (and sometimes different recipes and procedures) for the different work levels. In the following test of the effect of total work input on lipid binding a single mixer was used (Morton “Duplex”, variable speed, fitted with Kopp gears ; capacity 5.1 1 and procedures following mixing’ were identical for all doughs.

(P > 20 %).

Lipid binding in flour, dough and bread 151

The lipid contribution on a dry weight basis given by each component in the recipe (see Materials and methods : extractions as for flour) was as follows : flour, 1.54 % ; yeast, 0.02%; fat,O.81%; total, 2.37%. Total without fat 1.56%.

3.3.1. Doughs These were mixed at 300 revs/min to 2 and 6 Wh/lb alternately, with four replications of each work level. The work input required 75 seconds (s.D. 2.9 s) for 2 Wh/lb and 185 seconds (s.D. 6.2 s) for 6 Wh/lb. Five doughs were prepared from each mix. One mix omitting the fat was also performed for each work level (not used in the statistical calculations). Final proof was carried out to constant height (3.9 in), requiring 48 min (s.D. 1.5 min) at 2 Wh/lb and 47 min (s.D. 0.6 min) at 6 Wh/lb, the difference being clearly insignificant. Approximately 75-g samples were removed from each of two doughs for drying and extraction.

3.3.2. Baking The remaining three doughs were baked as described. Approximately 75-g aliquots of crumb were removed from two ofthe three baked loaves in each batch. Dough and bread samples were frozen rapidly in solid carbon dioxide, broken into small lumps and placed in jars each containing 0.05 ml of 0.001 % hydroquinone in ether, flushed with nitrogen and stored in solid carbon dioxide until all the samples had been collected. All samples were then freeze-dried, mixed with solid carbon dioxide, ground in a mill (Glen Creston) under nitrogen cover and redried overnight in uucuo.

3.3.3. Extraction Doughs, etc. were extracted by two operators, each extracting one sample of each pair. (Note : all solvents contained 0.001 % hydroquinone as an antioxidant.) The lyophilised, ground samples were extracted successively with ether as follows: (1) 20 ml/g, (2) 4 ml/g, (3 to 6) 2 ml/g each (approximately 24 h/extraction). For one dough from each mix yields of extractions 1 and 2 were bulked, but the crude yields of each of extractions 3 to 6 were recorded separately. Some ofthelatterextractscontainedmaterial which would not redissolve in ether and the crude yields were corrected accordingly. The yields became negligible only after six extractions. Only total ether-extractable lipid yields were determined for the remaining samples. The extracted dough and bread residues were desiccated in uacuo to remove the last traces of ether, and then extracted with WSB to give the total “bound lipid” yield. Samples of flour and yeast were extracted similarly to the dough and bread samples.

Purified lipid yields from flour and fromdough and bread made without added bakery fat are given (as % w/w of dried products) in Table 4.

TABLE 4. Purified lipid yields from flour, dough and bread with no added bakery fat (% w/w of dried products)

Dough Bread

Flour 2 Whjlb 6 Wh/lb 2 Whjlb 6 Whilb

Ether 1 .oo 0.53 0.48 0.41 0.37 WSB 0.59 1.18 1.29 1.33 1.25

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The corresponding yields obtained from fat-containing dough and bread are given

The corresponding “bound” lipid phosphorus values, each expressed as a percentage in Table 5.

of the total lipid phosphorus extracted, are given in Table 6.

TABLE 6. Bound lipid P as % of total lipid P extracted from flour, dough and bread

Without fat With fat

Flour 81.5 - Dough 2 Whjlb 98.7 98.3 Dough 6 Whjlb 98.5 98.0 Bread 2 Whilb 98.4 98.0 Bread 6 Wh/lb 98.4 91.1

4. Discussion

The work done on the dough during mixing varies with different breadmaking processes. Those depending on bulk fermentation use relatively low work levels, represented in the present study by 2 Wh/lb of dough, while mechanical development processes such as the CBP employ a work input of about 5 Wh/lb, to produce bread of optimal specific volume and crumb structure. In bulk fermentation processes, inclusion of fat often results in considerably increased loaf volume, but bread of acceptable quality can be obtained using recipes omitting this ingredient. In CBP and similar processes, omission of the fat or use of a fat with insufficient solid fat content results in loaves of poor specific volume and crumb structure, unacceptable as commercial products.

In the preliminary experiment A, the distribution of lipid was determined in the simplest possible dough, made from flour and water, and in the same dough to which fat had been added. The extensive binding of lipids and phospholipids on doughmaking, first reported by Olcott and Mecham4’ and later by others, is the most striking feature of Tables 1 and 2.

The addition of fat to the dough resulted in an increase in the ratio of “free” to “bound” lipid, and this is a feature of all the experiments described, though the importance of this change in breadmaking is still unknown.

Time of addition of the fat made no significant difference to the relative proportions of “free” and “bound” lipid extracted, and also had no effect on phospholipid distribution between “free” and “bound” fractions.

In the second experiment, B, using a commercial type of recipe, a fourfold difference in rates of work input caused no significant difference in the distribution of lipids between the “free” and “bound” fractions, so that under normal bakery conditions, even if lipid distribution were related to loaf properties, which is still an open question, variations in rate of work input would be unlikely to exert a significant influence.

The final experiment bears on a point which is of considerable importance both technologically and theoretically : total work input, in the range of normal doughmaking practice (about 2 to 6 Wh/lb) is known to have a substantial effect on the volume and


structure of bread from mechanically developed doughs containing fat. It is therefore important to establish whether changing total work input has an effect on the binding of lipid. Table 4 indicates that for these doughs, mixed in air in the same mixer, no significant difference could be established between the levels of “free” and “bound” lipid at work inputs of 2 and 6 Wh/lb (0.16 to 0.48 h.p. min/lb) respectively, and the same conclusion could be drawn from the data for bread. Consideration was given to the possibility that a true increase of “free” lipid with increasing work level (cf. reference 22) was missed in these experiments. This was discounted in view of the 95 % confidence limits of the “negative” difference (decrease in “free” lipid) actually observed, which was -0.01 & 0.025, i.e. -0.035 to + 0.015.

Neither variation in the work done on the dough in mixing nor the addition of fat to the dough resulted in any detectable change in distribution of lipid phosphorus in the doughs examined in these experiments.

The critical effect of fat in mechanical development processes cannot, in the light of the present work, be attributable to any change in the ratio of free to bound lipid as a result of the additional work input characteristic of mechanical development processes as compared with conventional doughmaking methods, nor to a change in the ratio of free to bound phospholipid. The possibility that a shift of distribution of other in- dividual types of lipid, e.g. triglyceride or glycolipid, may be implicated in the action of fat in mechanically developed doughs requires future study.

It must be emphasised that the results reported here are not directly comparable with those of other workers using different doughmaking recipes, since the additional ingredients (e.g. active lipo~ygenases,’~ sugar,42 skim milk 44 etc.) may modify the effects produced with the “lean” recipe. The major effects of fat on loaf volume and crumb structure and texture are exerted, however, in the absence of such ingredients,’8 and also in conditions (absence of oxygen) where the natural lipoxygenase of flour is inopera t i~e .~~, 46 Earlier studies” on doughs containing enzyme active soya, mixed in air or nitrogen to different work levels, indicate, in our opinion, that the effects of lipoxygenase on lipid binding at work levels normally used in doughmaking are slight, and the balance of the evidence at present available suggests to us that these enzymes are probably oflittleimportance in the essential mechanism oftheaction offat in baking.

Acknowledgements The financial support ofthe U.S. Department of Agriculture is gratefully acknowledged. Thanks are also due to Dr J. B. Hutchinson for statistical analyses; Mr T. H. Collins and his staff for dough mixing and breadmaking; to Mr P. Maple and Mr M. R. Bennett for skilled technical assistance.

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46. Fisher, N. f Collins, T. H.; Dodds, N. J. H. (to be published).

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Lipid binding in flour, dough and bread