- Email: [email protected]
The significance of fibrils produced by hydration of wheat proteins
Journal of Cereal Science 12 (1990) 207-221
The Significance of Fibrils Produced by Hydration of Wheat Proteins A. D. EVERS*, H. R. KERRt and 1. CASTLE Flour Milling and Baking Research Association, Chorley wood, Herts. WD35SH, U.K. Received 22 December 1989
Some characteristics of the microscopic protein fibrils generated spontaneously on contact between wheat endosperm particles and a water/air interface have been studied. An inverse relationship between particle concentration and the proportion of particles producing fibrils was discovered. In further experiments, in which particle concentration was carefully controlled, it was found that increase in surface pressure of the suspending medium led to proportionally fewer particles forming fibrils. No evidence was found for the involvement of spontaneously formed fibrils in the floc formation involved in sedimentation tests for flour quality. Surface pressure measurements confirmed the presence of one or more surfactants in flour, and a hypothesis is presented to account for spontaneous fibril formation. Surfactants may be important in determining dough properties but it is unlikely that their influence is mediated through spontaneous fibril formation as few would occur at the high concentrations of endosperm present in dough.
Introduction The cohesive and extensible properties of hydrated wheat endosperm proteins that allow light, leavened bread to be produced from wheaten flour have been recognized and exploited for hundreds of years. A related property has recently been noted by microscopists; the spontaneous formation of fine extended projections around flour particles when these contact an air/water interface. Spontaneous fibril formation was first observed by Seckinger and Wolf1 and a comprehensive description of the phenomenon was later provided by Bernardin and Kasarda 2 • The latter authors studied dynamic aspects of fibril formation. They showed that initiation occurred within 0·05 s of contact with the interface and that extension was complete in 5 s. Relating their observations of fibril formation to bread-making properties, they inferred that the properties of the gluten-forming proteins alone could account for the characteristic breadmaking properties of wheat flour, without involving interaction with lipids. Fibril formation was also observed when endosperm particles of rye and triticale >I<
To whom correspondence should be addressed.
t Present address: B.P. International Ltd, Sunbury Research Centre, Chertsey Road, Sunbury on Thames,
Middx. TW16 7LN, U.K.
0733-5210/90/060207 + 15 $03.00/0
© 1990 Academic Press Limited
A. D. EVERS ET AL.
208
(both cereals with some bread-making qualities) made contact with a water/air interface. Corn, rice and barley endosperm particles showed no tendency to produce fibrils. Muller and Bemardin 3 proposed that fibril formation was involved in flour behaviour, both in dough formation and in baking quality tests. They saw it as a possible mechanism for the floc formation that had been shown 4 to underlie the Zeleny sedimentation test 5• Adeyemi and Muller6 compared sedimentation values of flours with the tendencies of these flours to form fibrils. A strong positive correlation was obtained when fibrils were counted on particles withdrawn from the sedimentation fluid, but counts of fibrils in preparations made from dry flour and water gave a strong negative correlation with sedimentation values. The explanation offered for these anomalous findings was that the solvents present in the sedimentation solution appeared to rupture cell walls, thus facilitating emergence of fibrils 6 • The fibril counts made were considered to be semiquantitative and, in spite of the good agreement between them and other quality tests, no suggestion was made that fibril counting could be recommended as a routine test. The present work was undertaken to examine the possibility of refining the method into a rapid test offlour suitability for breadmaking. In this report we consider factors affecting the semi-quantitative assessments and the fundamental causes of fibril formation.
Experimental Materials Flours. Fibril counts were made on three flours: a Canadian Western Red Spring (CWRS) of excellent baking quality and two UK. cultivars, one of good (cv. Avalon) and one of poor (cv. Brigand) baking properties. These were milled by a standard practice, on Buhler experimental mills. Extraction rate Protein content (%) CWRS 76·7 14·9 cv. Brigand 8·8 75'5 cv. Avalon 10'9 76'8 Flours representing a similar quality range, but produced from a new set of millings were used for quantitative surface studies. These were: CWRS cv. Brock cv. Mercia
Extraction rate 74'5 72'3 74'5
Protein content (%)
14-1 6·8
7·1
Glutens. Vital gluten particles, prepared by washing hydrated insoluble protein from wheat flour and subsequently drying and grinding it, retain many of the qualities of the original flour protein. When hydrated, gluten is capable of forming an elastic, coherent mass similar to that which can be obtained by removing the starch from a wheat flour. Since it is assumed that protein is the flour component responsible for formation of fibrils we examined vital gluten particles suspended in water for fibril fonnation by the microscopic technique described for use with flours. Glutens from three u.K. wheat cultivars of varying breadmaking properties were used. For quantitative surface
WHEAT PROTEIN FIBRILS
209
studies glutens prepared from the CWRS, cv. Brock and cv. Mercia set of flours described above were used. The protein contents of the glutens were CWRS 66·7 %, Brock 66'1 % and Mercia 63·1 %. Detail of the laboratory scale preparation of glutens is given in the flow diagram below. 300 \l flour },
Hobart mixer, with flat beater
215ml H20
2 min, speed I, 3 min, speed 2
I
Dry gluten
Ground in coffee grinder
Dout,lh
5 min speed I Freeze dry
Wet t,lluten
l
Starch
~----------+~ milk and
solubles
Powdered t,lluten
Chemicals. Solvents and other chemicals used were of general purpose grade. Microscopy. A Nikon SKe microscope fitted with brightfield and phase contrast optics was used for transmitted light microscopy. For reflected light microscopy an Olympus SZ-Tr Stereo-zoom microscope was used. Scanning electron micrographs were taken with a Cambridge S600 microscope. Light micrographs were taken on Ilford FP4 35 mm black and white film, using an Olympus OM2m camera, relying on the built-in light metering system for exposure control. Scanning micrographs were taken on Polaroid Type 55 positive/negative film with a 90 x 120 rom format.
Examination (a) Aqueous mounts. The first slides examined were offiour and water and were prepared according to Adeyemi and Muller? It was noted, however, that the relative amounts of water and fiour varied in an uncontrolled manner. Further, there seemed to be a negative relationship between particle concentration and the proportion of particles forming fibrils. The relationship was investigated by examining slides prepared from carefully controlled quantities of the two components. Flour (50 J.Lg to 5 mg) was placed on a pre-weighed, clean, dry, glass slide on a balance pan and freshly distilled water (501-11) was carefully added from an Ultipette (Barky Instruments Ltd, Folkestone, U.K.). The masses of flour and water were recorded to an accuracy of 10 I-Ig. A cover glass was gently placed over the suspension on the slide, which was then taken to the microscope and examined under phase contrast conditions. The addition of the cover glass was recognized as undesirable as it introduced the risk of additional fibril formation through possible shearing of suspended particles. We were alerted to this possibility by observing many fibrils preferentially orientated in one direction, following application of a cover glass in some cases. It was nevertheless necessary to minimize the danger of mishap during transit from the balance on which the slide was prepared to the remote microscope. When the significance of particle concentration had been established, it became clear that valid comparisons could be made with relatively few preparations, provided that the range of solid concentrations was confined within close limits. Thus a routine method was adopted in which a standard concentration of 1% ±0'3 w/v was used. For the experiment in which the effects of flour extracts on fibril formation were examined, the suspending medium was added to the slide when it was in position on the microscope stage. No cover glass was used with these preparations.
A. D. EVERS ET AL.
210
100.-----------------------, ( 0)
....
% Solids w/w
FIGURE L The proportion of particles that produced fibrils, as a function of solids concentration in the aqueous suspensions examined. (a) Flours from: ... cv. Brigand, • cv. Avalon and. CWRS. (b) Glutens from: • cv. Bounty, ... cv. Longbow, • cv. Highbury. Although techniques changed as the work progressed all results included within a data set were obtained by a single method. Throughout, each slide was examined by microscopists with no knowledge of the identity of the sample. Although they knew that replication was involved, the nature of the replication was not divulged until the counting of a complete set was finished. Counts were continuously recorded on two' tally counters' - one for non fibril-forming particles and one for fibril-fanning particles. Every particle seen on a slide was categorized. (b) Mounts in sedimentation test reagents. Published results 7 suggested that assessment of fibrilforming capacity in fiocs present in the lactic acidjpropan-2-01 solution of the Zeleny test 5 may give a better indication offiour quality than counting in aqueous suspension. Positive correlations had been found between putative flour quality and the proportion of particles producing fibrils in the organic medium. To investigate this relationship, we counted fibril-forming particles in aqueous mounts to which had been added varying amounts of the individual Zeleny test components. Thus counts were made in solutions containing between 3 and 75 % propan-2-01 and between 0·25 and 88 % lactic acid. The Zeleny test has never become adopted as a routine means of evaluating wheat quality in the U.K. since it has not been shown to rank British wheats with sufficient precision. An alternative
WHEAT PROTEIN FIBRILS
211
sedimentation testS to the Zeleny test has been introduced since the work of Adeyemi and MulIers, 7 was published, and this has been widely adopted. Like the Zeleny test the alternative test involves shaking a sample of flour (or meal) first with water and then with a second solution, followed by a timed sedimentation in a measuring cylinder. In the Zeleny test the second solution contains lactic acid and propan-2-ol while in the alternative test it contains lactic acid and sodium dodecylsulphate (SDS). The test is frequently referred to as the SDS Sedimentation test. In view of the similarities of the two tests it might be inferred that their mechanisms are similar. Hence the effects of SDS sedimentation reagents on the tendency of endosperm particles to form fibrils was investigated.
Protein determination Protein was determined by Kjeldahl (N x 5'7) and expressed on a 14% moisture basis.
Surface pressure experiments A Joyce.Loebl Langmuir mini trough with thermostatted bath was used for surface pressure (11) measurements. (1) Equilibrium measurements. Aqueous fiour extracts (0'05, 1, 4 or 10 % w(v) were prepared by gently stirring the flour into double-distilled de-ionized water and leaving the suspension for 20 min in an ice-bath (to minimize enzyme activity). Solids were removed by centrifugation at 2500 rev(min (1100 g) and decantation of supernatant into a trough. Air bubbles or dust particles were removed with a clean glass pipette. The surface area of the trough was fixed at 0·056 m 2 and the surface tension (y) of the water used to prepare the extracts was 70 mN(m. Following equilibration at 20°C (30 min), values of y were measured by the Wilhelmy plate method using a pre-wetted clean filter paper strip 10 mm wide. From these, surface pressure (11) values were calculated as the reduction in y. All experiments were duplicated. (2) Dynamic measurements. A small amount offiour (c. 0·01 g) was sprinkled on to the surface of water in the corner of the trough furthest from the Wilhelmy plate. The progressive increase in 11 was noted for the three flours and for the glutens extracted from the flours.
Results and Discussion Concentration and particle-size effects
In Fig. lea) the proportion of flour particles producing fibrils is shown as a function of particle concentration, for flours of several types, separately suspended in aqueous mounts. The single exponential curve fitted to the complete data set demonstrates the critical dependence of fibril formation on concentration and the absence of consistent differences in that relationship among flour types. During the examinations an impression was gained that smaller particles showed a greater tendency to form fibrils and since soft flours contain a greater proportion of these, there was a possibility that variation due to endosperm texture was masking differences in tendency to form fibrils. We investigated this possibility by sieving samples and assessing fibril formation only on particles greater than 63 11m. The results (data not shown) were similar to those obtained with the complete flour. Clearly, the initial impression of an association between size and fibril-forming capacity of flour particles was erroneous. However, the relationship between concentration and fibril formation was endorsed by the results with the larger particles, reaffirming the necessity to control
A. D. EVERS ET AL.
212
FIGURE 2. Light micrographs of fibril-producing particles of (a) flour and (b) gluten.
particle/water relationships in making comparisons among flours. The validity of published semi-quantitative results in which no such control was exerted must thus be questioned. Fibrils in vital gluten
As with flours, the proportion of gluten particles forming fibrils declined as particle concentration increased [Fig. l(b)] but the decline was slower and fibrils formed at higher concentrations than in flour. No systematic differences were apparent among the three types of gluten. Fibrils formed on gluten particles were shorter and thicker than those on flour and there were fewer of them. A comparison is provided in Fig. 2. Effects of sedimentation-test reagents on fibril formation
Results of counts in solutions of propan-2-o1 and lactic acid are shown in Fig. 3, from which it is clear that both propan-2-o1 and lactic acid led not to an increase but a marked decline in fibril formation. No fibrils at all were observed at SDS concentrations above 0·14 %, in spite of the fact that the proportion of solids in all suspensions was kept below 1·25% w/v. SDS is a detergent and consequently it brings about a marked increase in II when added, even in minute quantities, to water. The Zeleny reagents also bring about an increase in II, though their effects are less marked. Bernardin and Kasarda 2 recognized that fibril formation may depend, to a degree, on surface activity and the results here accord with that view. Effect of decreasing surface pressure
If increasing the IT of the suspending medium reduces the tendency of added flour particles to form fibrils, then it might logically be expected that decreased IIwould have the opposite effect. Demonstration of this would provide good evidence for the involvement of IT effects in the fibril-forming mechanism. Few substances are capable of
WHEAT PROTEIN FIBRILS
::g; :g
0
~c
80
I
(,) I
.
!
!
80
60
40
20
213
• 100
% Lactic acid v/v ( b)
a.
.!2
";:
.c G: ~
I
• 0
20
40
60
I
80
100
% Propan-2-ol v/v
FIGURE 3. The proportion of flour particles that formed fibrils, as a function of: (a) lactic acid; and (b) propan-2-ol concentration in the suspending medium. All suspensions contained c. 1 % solids w/w. Arrows indicate approximate concentrations in a suspension for the Zeleny test.
increasing y in water; we examined two that are: NaCl and sucrose. A 5 M solution of NaCI has a y value 9 mN/m higher than pure water at 20°C 9. We examined several mounts of flour particles in a 5 M NaCl solution. No fibrils were detected. In aiM solution some fibrils were observed but the proportion was 50 % less than found in pure water mounts (Fig. 4). Sucrose also increases y of water, a 1·2 M solution having approximately the same value as aiM NaCl solution9 • A reduction in fibril-forming capacity was again noted, but whereas counts in 1M NaCI were 50% lower than those in water, 1·2 M sucrose showed only a 20 % reduction. Clearly, these results did not support the hypothesis that fibril formation results from IT effects, but fibril formation may have been inhibited by chemical effects on endosperm proteins in the case of NaCI and bulk viscosity increase in the case of sucrose. Indeed it is unlikely that addition of solutes that modify IT can be achieved without some physico-chemical change in the nature of the particles, the suspending medium or the interaction between the two. An alternative strategy for examining the relationship between IT and the tendency to form fibrils is the separate measurement of these parameters in water to which flour extracts are added. Effect offlour extract on fibril formation
The results of examinations of 1% flour suspensions in water and in a series of aqueous flour extracts are shown in Table 1.
214
A. D. EVERS ET AL.
100
% Solids w/w
FIGURE 4. The proportions of flour particles that fonned fibrils as a function of flour concentration in • aqueous and. 1 M NaCI suspensions. TABLE 1. Proportions (%) of wheat flour particles fonning fibrils when suspended in water, or flour extract prepared from suspensions of 1-10 % flour in water. Mean proportion of fibrils in 1 % suspension and S.D. of mean. Flour extract
Microscopist (l) Microscopist (2)
Water
1%
2%
5%
10%
65·5±5 52·5±5
50'5±7 42'5±9
13-8± 14 2'3±4
5'2±4 5'8±5
o o
Although agreement between microscopists was not good (there is a subjective element in counting fibrils) both microscopists registered a marked decline in the proportion of fibril-formation with increase in strength of flour extract, and no fibrils were seen in the 10 % extract. Effect offlour extract concentration on surface pressure
The relationship between IT and extract concentration was measured. Relationships for all three flours (shown in Fig. 5) were similar. Linearity with log % extract indicates an exponential relationship between IT and extract concentration.
215
WHEAT PROTEIN FIBRILS 30
"
:€z
~ ~
'" Ul Ul
~
20
CO! U
~:>
(f)
10
-I
0 10910 (% extract)
FIGURE 5. Surface pressure of aqueous flour extracts as a function of loglO flour weight extracted per lao ml water. Flours from three wheats indicated by: ~ CWRS, • cv. Mercia, .. cv. Brock.
The flour with best baking properties was associated with the highest Il, but little difference existed among all types. In this context it may be noted that Lindahl 10 was unable to observe any significant differences between surface properties of his aqueous flour extracts, even when their tertiary structures were known to be different. The exponential decline in proportion of fibril-forming particles, with percent solids (Fig. I) and the similar increase in IT with increasing extract concentration are compatible with a causal relationship. Although a mechanism is unclear, the following observations are relevant: size differences have been ruled out and no other physical differences were obvious between the particles that give rise to fibrils and those that do not. This suggests that fibril formation is less a function of individual particle properties than of the individual relationships between particles and the suspending medium. The most likely way in which such relationships may differ is in time of contact. Whatever the manner of adding particulate material to a liquid surface it is almost inevitable that contact occurs non-uniformly. Although the interval between first and last particles may be short it is possible that changes induced by a contact with the first particles can influence relationships between the liquid and the latter particles. For example, a soluble surfaceactive component of the first particles may rapidly disperse, so that surface-active extracts from later particles have to spread against an existing surface pressure, thus reducing their own n. For such a sequence of events to apply here the effects of the extracted surfactant component would have to be extremely rapid. Such speed is characteristic of surfactants, but to detect evidence of one or more being present in flour we examined changes in surface pressure as a function of time after the addition of flour particles to water in a trough. The results are shown in Fig. 6(a). Near-maximum surface pressure was achieved within one minute, in accord with the findings of Lundh et aZ,u who showed that an equilibrium value was achieved after 30 min, but was closely approached within a few seconds. Such a rate of change is compatible with extremely rapid and energetic changes in the immediate vicinity of particles striking pure water. As in the earlier surface-pressure experiment the CWRS
A. D. EVERS ET AL.
216
25.-----------------, ~
~
20
..",
T
Y ''If
V
.,. T V ' "
"
.,. ............ -.: •• • • : : : • ~ Ir..
• •
••
It
J.
(a)
15 "!'2••
•
10
~
0"'___ _ --'-_ _ --'-_ _
.1--_---'
--1
iil '" E c.
'" ~ :J
.. :".,..,...,
• t. .. ~.
20f-
en
•
,:1"-
•
...
T
...
..
•
•
... .......
(b)
•
o
•
".,..,.
'. ~
••
10':·
!'r0.
'I": o
I
10
20
I
I
30
40
50
Time (min)
FIGURE 6. Surface pressure ofa film spread from particles as a function of time. (a) Flours from three wheat types. (b) Gluten from the same flours. (Symbols as in Fig. 5.)
flour exerted the greatest n. In the present case, the U.K. wheats reflected this quality ranking also, the better baking cultivar having the higher surface activity. In a similar experiment with gluten samples prepared from the flours used above, differences in sample behaviour were smaller [Fig. 6(b)]; probably because a surface-active fraction was partially removed during gluten extraction. The ranking of limiting surface pressures was the reverse of that found with the flours [Fig. 6(b)] but no explanation for this can be offered. Compared with flours, gluten particles exerted the same ultimate n but the time to maximum was much longer (approximately 5 min for flours and 20 min for glutens). The apparently lower energy in the vicinity of gluten particles compared with flour is compatible with the shorter fibrils and fewer fibril-forming particles characteristics of gluten. Further evidence for surface factors being involved in fibril formation came from experiments involving a visual manifestation of surface pressure.
217
WHEAT PROTEIN FIBRILS
2
3
FIGURE 7. Sequence of events in demonstration of surface pressure induced by flour particles on a water drop. (I) Water drop on slide. (2) 'Monolayer' of spores on drop surface. (3) Clear area surrounding fibrillar flour particle,
Microscopical evidence for the involvement of surface activity in fibril formation
Small quantities of flour deposited on the meniscus surface of a water-drop rapidly spread over that surface. The spreading occurs with such a speed that few details of particle behaviour can be observed. Also, any changes in surface pressure remain undetected since areas of different surface tension are not optically different. It is possible to create optical contrast by arranging for a coloured or opaque monolayer to cover the water surface before contact by a surface-active agent is made. When contrast subsequently occurs, the surfactant itself tends to form a monolayer, thus displacing the opaque marker radially. The familiar toy' camphor boat' can be shown to be motivated by surface pressure if a monolayer of oxidized oil is allowed to spread over the water surface before the boat is floated. When the boat is placed, a clear camphor monolayer spreads and the boat, which remains at the boundary of water surface (indicated by oxidized oil film) and camphor monolayer, is drawn by the contracting water surface. A similar demonstration of surface activity associated with flour particles was performed. Oxidized oil may be used but clearer demarcation has been achieved with the tiny spores of puff-balls. Addition of one or a few flour particles to a monolayer of the spores on a water drop on a microscope slide rapidly induced a clear area around the particles, which invariably developed long fibrils. The sequence is illustrated in Fig. 7. Deposition of further flour particles on the surface led to a progressive reduction in the rate of spread by the zone and the later added particles appeared to show a diminishing tendency to produce fibrils although this was difficult to confirm quantitatively because particles could not be identified as having been added earlier or later. Further microscopical evidence of the involvement of IT in fibril formation was sought in the examination of the directions in which fibrils radiate from flour particles. In light microscopical examination of fibrillar particles the optical axis is almost perpendicular to most of the surface of a water drop. Consequently fibrils formed in the plane of the surface can be seen clearly. Should fibrils be produced into the solid angle between the surface plane and the optical axis they would be in different focal planes and, because of the limited depth offield of the light microscope, they would not be clearly seen. Also,
218
A. D. EVERS ET AL.
FIGURE 8. Micrographs of the same flour particle with fibrils under different conditions. (a) Suspended on the surface of a water drop - phase contrast. (b) and (c) Dry, after evaporation of water; imaged by (b) phase contrast and (c) scanning electron microscope.
they would be largely obscured by the particle itself and any fibrils in the surface plane. Because of these difficulties an impression is gained that fibrils radiate only in the surface plane, a condition compatible with the concept of their surface-dependent origin. Bernardin and Kasarda 2 were of the opinion, however, that most of the endosperm contents were dispersed under the surface of the droplet, although they too must have experienced difficulties similar to those described in confirming this visually. The method adopted in the present work, for seeking fibrils other than those in the surface plane, was first a photograph a fibrillar particle on the surface of water. The water was then allowed to evaporate so that the particle was deposited on the slide. A further photograph was then taken so that any structures which had been brought into the same focal plane as those previously recorded, could be identified by comparison of the two images. For more thorough scrutiny the dry mount was also examined by scanning electron microscope. No evidence of additional fibrils in the 'stranded' particles were seen by either technique used. Examples of the three types of image are shown in Fig. 8. It may be deduced from the above that all fibrils radiate from particles in the surface plane, and consequently this provides further evidence of the involvement of surface phenomena in fibril formation.
WHEAT PROTEIN FIBRILS
219
General discussion None of the evidence deriving from the work described here or elsewhere in the literature conclusively demonstrates that fibril formation is totally dependent upon surface phenomena. Much of it, however, is totally compatible with such a relationship, and this provides a reasonable basis for an hypothesis on the mechanism of fibril formation. The hypothesis suggested requires that particles of flour coming into contact with a pure water surface do so, not simultaneously, but sequentially. The first particles to make contact experience two changes relevant to fibril development: (a) the insoluble proteins become very rapidly hydrated, allowing them to exhibit the properties of coherence and extensibility well-known to be associated with gluten, and (b) dispersible, surface-active components rapidly become extracted. The surface pressure exerted by the flour extract as it 'expands' over the waterI air interface causes soluble components to be drawn radially outwards from the flour particle in the plane of the surface. Solid components may also be 'drawn out' in this current. Some starch granules float freely away while others, which are closely associated with the protein matrix of the endosperm, draw out threads of that protein, which, as already described, has become hydrated. The local effect of extracts from particles which make contact later is less marked because of the increased IT over the whole surface, already brought about by extracts from earlier particles. Thus, insufficient IT is generated around later particles to produce fibrils. The role of the solid component in the drawing of the protein strands is not certain, but in many cases starch granules of various sizes are seen at the extremes and along the length offibrils. Their presence may explain why the insoluble proteins are drawn as thin strands rather than a continuous sheet. Certainly the fibrils associated with vital gluten particles, which obviously contain fewer starch granules, are shorter and thicker. In passing it is also worth noting another difference between vital gluten and flour. The rate of decline, with increasing solids content, of fibrillar particle occurrence was more gradual for gluten than for flour. Water is used in the production of gluten, thus reducing the amount of surface-active component in the product. The amount of water used is limited, however, and it appears that some surfactant remains. Because of the extensibility of the hydrated insoluble proteins, fibrils can be produced by imposing stresses other than II. Movement of a cover-glass on a suspension can lead to the formation of proteinaceous projections extending in the direction of cover-glass movement. Apart from their directional bias, these fibrils appear very similar to the spontaneously generated fibrils so far considered. The behaviour of the hydrated proteins appears to be the same in both cases, although the driving forces differ. In considering the importance of fibril formation to technological properties of flours and doughs, the two characteristics of wheat proteins involved in the process should be considered separately. The hydration of insoluble protein to form a coherent, extensible network structure is already recognized as an important factor in producing a light gasretaining structure, but the study of fibrils has revealed a remarkable rate of hydration. The role of surface-active components merely in producing fibrils should not be exaggerated in the context of commercial dough-mixing. Spontaneous fibril formation has been shown to be essentially a feature of very dilute suspensions of flour, the
220
A. D. EVERS ET AL.
proportion of fibrillar particles declining steeply with increasing concentration. In doughs the solids content is about 40 %. Fibril formation has not been studied microscopically at this concentration because of excessive particle density, but it is clear from the rapid decline in fibril formation at much lower concentrations that the spontaneous fibril formation with which we have been concerned here plays little or no part. Perhaps the most interesting aspect of fibril studies, in the commercial context, is the focus they bring to the surfactant component or components present in wheat flour. This has received more attention in recent years. However, we are not yet in a position to identify the component or components responsible for effecting the observed marked change in II. Lipids are known to disperse from flours and this class of compounds has members with the amphiphilic properties characteristic of surfactants. Our exploratory experiments with thin-layer chromatography using chloroform methanol extracts from the aqueous extracts of flours showed traces of lipids. None of these was of a type which might be expected to exhibit surfactant properties, nor did a re-dispersal of the concentrate significantly reduce the fibril-forming capacity of flour particles. Clearly, a more sophisticated approach whereby the surface film is concentrated and analysed is required. Other classes of compounds such as soluble proteins should be considered.
Conclusions Improved methods for preparing samples for microscopical counting have enabled concentration effects to be established. It has been shown that the formation of spontaneously generated protein fibrils, when flour particles contact a water/air interface, occurs most prolifically at low concentration. It is postulated that the decline in fibril formation with increasing concentration is related to the development of a II maximum by an extractable endosperm surfactant, the latter phenomenon producing the driving force for fibril formation. No evidence has been found to support the suggestion that entanglement of fibrils on flour particles is responsible for the floc formation which occurs during sedimentation tests of flour quality. Indeed few particles suspended in the liquids used in those tests produced fibrils. The ability of hydrated insoluble wheat endosperm proteins to be drawn into long narrow strands is an important characteristic in relation both to fibril formation and dough mixing. However, the spontaneous formation of fibrils is unlikely to feature significantly in dough mixing because of the low proportion of particles forming fibrils at the concentrations used in doughs. Note added in revision
We are grateful to the referee who drew our attention to a very recent publication on this subject. The conclusions published here are in close accord with those of Amend and Belitz12 although they were arrived at entirely independently and, in some cases, by different approaches. We are grateful to Heather Riley and Simon Taylor for careful technical assistance. The work was supported by the Ministry of Agriculture, Fisheries and Food and this report, based on MAFF report no. M17, 1986 is published with permission of that Authority.
WHEAT PROTEIN FIBRILS
221
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Seckinger, H. L. and Wolf, M. 1. Cereal Chern. 47 (1970) 236--243. Bernardin, J. E. and Kasarda, D. D. Cereal Chern. 50 (1973) 529-536. Muller, H. G. and Bernardin, J. E. Cereal Chern. 52 (1975) 122-124. Frazier, P. PhD thesis, Leeds University (1971). AACC. Approved methods of the AACC. Test 56.60. American Association of Cereal Chemists, St Paul, MN (1962). Adeyemi, 1. A. and Muller, H. G. in 'Food Texture and Rheology' (p. Sherman, ed.), Academic Press, London (1977) pp 325-341. Adeyemi, 1. A. and Muller, H. G. J. Sci. Fd. Agric. 26 (1975) 544-545. Axford, D. W. E. A., McDermott, E. E. and Redman, D. G. Cereal Chern. 56 (1979) 582-584. Weast, R. C. (ed.). 'Handbook of Chemistry and Physics', 68th edn. CRC Press, Boca Raton, FL (1987). Lindahl, L. 1. J. Dispn. Sci. Technol. 8 (1987) 309-319. Lundh, G., Eliasson, A. C. and Larsson, K. J. Cereal Sci. 7 (1988) 1-9. Amend, T. and Belitz, H. D. Z. Lebensrn. Unters. Forsch. 189 (1989) 103-109.
The Significance of Fibrils Produced by Hydration of Wheat Proteins A. D. EVERS*, H. R. KERRt and 1. CASTLE Flour Milling and Baking Research Association, Chorley wood, Herts. WD35SH, U.K. Received 22 December 1989
Some characteristics of the microscopic protein fibrils generated spontaneously on contact between wheat endosperm particles and a water/air interface have been studied. An inverse relationship between particle concentration and the proportion of particles producing fibrils was discovered. In further experiments, in which particle concentration was carefully controlled, it was found that increase in surface pressure of the suspending medium led to proportionally fewer particles forming fibrils. No evidence was found for the involvement of spontaneously formed fibrils in the floc formation involved in sedimentation tests for flour quality. Surface pressure measurements confirmed the presence of one or more surfactants in flour, and a hypothesis is presented to account for spontaneous fibril formation. Surfactants may be important in determining dough properties but it is unlikely that their influence is mediated through spontaneous fibril formation as few would occur at the high concentrations of endosperm present in dough.
Introduction The cohesive and extensible properties of hydrated wheat endosperm proteins that allow light, leavened bread to be produced from wheaten flour have been recognized and exploited for hundreds of years. A related property has recently been noted by microscopists; the spontaneous formation of fine extended projections around flour particles when these contact an air/water interface. Spontaneous fibril formation was first observed by Seckinger and Wolf1 and a comprehensive description of the phenomenon was later provided by Bernardin and Kasarda 2 • The latter authors studied dynamic aspects of fibril formation. They showed that initiation occurred within 0·05 s of contact with the interface and that extension was complete in 5 s. Relating their observations of fibril formation to bread-making properties, they inferred that the properties of the gluten-forming proteins alone could account for the characteristic breadmaking properties of wheat flour, without involving interaction with lipids. Fibril formation was also observed when endosperm particles of rye and triticale >I<
To whom correspondence should be addressed.
t Present address: B.P. International Ltd, Sunbury Research Centre, Chertsey Road, Sunbury on Thames,
Middx. TW16 7LN, U.K.
0733-5210/90/060207 + 15 $03.00/0
© 1990 Academic Press Limited
A. D. EVERS ET AL.
208
(both cereals with some bread-making qualities) made contact with a water/air interface. Corn, rice and barley endosperm particles showed no tendency to produce fibrils. Muller and Bemardin 3 proposed that fibril formation was involved in flour behaviour, both in dough formation and in baking quality tests. They saw it as a possible mechanism for the floc formation that had been shown 4 to underlie the Zeleny sedimentation test 5• Adeyemi and Muller6 compared sedimentation values of flours with the tendencies of these flours to form fibrils. A strong positive correlation was obtained when fibrils were counted on particles withdrawn from the sedimentation fluid, but counts of fibrils in preparations made from dry flour and water gave a strong negative correlation with sedimentation values. The explanation offered for these anomalous findings was that the solvents present in the sedimentation solution appeared to rupture cell walls, thus facilitating emergence of fibrils 6 • The fibril counts made were considered to be semiquantitative and, in spite of the good agreement between them and other quality tests, no suggestion was made that fibril counting could be recommended as a routine test. The present work was undertaken to examine the possibility of refining the method into a rapid test offlour suitability for breadmaking. In this report we consider factors affecting the semi-quantitative assessments and the fundamental causes of fibril formation.
Experimental Materials Flours. Fibril counts were made on three flours: a Canadian Western Red Spring (CWRS) of excellent baking quality and two UK. cultivars, one of good (cv. Avalon) and one of poor (cv. Brigand) baking properties. These were milled by a standard practice, on Buhler experimental mills. Extraction rate Protein content (%) CWRS 76·7 14·9 cv. Brigand 8·8 75'5 cv. Avalon 10'9 76'8 Flours representing a similar quality range, but produced from a new set of millings were used for quantitative surface studies. These were: CWRS cv. Brock cv. Mercia
Extraction rate 74'5 72'3 74'5
Protein content (%)
14-1 6·8
7·1
Glutens. Vital gluten particles, prepared by washing hydrated insoluble protein from wheat flour and subsequently drying and grinding it, retain many of the qualities of the original flour protein. When hydrated, gluten is capable of forming an elastic, coherent mass similar to that which can be obtained by removing the starch from a wheat flour. Since it is assumed that protein is the flour component responsible for formation of fibrils we examined vital gluten particles suspended in water for fibril fonnation by the microscopic technique described for use with flours. Glutens from three u.K. wheat cultivars of varying breadmaking properties were used. For quantitative surface
WHEAT PROTEIN FIBRILS
209
studies glutens prepared from the CWRS, cv. Brock and cv. Mercia set of flours described above were used. The protein contents of the glutens were CWRS 66·7 %, Brock 66'1 % and Mercia 63·1 %. Detail of the laboratory scale preparation of glutens is given in the flow diagram below. 300 \l flour },
Hobart mixer, with flat beater
215ml H20
2 min, speed I, 3 min, speed 2
I
Dry gluten
Ground in coffee grinder
Dout,lh
5 min speed I Freeze dry
Wet t,lluten
l
Starch
~----------+~ milk and
solubles
Powdered t,lluten
Chemicals. Solvents and other chemicals used were of general purpose grade. Microscopy. A Nikon SKe microscope fitted with brightfield and phase contrast optics was used for transmitted light microscopy. For reflected light microscopy an Olympus SZ-Tr Stereo-zoom microscope was used. Scanning electron micrographs were taken with a Cambridge S600 microscope. Light micrographs were taken on Ilford FP4 35 mm black and white film, using an Olympus OM2m camera, relying on the built-in light metering system for exposure control. Scanning micrographs were taken on Polaroid Type 55 positive/negative film with a 90 x 120 rom format.
Examination (a) Aqueous mounts. The first slides examined were offiour and water and were prepared according to Adeyemi and Muller? It was noted, however, that the relative amounts of water and fiour varied in an uncontrolled manner. Further, there seemed to be a negative relationship between particle concentration and the proportion of particles forming fibrils. The relationship was investigated by examining slides prepared from carefully controlled quantities of the two components. Flour (50 J.Lg to 5 mg) was placed on a pre-weighed, clean, dry, glass slide on a balance pan and freshly distilled water (501-11) was carefully added from an Ultipette (Barky Instruments Ltd, Folkestone, U.K.). The masses of flour and water were recorded to an accuracy of 10 I-Ig. A cover glass was gently placed over the suspension on the slide, which was then taken to the microscope and examined under phase contrast conditions. The addition of the cover glass was recognized as undesirable as it introduced the risk of additional fibril formation through possible shearing of suspended particles. We were alerted to this possibility by observing many fibrils preferentially orientated in one direction, following application of a cover glass in some cases. It was nevertheless necessary to minimize the danger of mishap during transit from the balance on which the slide was prepared to the remote microscope. When the significance of particle concentration had been established, it became clear that valid comparisons could be made with relatively few preparations, provided that the range of solid concentrations was confined within close limits. Thus a routine method was adopted in which a standard concentration of 1% ±0'3 w/v was used. For the experiment in which the effects of flour extracts on fibril formation were examined, the suspending medium was added to the slide when it was in position on the microscope stage. No cover glass was used with these preparations.
A. D. EVERS ET AL.
210
100.-----------------------, ( 0)
....
% Solids w/w
FIGURE L The proportion of particles that produced fibrils, as a function of solids concentration in the aqueous suspensions examined. (a) Flours from: ... cv. Brigand, • cv. Avalon and. CWRS. (b) Glutens from: • cv. Bounty, ... cv. Longbow, • cv. Highbury. Although techniques changed as the work progressed all results included within a data set were obtained by a single method. Throughout, each slide was examined by microscopists with no knowledge of the identity of the sample. Although they knew that replication was involved, the nature of the replication was not divulged until the counting of a complete set was finished. Counts were continuously recorded on two' tally counters' - one for non fibril-forming particles and one for fibril-fanning particles. Every particle seen on a slide was categorized. (b) Mounts in sedimentation test reagents. Published results 7 suggested that assessment of fibrilforming capacity in fiocs present in the lactic acidjpropan-2-01 solution of the Zeleny test 5 may give a better indication offiour quality than counting in aqueous suspension. Positive correlations had been found between putative flour quality and the proportion of particles producing fibrils in the organic medium. To investigate this relationship, we counted fibril-forming particles in aqueous mounts to which had been added varying amounts of the individual Zeleny test components. Thus counts were made in solutions containing between 3 and 75 % propan-2-01 and between 0·25 and 88 % lactic acid. The Zeleny test has never become adopted as a routine means of evaluating wheat quality in the U.K. since it has not been shown to rank British wheats with sufficient precision. An alternative
WHEAT PROTEIN FIBRILS
211
sedimentation testS to the Zeleny test has been introduced since the work of Adeyemi and MulIers, 7 was published, and this has been widely adopted. Like the Zeleny test the alternative test involves shaking a sample of flour (or meal) first with water and then with a second solution, followed by a timed sedimentation in a measuring cylinder. In the Zeleny test the second solution contains lactic acid and propan-2-ol while in the alternative test it contains lactic acid and sodium dodecylsulphate (SDS). The test is frequently referred to as the SDS Sedimentation test. In view of the similarities of the two tests it might be inferred that their mechanisms are similar. Hence the effects of SDS sedimentation reagents on the tendency of endosperm particles to form fibrils was investigated.
Protein determination Protein was determined by Kjeldahl (N x 5'7) and expressed on a 14% moisture basis.
Surface pressure experiments A Joyce.Loebl Langmuir mini trough with thermostatted bath was used for surface pressure (11) measurements. (1) Equilibrium measurements. Aqueous fiour extracts (0'05, 1, 4 or 10 % w(v) were prepared by gently stirring the flour into double-distilled de-ionized water and leaving the suspension for 20 min in an ice-bath (to minimize enzyme activity). Solids were removed by centrifugation at 2500 rev(min (1100 g) and decantation of supernatant into a trough. Air bubbles or dust particles were removed with a clean glass pipette. The surface area of the trough was fixed at 0·056 m 2 and the surface tension (y) of the water used to prepare the extracts was 70 mN(m. Following equilibration at 20°C (30 min), values of y were measured by the Wilhelmy plate method using a pre-wetted clean filter paper strip 10 mm wide. From these, surface pressure (11) values were calculated as the reduction in y. All experiments were duplicated. (2) Dynamic measurements. A small amount offiour (c. 0·01 g) was sprinkled on to the surface of water in the corner of the trough furthest from the Wilhelmy plate. The progressive increase in 11 was noted for the three flours and for the glutens extracted from the flours.
Results and Discussion Concentration and particle-size effects
In Fig. lea) the proportion of flour particles producing fibrils is shown as a function of particle concentration, for flours of several types, separately suspended in aqueous mounts. The single exponential curve fitted to the complete data set demonstrates the critical dependence of fibril formation on concentration and the absence of consistent differences in that relationship among flour types. During the examinations an impression was gained that smaller particles showed a greater tendency to form fibrils and since soft flours contain a greater proportion of these, there was a possibility that variation due to endosperm texture was masking differences in tendency to form fibrils. We investigated this possibility by sieving samples and assessing fibril formation only on particles greater than 63 11m. The results (data not shown) were similar to those obtained with the complete flour. Clearly, the initial impression of an association between size and fibril-forming capacity of flour particles was erroneous. However, the relationship between concentration and fibril formation was endorsed by the results with the larger particles, reaffirming the necessity to control
A. D. EVERS ET AL.
212
FIGURE 2. Light micrographs of fibril-producing particles of (a) flour and (b) gluten.
particle/water relationships in making comparisons among flours. The validity of published semi-quantitative results in which no such control was exerted must thus be questioned. Fibrils in vital gluten
As with flours, the proportion of gluten particles forming fibrils declined as particle concentration increased [Fig. l(b)] but the decline was slower and fibrils formed at higher concentrations than in flour. No systematic differences were apparent among the three types of gluten. Fibrils formed on gluten particles were shorter and thicker than those on flour and there were fewer of them. A comparison is provided in Fig. 2. Effects of sedimentation-test reagents on fibril formation
Results of counts in solutions of propan-2-o1 and lactic acid are shown in Fig. 3, from which it is clear that both propan-2-o1 and lactic acid led not to an increase but a marked decline in fibril formation. No fibrils at all were observed at SDS concentrations above 0·14 %, in spite of the fact that the proportion of solids in all suspensions was kept below 1·25% w/v. SDS is a detergent and consequently it brings about a marked increase in II when added, even in minute quantities, to water. The Zeleny reagents also bring about an increase in II, though their effects are less marked. Bernardin and Kasarda 2 recognized that fibril formation may depend, to a degree, on surface activity and the results here accord with that view. Effect of decreasing surface pressure
If increasing the IT of the suspending medium reduces the tendency of added flour particles to form fibrils, then it might logically be expected that decreased IIwould have the opposite effect. Demonstration of this would provide good evidence for the involvement of IT effects in the fibril-forming mechanism. Few substances are capable of
WHEAT PROTEIN FIBRILS
::g; :g
0
~c
80
I
(,) I
.
!
!
80
60
40
20
213
• 100
% Lactic acid v/v ( b)
a.
.!2
";:
.c G: ~
I
• 0
20
40
60
I
80
100
% Propan-2-ol v/v
FIGURE 3. The proportion of flour particles that formed fibrils, as a function of: (a) lactic acid; and (b) propan-2-ol concentration in the suspending medium. All suspensions contained c. 1 % solids w/w. Arrows indicate approximate concentrations in a suspension for the Zeleny test.
increasing y in water; we examined two that are: NaCl and sucrose. A 5 M solution of NaCI has a y value 9 mN/m higher than pure water at 20°C 9. We examined several mounts of flour particles in a 5 M NaCl solution. No fibrils were detected. In aiM solution some fibrils were observed but the proportion was 50 % less than found in pure water mounts (Fig. 4). Sucrose also increases y of water, a 1·2 M solution having approximately the same value as aiM NaCl solution9 • A reduction in fibril-forming capacity was again noted, but whereas counts in 1M NaCI were 50% lower than those in water, 1·2 M sucrose showed only a 20 % reduction. Clearly, these results did not support the hypothesis that fibril formation results from IT effects, but fibril formation may have been inhibited by chemical effects on endosperm proteins in the case of NaCI and bulk viscosity increase in the case of sucrose. Indeed it is unlikely that addition of solutes that modify IT can be achieved without some physico-chemical change in the nature of the particles, the suspending medium or the interaction between the two. An alternative strategy for examining the relationship between IT and the tendency to form fibrils is the separate measurement of these parameters in water to which flour extracts are added. Effect offlour extract on fibril formation
The results of examinations of 1% flour suspensions in water and in a series of aqueous flour extracts are shown in Table 1.
214
A. D. EVERS ET AL.
100
% Solids w/w
FIGURE 4. The proportions of flour particles that fonned fibrils as a function of flour concentration in • aqueous and. 1 M NaCI suspensions. TABLE 1. Proportions (%) of wheat flour particles fonning fibrils when suspended in water, or flour extract prepared from suspensions of 1-10 % flour in water. Mean proportion of fibrils in 1 % suspension and S.D. of mean. Flour extract
Microscopist (l) Microscopist (2)
Water
1%
2%
5%
10%
65·5±5 52·5±5
50'5±7 42'5±9
13-8± 14 2'3±4
5'2±4 5'8±5
o o
Although agreement between microscopists was not good (there is a subjective element in counting fibrils) both microscopists registered a marked decline in the proportion of fibril-formation with increase in strength of flour extract, and no fibrils were seen in the 10 % extract. Effect offlour extract concentration on surface pressure
The relationship between IT and extract concentration was measured. Relationships for all three flours (shown in Fig. 5) were similar. Linearity with log % extract indicates an exponential relationship between IT and extract concentration.
215
WHEAT PROTEIN FIBRILS 30
"
:€z
~ ~
'" Ul Ul
~
20
CO! U
~:>
(f)
10
-I
0 10910 (% extract)
FIGURE 5. Surface pressure of aqueous flour extracts as a function of loglO flour weight extracted per lao ml water. Flours from three wheats indicated by: ~ CWRS, • cv. Mercia, .. cv. Brock.
The flour with best baking properties was associated with the highest Il, but little difference existed among all types. In this context it may be noted that Lindahl 10 was unable to observe any significant differences between surface properties of his aqueous flour extracts, even when their tertiary structures were known to be different. The exponential decline in proportion of fibril-forming particles, with percent solids (Fig. I) and the similar increase in IT with increasing extract concentration are compatible with a causal relationship. Although a mechanism is unclear, the following observations are relevant: size differences have been ruled out and no other physical differences were obvious between the particles that give rise to fibrils and those that do not. This suggests that fibril formation is less a function of individual particle properties than of the individual relationships between particles and the suspending medium. The most likely way in which such relationships may differ is in time of contact. Whatever the manner of adding particulate material to a liquid surface it is almost inevitable that contact occurs non-uniformly. Although the interval between first and last particles may be short it is possible that changes induced by a contact with the first particles can influence relationships between the liquid and the latter particles. For example, a soluble surfaceactive component of the first particles may rapidly disperse, so that surface-active extracts from later particles have to spread against an existing surface pressure, thus reducing their own n. For such a sequence of events to apply here the effects of the extracted surfactant component would have to be extremely rapid. Such speed is characteristic of surfactants, but to detect evidence of one or more being present in flour we examined changes in surface pressure as a function of time after the addition of flour particles to water in a trough. The results are shown in Fig. 6(a). Near-maximum surface pressure was achieved within one minute, in accord with the findings of Lundh et aZ,u who showed that an equilibrium value was achieved after 30 min, but was closely approached within a few seconds. Such a rate of change is compatible with extremely rapid and energetic changes in the immediate vicinity of particles striking pure water. As in the earlier surface-pressure experiment the CWRS
A. D. EVERS ET AL.
216
25.-----------------, ~
~
20
..",
T
Y ''If
V
.,. T V ' "
"
.,. ............ -.: •• • • : : : • ~ Ir..
• •
••
It
J.
(a)
15 "!'2••
•
10
~
0"'___ _ --'-_ _ --'-_ _
.1--_---'
--1
iil '" E c.
'" ~ :J
.. :".,..,...,
• t. .. ~.
20f-
en
•
,:1"-
•
...
T
...
..
•
•
... .......
(b)
•
o
•
".,..,.
'. ~
••
10':·
!'r0.
'I": o
I
10
20
I
I
30
40
50
Time (min)
FIGURE 6. Surface pressure ofa film spread from particles as a function of time. (a) Flours from three wheat types. (b) Gluten from the same flours. (Symbols as in Fig. 5.)
flour exerted the greatest n. In the present case, the U.K. wheats reflected this quality ranking also, the better baking cultivar having the higher surface activity. In a similar experiment with gluten samples prepared from the flours used above, differences in sample behaviour were smaller [Fig. 6(b)]; probably because a surface-active fraction was partially removed during gluten extraction. The ranking of limiting surface pressures was the reverse of that found with the flours [Fig. 6(b)] but no explanation for this can be offered. Compared with flours, gluten particles exerted the same ultimate n but the time to maximum was much longer (approximately 5 min for flours and 20 min for glutens). The apparently lower energy in the vicinity of gluten particles compared with flour is compatible with the shorter fibrils and fewer fibril-forming particles characteristics of gluten. Further evidence for surface factors being involved in fibril formation came from experiments involving a visual manifestation of surface pressure.
217
WHEAT PROTEIN FIBRILS
2
3
FIGURE 7. Sequence of events in demonstration of surface pressure induced by flour particles on a water drop. (I) Water drop on slide. (2) 'Monolayer' of spores on drop surface. (3) Clear area surrounding fibrillar flour particle,
Microscopical evidence for the involvement of surface activity in fibril formation
Small quantities of flour deposited on the meniscus surface of a water-drop rapidly spread over that surface. The spreading occurs with such a speed that few details of particle behaviour can be observed. Also, any changes in surface pressure remain undetected since areas of different surface tension are not optically different. It is possible to create optical contrast by arranging for a coloured or opaque monolayer to cover the water surface before contact by a surface-active agent is made. When contrast subsequently occurs, the surfactant itself tends to form a monolayer, thus displacing the opaque marker radially. The familiar toy' camphor boat' can be shown to be motivated by surface pressure if a monolayer of oxidized oil is allowed to spread over the water surface before the boat is floated. When the boat is placed, a clear camphor monolayer spreads and the boat, which remains at the boundary of water surface (indicated by oxidized oil film) and camphor monolayer, is drawn by the contracting water surface. A similar demonstration of surface activity associated with flour particles was performed. Oxidized oil may be used but clearer demarcation has been achieved with the tiny spores of puff-balls. Addition of one or a few flour particles to a monolayer of the spores on a water drop on a microscope slide rapidly induced a clear area around the particles, which invariably developed long fibrils. The sequence is illustrated in Fig. 7. Deposition of further flour particles on the surface led to a progressive reduction in the rate of spread by the zone and the later added particles appeared to show a diminishing tendency to produce fibrils although this was difficult to confirm quantitatively because particles could not be identified as having been added earlier or later. Further microscopical evidence of the involvement of IT in fibril formation was sought in the examination of the directions in which fibrils radiate from flour particles. In light microscopical examination of fibrillar particles the optical axis is almost perpendicular to most of the surface of a water drop. Consequently fibrils formed in the plane of the surface can be seen clearly. Should fibrils be produced into the solid angle between the surface plane and the optical axis they would be in different focal planes and, because of the limited depth offield of the light microscope, they would not be clearly seen. Also,
218
A. D. EVERS ET AL.
FIGURE 8. Micrographs of the same flour particle with fibrils under different conditions. (a) Suspended on the surface of a water drop - phase contrast. (b) and (c) Dry, after evaporation of water; imaged by (b) phase contrast and (c) scanning electron microscope.
they would be largely obscured by the particle itself and any fibrils in the surface plane. Because of these difficulties an impression is gained that fibrils radiate only in the surface plane, a condition compatible with the concept of their surface-dependent origin. Bernardin and Kasarda 2 were of the opinion, however, that most of the endosperm contents were dispersed under the surface of the droplet, although they too must have experienced difficulties similar to those described in confirming this visually. The method adopted in the present work, for seeking fibrils other than those in the surface plane, was first a photograph a fibrillar particle on the surface of water. The water was then allowed to evaporate so that the particle was deposited on the slide. A further photograph was then taken so that any structures which had been brought into the same focal plane as those previously recorded, could be identified by comparison of the two images. For more thorough scrutiny the dry mount was also examined by scanning electron microscope. No evidence of additional fibrils in the 'stranded' particles were seen by either technique used. Examples of the three types of image are shown in Fig. 8. It may be deduced from the above that all fibrils radiate from particles in the surface plane, and consequently this provides further evidence of the involvement of surface phenomena in fibril formation.
WHEAT PROTEIN FIBRILS
219
General discussion None of the evidence deriving from the work described here or elsewhere in the literature conclusively demonstrates that fibril formation is totally dependent upon surface phenomena. Much of it, however, is totally compatible with such a relationship, and this provides a reasonable basis for an hypothesis on the mechanism of fibril formation. The hypothesis suggested requires that particles of flour coming into contact with a pure water surface do so, not simultaneously, but sequentially. The first particles to make contact experience two changes relevant to fibril development: (a) the insoluble proteins become very rapidly hydrated, allowing them to exhibit the properties of coherence and extensibility well-known to be associated with gluten, and (b) dispersible, surface-active components rapidly become extracted. The surface pressure exerted by the flour extract as it 'expands' over the waterI air interface causes soluble components to be drawn radially outwards from the flour particle in the plane of the surface. Solid components may also be 'drawn out' in this current. Some starch granules float freely away while others, which are closely associated with the protein matrix of the endosperm, draw out threads of that protein, which, as already described, has become hydrated. The local effect of extracts from particles which make contact later is less marked because of the increased IT over the whole surface, already brought about by extracts from earlier particles. Thus, insufficient IT is generated around later particles to produce fibrils. The role of the solid component in the drawing of the protein strands is not certain, but in many cases starch granules of various sizes are seen at the extremes and along the length offibrils. Their presence may explain why the insoluble proteins are drawn as thin strands rather than a continuous sheet. Certainly the fibrils associated with vital gluten particles, which obviously contain fewer starch granules, are shorter and thicker. In passing it is also worth noting another difference between vital gluten and flour. The rate of decline, with increasing solids content, of fibrillar particle occurrence was more gradual for gluten than for flour. Water is used in the production of gluten, thus reducing the amount of surface-active component in the product. The amount of water used is limited, however, and it appears that some surfactant remains. Because of the extensibility of the hydrated insoluble proteins, fibrils can be produced by imposing stresses other than II. Movement of a cover-glass on a suspension can lead to the formation of proteinaceous projections extending in the direction of cover-glass movement. Apart from their directional bias, these fibrils appear very similar to the spontaneously generated fibrils so far considered. The behaviour of the hydrated proteins appears to be the same in both cases, although the driving forces differ. In considering the importance of fibril formation to technological properties of flours and doughs, the two characteristics of wheat proteins involved in the process should be considered separately. The hydration of insoluble protein to form a coherent, extensible network structure is already recognized as an important factor in producing a light gasretaining structure, but the study of fibrils has revealed a remarkable rate of hydration. The role of surface-active components merely in producing fibrils should not be exaggerated in the context of commercial dough-mixing. Spontaneous fibril formation has been shown to be essentially a feature of very dilute suspensions of flour, the
220
A. D. EVERS ET AL.
proportion of fibrillar particles declining steeply with increasing concentration. In doughs the solids content is about 40 %. Fibril formation has not been studied microscopically at this concentration because of excessive particle density, but it is clear from the rapid decline in fibril formation at much lower concentrations that the spontaneous fibril formation with which we have been concerned here plays little or no part. Perhaps the most interesting aspect of fibril studies, in the commercial context, is the focus they bring to the surfactant component or components present in wheat flour. This has received more attention in recent years. However, we are not yet in a position to identify the component or components responsible for effecting the observed marked change in II. Lipids are known to disperse from flours and this class of compounds has members with the amphiphilic properties characteristic of surfactants. Our exploratory experiments with thin-layer chromatography using chloroform methanol extracts from the aqueous extracts of flours showed traces of lipids. None of these was of a type which might be expected to exhibit surfactant properties, nor did a re-dispersal of the concentrate significantly reduce the fibril-forming capacity of flour particles. Clearly, a more sophisticated approach whereby the surface film is concentrated and analysed is required. Other classes of compounds such as soluble proteins should be considered.
Conclusions Improved methods for preparing samples for microscopical counting have enabled concentration effects to be established. It has been shown that the formation of spontaneously generated protein fibrils, when flour particles contact a water/air interface, occurs most prolifically at low concentration. It is postulated that the decline in fibril formation with increasing concentration is related to the development of a II maximum by an extractable endosperm surfactant, the latter phenomenon producing the driving force for fibril formation. No evidence has been found to support the suggestion that entanglement of fibrils on flour particles is responsible for the floc formation which occurs during sedimentation tests of flour quality. Indeed few particles suspended in the liquids used in those tests produced fibrils. The ability of hydrated insoluble wheat endosperm proteins to be drawn into long narrow strands is an important characteristic in relation both to fibril formation and dough mixing. However, the spontaneous formation of fibrils is unlikely to feature significantly in dough mixing because of the low proportion of particles forming fibrils at the concentrations used in doughs. Note added in revision
We are grateful to the referee who drew our attention to a very recent publication on this subject. The conclusions published here are in close accord with those of Amend and Belitz12 although they were arrived at entirely independently and, in some cases, by different approaches. We are grateful to Heather Riley and Simon Taylor for careful technical assistance. The work was supported by the Ministry of Agriculture, Fisheries and Food and this report, based on MAFF report no. M17, 1986 is published with permission of that Authority.
WHEAT PROTEIN FIBRILS
221
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Seckinger, H. L. and Wolf, M. 1. Cereal Chern. 47 (1970) 236--243. Bernardin, J. E. and Kasarda, D. D. Cereal Chern. 50 (1973) 529-536. Muller, H. G. and Bernardin, J. E. Cereal Chern. 52 (1975) 122-124. Frazier, P. PhD thesis, Leeds University (1971). AACC. Approved methods of the AACC. Test 56.60. American Association of Cereal Chemists, St Paul, MN (1962). Adeyemi, 1. A. and Muller, H. G. in 'Food Texture and Rheology' (p. Sherman, ed.), Academic Press, London (1977) pp 325-341. Adeyemi, 1. A. and Muller, H. G. J. Sci. Fd. Agric. 26 (1975) 544-545. Axford, D. W. E. A., McDermott, E. E. and Redman, D. G. Cereal Chern. 56 (1979) 582-584. Weast, R. C. (ed.). 'Handbook of Chemistry and Physics', 68th edn. CRC Press, Boca Raton, FL (1987). Lindahl, L. 1. J. Dispn. Sci. Technol. 8 (1987) 309-319. Lundh, G., Eliasson, A. C. and Larsson, K. J. Cereal Sci. 7 (1988) 1-9. Amend, T. and Belitz, H. D. Z. Lebensrn. Unters. Forsch. 189 (1989) 103-109.