J Sci Food Agric 1998, 78, 423È428

Wheat Flour Proteins: Isolation and Functionalityof Gliadin and EnrichedHMW-GluteninFractions

Brahim Mimouni,¤ Jean Michel Robin, Jean-Louis Azanza and Jacques Raymond*

ISTAB, Laboratoire de Biochimie et Technologie des Aliments, Universite� Bordeaux I, Avenue desFaculte� s, 33 405 Talence cedex, France

(Received 8 May 1997 ; revised version received 11 December 1997 ; accepted 11 March 1998)

Abstract : The amount of glutenin subunits of high molecular weight (HMW-glutenins) appears to be closely related to breadmaking. The relationship of theirspeciÐc functional properties and gluten quality has not been demonstrated. Thedifficulty in isolating non-denatured HMW-glutenins explains largely the lack ofinformation. Investigations have been conducted to isolate HMW-gluteninswithout using denaturing agents such as SDS or urea. Using wholemeal treatedin mild reducing conditions with the procedure uses solubilisation inNa2SO3 ,hot aqueous ethanol with the subsequent speciÐc precipitation of HMW-glutenins at low temperature. Proteins present in the various extracts wereanlaysed by SDS-PAGE and reverse-phase HPLC. The HMW-glutenins wereobtained by precipitation at low temperature from ethanol extract (puriÐcationfactor D12), whereas the supernatant mainly contained gliadins and LMW-glutenins. After addition of these fractions to meal the modiÐcations of the rheol-ogical properties of the resulting dough were investigated. A model explainingthe participation of the di†erent fractions to the structure and the solubility ofgluten is suggested. Society of Chemical Industry.( 1998

J Sci Food Agric 78, 423È428 (1998)

Key words : gluten ; HMW-glutenins ; gliadins ; wheat Ñour proteins ; functionality

ABBREVIATIONS

ACN AcetonitrileEtOH EthanolG Swelling of the doughHMW High molecular weightL Extensibility of the doughLMW Low molecular weight2-ME b-mercaptoethanolP Tenacity of the dough

* To whom correspondence should be addressed.¤ Present address : Faculte� des Sciences et Techniques, De� par-tement de Biologie, Avenue A El Khattabi, B P 618, Marra-kech, Morocco.

RP-HPLC Reverse-phase high performance liquidchromatography

SDS-PAGE Sodium dodecyl sulphate polyacrylamidegel electrophoresis

TFA TriÑuoroacetic acidW Strength of the dough

INTRODUCTION

Wheat proteins are the best known proteins of cerealgrains, and the gluten complex plays the main role indetermining the technological quality of wheat

4231998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

424 B Mimouni et al

(MacRitchie 1992, 1994 ; Shewry 1995 ; Shewry et al1994, 1995 ; Shewry and Tatham 1997). Based on di†er-ent crude analyses (Dacosta 1986), gluten contains 77%proteins, 16% carbohydrates, 6% lipids and 1% ashes(w/w, dry basis). Gluten proteins may be dividedaccording to their solubility (Osborne 1924) or theirmolecular characteristics (Kreis and Shewry 1989). Glia-dins constitute a very heterogeneous group with relativemolecular masses between 11 000 and 80 000, and(Mr)four fractions (a, b, c, u) can be distinguished by PAGE.(Woychik et al 1961 ; Autran 1990). Glutenins are char-acterised by their non-solubility in the usual solvents,and di†erent models have been proposed to explaintheir structure (Kasarda et al 1976 ; Ewart 1979) andaggregation states (Bietz and Wall 1972). 2-ME-SDS-PAGE electrophoresis permits the separation of twogroups of glutenins : LMW-glutenins (low subunits)Mrand HMW-glutenins (high subunits). The LMW-Mrglutenins are partially soluble in 70% ethanol and areanalogous with some gliadins (Payne and Cor-high-MrÐeld 1979), while about 20 HMW-glutenin subunitshave been identiÐed and classiÐed according to theirelectrophoretic mobilities (Payne et al 1981). HMW-glutenins have a key role in the functionality of gluten.As strong denaturing conditions are required to isolatefully gluten components, mild reducing conditions wereused to obtain enriched HMW-glutenins and gliadinfractions. The extraction process allows for preparationfrom large quantities of meal, and enough protein isobtained for Chopin Alveograph tests. Some hypothesesare advanced concerning the solubility properties of themain gluten components and their relationships to eachother.

MATERIALS AND METHODS

Gluten (75% w/w protein, dry basis) was obtained afterlixiviation from high-strength Ñour (W \ 260). Crudegluten was then lyophilised and used for the prep-aration of enriched fractions. The functional propertiesof the samples were identical to those of commercial

gluten produced by the Martin process (not shown) andprovided by Amylum Aquitaine (Bordeaux, France).

A low-strength Ñour (W \ 140) was tested as refer-ence in the Chopin Alveograph tests. SDS-PAGE wereperformed according to Laemmli (1970), in the presenceof 2-ME on slab gels (160/140/7É5 mm) using a linearacrylamide gradient (10È20% w/v). Flour quality testswere determined with the Chopin MA 87 Alveograph toobtain the characteristics of strength (W ), tenacity (P),swelling (G) and extensibility (L ) of the dough. Datacorrespond to the mean of Ðve Alveogram tests.RP-HPLC was performed on a Synchropak RP-P, C18column (250] 4É6 mm, porosity on reduced and300 Ó)alkylated fractions. For alkylation, samples were incu-bated for 15 min in the dark in SDS sample bu†er(50 mM Tris/HCl, pH 6É8, 2% (mass/vol) SDS) contain-ing 100 mM iodacetamide. Protein content was deter-mined by the micro Kjeldahl procedure (Pearson 1973)and moisture by heating 90 min at 130¡C (Norme ISOVO 3 701 AFNOR). To calculate the amount of proteinpresent, the nitrogen content is multiplied by a factor of5É70. The extraction procedure is composed of twosteps.

(1) A sulphitolysis in the presence of 25 mM Na2SO3(repeated in the same conditions).

(2) A solubilisation in EtOH 70% followed by acold precipitation of the HMW-glutenins. Aftercentrifugation, the remaining supernatant isdiluted with 1É5% NaCl, according to Popineau(1985), to yield the gliadin-LMW-gluteninenriched fraction. The EtOH step is also repeat-ed, with only one minor change in the tem-perature of solubilisation.

RESULTS AND DISCUSSION

Process for the preparation of enriched fractions

All the procedures of the puriÐcation process with thecorresponding electrophoretic patterns are given in Figs

TABLE 1Protein (on dried basis) and moisture in the experimental material

L ow-strength High-strength Gluten Enriched EnrichedÑour Ñour gliadin-L MW -glutenin HMW -glutenin

fraction fraction

Moisture 138 118 53 27 54g kg~1Protein 93 110 652 760 721g kg~1

Functionality of gliadin and HMW -glutenin enriched fractions 425

Fig 1. Diagram of the di†erent steps of the extraction process.

1 and 2. The staring material is 400 g of Ñour. Moistureand protein determination are reported in Table 1. ThepuriÐcation factor determined by RP-HPLC for theHMW-glutenin enriched fraction is 12 when comparedto gluten, and the recovery is about 10% of the totalproteins of the Ñour. The chromatographic proÐles ofthe fractions obtained after the second precipitation (1 hat 0¡C) are shown in Fig. 3A. The ratio of HMW-glutenins to non-HMW-glutenins proteins increases bya factor 3 and 4, respectively, as a result of the Ðrst andsecond precipitations (Fig 3B).

Functionality of the enriched fractions

Low-W wheat Ñour supplemented with 0É5È2% of thedi†erent enriched fractions (alone or mixed) was tested.Low-W wheat Ñour supplemented with native glutenwas used as a standard. Results are shown in Fig 4(a)for gluten addition and Fig 4(b) for addition of gliadin-LMW-glutenin enriched fractions. Figure 5 shows theresults of supplementation with HMW-gluteninenriched fractions (a) and with a 2% addition of amixture of the two enriched fractions (b). These resultsshow that the addition of the enriched fractions give an

Fig 2. 2-ME-SDS-PAGE analyses of the obtained fractions.Numbers correspond to the steps shown in Fig. 1. Wholewheat proteins pattern and reference (kDa) are indicated.Mr

increase in tenacity (measured by the overpressure P,mm) and a decrease in swelling index G (ml) andstrength (measured by the deformation energy W ,10~4] J). The decrease in W , despite the increase inprotein concentration, indicates a loss in the viscoelasticproperties.

The results also show the mixture of the two enrichedfractions does not produce a recovery of any viabilitysuggesting that there are no more functional inter-actions between glutenins and gliadins. The viscoelasticcharacter is lost, despite a great number of renaturationtests being conducted in di†erent experimental condi-tions (results not shown). BrieÑy, the HMW-gluteninand gliadin-LMW-glutenin enriched fractions weremixed in various proportions in acidic, alkaline orEtOH solutions, then neutralised, dialysed, lyophilisedand tested for functionality in the supplementation tests.As gluten functionality is never recovered, it is con-cluded that sulphitolysis and/or ethanol fractionationsteps are responsible for gluten inactivation.

To obtain information about which stage of theextraction process may have been responsible for theloss in the viscoelastic properties, the gluten recovered

426 B Mimouni et al

Fig 3. (A) Chromatograms of the HMW-glutenin pellet (left)and of the remaining supernatant (right) after 1 h of precipi-tation at 0¡C (second precipitation) ; column synchropackRP-P-C18 (250] 4É6 mm) ; 1 mg of reduced and alkylatedproteins were dissolved in 200 ll of 30% ACN, 0É1% TFA intri-distilled water before injection ; Ñow rate 1 ml min~1 ;elution, ACN gradient (30È70%) ; (1) HMW-glutenin group,(2) LMW-glutenin-gliadin group. (B) QuantiÐcation of thechromatographic proÐle showing the ratio of HMW-gluteninsto non-HMW-glutenin proteins in the pellet (hatched bar) and

the supernatant (unÐlled bar).

after sulphitolysis (gluten S) was tested. When 0É5% ofgluten S is added to the Ñour, a stabilisation of theparameter G and an increase in P and W are observed.This gluten still possesses viscoelastic properties whencompared to the reference (Fig 5c), indicating that theEtOH fractionation step was mainly responsible forgluten inactivation.

The enriched fractions present a high aggregationstate, as shown by the very low solubility of these frac-tions in SDS (Fig 6A, lane 2 and 3 right). Such resultsare very similar to those obtained by thermal denatur-ation (Dacosta 1986). This result shows that, afterEtOH treatment and precipitation, the enriched frac-tions are aggregated, which may explain their loss in

Fig 4. InÑuence of gluten addition (a) or gliadin-LMW-glu-tenin addition (b) on the alveogram proÐles and on the visco-

elastic parameters. Percentage addition are indicated.

Fig 5. InÑuence of HMW-glutenin addition at the indicatedpercentage (a) and of a 2% mixture of the enriched fractions(b) on the Alveogram proÐles and on the viscoelastic param-eters Alveogram of sulphitolysed gluten (S gluten) compared

with a low-W reference dough (c).

Functionality of gliadin and HMW -glutenin enriched fractions 427

Fig 6. (A) 2-ME-SDS-PAGE analyses of aggregated proteinsremaining in the pellet (left) or solubilised in the supernatant(right) after SDS treatment ; (1) high-strength Ñour, (2) gliadin-LMW-glutenin enriched fraction, (3) HMW-glutenin enrichedfraction, (4) gluten, (5) S gluten. (B) 2-ME-SDS-PAGEanalyses of two successive NaCl extractions (lanes 1 and 2) ;

extract obtained after the two NaCl extractions (laneNa2SO33) ; standard proteins (E), whole gluten (GT) and (kDa) areMrindicated.

vitality. To understand the role of sulphitolysis, twosuccessive salt extractions were carried out on wheatÑour to remove the albumins and globulins (Fig 6B,lane 1 and 2 left). Then, sulphitolysis was performedand, after centrifugation, proteins were analysed by twodimensional SDS-PAGE. As shown in Fig 6B (lane 3,right), sulphitolysis is responsible for the solubilisationof u gliadins and some LMW-glutenins. Also observedare traces of other gliadins and a strong albumindoublet which was not solubilised after the two NaCltreatments. u-Gliadins and LMW-glutelins seem tohave a key role in the structure and properties of thewhole gluten, as without sulphitolysis, only gliadins areextracted with EtOH and, after sulphitolysis, gliadinsand HMW-glutenins are extracted with EtOH.

The extracts adversely a†ect the functionality of thebase Ñour, and this is attributable to the separated

gluten components, although the presence of chemicalresidues resulting from the process cannot be ruled out.

CONCLUSIONS

Despite negative results in term of functionality, thepreparation of the enriched fractions and their solubilitybehaviour may give interesting information aboutgluten structure and the functional relationship betweenits main components. As a result of the Ðndings, amodel explaining the behaviour of the gluten proteinsaccording to their extraction by di†erent solvents is sug-gested. The model is based on variations in thehydrophilic/hydrophobic balance of the gluten mol-ecules, in which four main types of molecules are con-sidered. Some LMW-glutenins and u-gliadins would bewater-soluble proteins but remain bound to the glutenby disulphide bridges partly with a-, b- and c-gliadinsand partly with HMW-glutenins. These intermolecularlinks are accessible as sulphitolysis easily allows theirsolubilisation in water. In contrast, they are not solu-bilised in salt solutions in the absence of sulphitolysis. Agreat part of the remaining gliadins (a, b, c) would bestrongly hydrophobic and are non-covalently bound inthe whole gluten as they are extracted in EtOH. A frac-tion of LMW-glutenins and some u-gliadins are alsoextracted, although they are hydrophilic, because theyare linked by disulphide bonds to the gliadin fraction.

In the model, HMW-glutenins would present amphi-philic properties with double potentiality to link thehydrophobic gliadin fraction by hydrophobic inter-actions and the water-soluble fraction of LMW-glutenins and u gliadins by disulphide bonds. So, inaqueous solutions, the behaviour of gluten is explainedby the favourable hydrophobic balance of its com-ponents, and no proteins are soluble. In EtOH, only thehydrophobic fraction is solubilised, as low-energy bondswithin hydrophobic interactions are disrupted when thedielectric constant of the media decreases. The remain-ing amphiphilic HMW-glutenins, having a positivehydrophilic balance, remain insoluble in EtOH.

Sulphitolysis is responsible for the solubilisation of u-gliadins and some LMW-glutenins and, as a conse-quence, the balance of the remaining gluten (S gluten)would become hydrophobic, explaining the solu-bilisation in hot EtOH of the HMW-glutenins in addi-tion to the a-, b- and c-gliadins. Further precipitation ofHMW-glutenins at low temperature may result fromstrong hydrogen bonds which are stabilised by adecrease in temperature.

The functional properties of S gluten appear to beclose to those of the native gluten. This indicates thatthe proteins separated after mild sulphitolysis do notbind the basic structure of the gluten, particularlyinvolving the HMW-glutenin/gliadin interaction. Theproposed model, summarised in Fig 7, is also in accord-ance with this observation. The solubilisation of gluten

428 B Mimouni et al

Fig 7. Proposed model for the associationÈdissociation processes occurring in gluten according to the solubilisation media : a-,(L)b- and c-gliadins ; u-gliadin ; HMW-glutenins ; LMW-glutenins.(…) (K) (==)

in EtOH and the resulting loss in the viscoelasticproperties would be due to irreversible biochemical andstructural changes. The (HMW-glutenins/gliadin-LMW-glutenins) structure of gluten would be quickly

xmodiÐed, leading to highly aggregated and polymerised(HMW-glutenins/HMW-glutenins)

m] (gliadins-LMW-

structures. Numerous SÈS interchanges mustglutenins)n

be involved, in addition to other low energetic bonds, toexplain the observed irreversible denaturation.

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