Effect of Puroindolines on the Breadmaking Properties of Wheat Flour Laurence Dubreil,1,2 Sabine Méliande,1 Hubert Chiron,1 Jean-Pierre Compoint,1 Laurence Quillien,1 Gérard Branlard,3 and Didier Marion1,4
ABSTRACT
Cereal Chem. 75(2):222–229
The role of lipid-binding proteins from wheat seed (puroindolines) on the breadmaking properties of wheat flour was investigated by determining the relationship between breadmaking quality and puroindoline content in samples of 32 wheat cultivars. An inverse relationship was mainly explained by the link between hardness and puroindoline contents. This link is in agreement with previous results which have shown a close structural identity between basic friabilins and puroindolines. Next, the effect of puroindolines in breadmaking was investigated by performing reconstitution experiments with two puroindoline-free hard cultivars
of opposite quality (Florence Aurore and Ecrin) as indicated in the screened wheat sample. Addition of 0.1% puroindolines to these flours drastically modified both the rheological properties of doughs and the structure of the bread crumb. Puroindolines are essential to the foaming properties of dough liquor, and a close relationship was found between the fine grain crumb provided by reconstituted flours with puroindolines and the fine structure of corresponding dough liquor foams. The effect of puroindolines on bread volume was mainly related to the rheological properties of wheat doughs.
It is now well accepted that wheat lipids play an essential role in the breadmaking process. Especially, lipid reconstitution of defatted flours has shown that wheat polar lipids (phospholipids and glycolipids) are bread volume improvers, while nonpolar lipids (triglycerides and free fatty acids) are detrimental to bread volume (MacRitchie and Gras 1973). These effects are the result of a complex mechanism involving the surface properties of polar lipids and proteins dispersed in the aqueous phase of dough, the competitive adsorption between lipids and proteins which is essential for the formation and stability of dough foam (Gan et al 1995; Marion and Clark, in press). This foam controls gas retention and expansion during mixing, proving, and the early stages of baking (Hoseney 1984; Bloksma 1990a,b). Recently, it has been suggested that puroindolines, the lipid-binding proteins from wheat flour, could contribute to the formation and stability of dough foams (Dubreil et al 1997). Puroindolines are basic proteins that contain five disulfide bridges and a unique tryptophan-rich domain that is involved in lipid recognition (Blochet et al 1993, Dubreil et al 1997). Two isoforms have been isolated from wheat flour: puroindoline-a (PIN-a, the major isoform) and puroindoline-b (PIN-b, the minor isoform) (Blochet et al 1991, Gautier et al 1994). PIN-a and PIN-b exhibit ≈60% homology in their sequence, but the tryptophan-rich domain of PIN-a (Trp-Arg-Trp-Trp-Lys-TrpTrp-Lys) is truncated in PIN-b (Trp-Pro-Thr-Trp-Trp-Lys). These proteins are capable of preventing the destabilization of protein foams by lipids (Clark et al 1994, Husband et al 1995). In some cases, the puroindoline-lipid complexes exhibit higher surface properties (Wilde et al 1993, Dubreil et al 1997). Furthermore, it has been shown that puroindolines are identical to basic friabilins (Morris et al 1994, Rahman et al 1994), the proteins found at the surface of starch granules that are biochemical markers for grain softness and hardness (Greenwell and Schofield 1986, Jolly et al 1993, Bettge et al 1996). The binding of friabilins to the starch granule surface is mediated by lipids (Greenblatt et al 1994). Even if these proteins do not quantitatively partition onto starch (Jolly et al 1993), such protein-lipidstarch interactions could contribute to dough cohesiveness.
The present work was undertaken to evaluate the role of puroindolines in breadmaking. To complete this objective, we investigated the variability of PIN-a content among samples of 32 wheat cultivars with different breadmaking qualities. Next, the effects of puroindolines on bread crumb structure and bread volume were studied by reconstitution experiments with puroindoline-free wheat cultivars.
1 INRA
Laboratoire de Biochimie et Technologie des Protéines BP 71627 44316 Nantes cedex 03, France. 2 Groupe Danone, Centre Jean Thèves, BP 16, 91207 Athis-Mons cedex and TEPRAL, 67037 Strasbourg cedex 02, France. 3 INRA Station de Génétique et d’Amélioration des Plantes, Domaine de Crouelle 63039 Clermont Ferrand cedex 02, France. 4 Corresponding author. E-mail:
[email protected] Publication no. C-1998-0211-03R. © 1998 by the American Association of Cereal Chemists, Inc.
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MATERIALS AND METHODS Plant Material Variability of PIN-a content was performed on samples of 32 individual Triticum aestivum cultivars with different breadmaking qualities. Wheat seeds were harvested in the experimental station of INRA (Clermont-Ferrand, France). The breadmaking value of the different cultivars was obtained from the French National trials, where any new released cultivar is experimented on for two years in four main cereal areas. The quality classes A, B1, B2, C1, C2, and D result from baking performances in a standard French baking test (CNERNA NE V03-716). They were used to rank any cultivar from extra strong (class A), to very good (B1), or very bad (D) breadmaking levels. Grain hardness was assessed using near-infrared reflectance (NIR) according to Approved Methods (AACC 1995). Five classes of hardness (soft, medium soft, medium hard, hard, and very hard) were considered in this work. This classification based on the hardness index obtained by NIR: soft (16– 30), medium soft (31–45), medium hard (46–65), hard (66–85), and very hard (>85). Breadmaking was performed with three puroindoline-free flours with good (Florence Aurore [FA]), medium (mixture of FA and Ecrin [EC] cultivars [1:1 w/w]), and poor (EC) breadmaking qualities. Reconstituted flours were obtained by mixing 200 g of wheat flour with freeze-dried puroindolines or egg white proteins (Ovomousse M251, Epi Bretagne, France) in a Chopin mixer (Tripette et Renaud, France). Purification of Puroindolines For immunochemical studies, PIN-a and PIN-b were purified as previously described (Blochet et al 1993, Dubreil et al 1997). The same batch of purified PIN-a was used for immunization of rabbits and for immunoassays. For baking experiments, a crude puroindoline fraction was used. This fraction was obtained from large-scale cation exchange chromatography and contained >95% puroindolines with ≈80% PIN-a and 20% PIN-b as determined by reversed-phase HPLC on a C4 Nucleosil column (Blochet et al 1991).
Production of Anti-PIN-a Antibodies Three rabbits were intracutaneously injected with 500 µg of PIN-a emulsified with Freund adjuvant (complete Freund adjuvant for the first injection and incomplete adjuvant for boosters). Sera were collected after the fourth injection (two weeks). Sera were desalted by gel-filtration on a column packed with Trysacryl GF05, and total immunoglobulins were purified by ion-exchange chromatography on a column packed with DEAE Trysacryl M. The specificity of antibodies was tested by Western blotting on a total protein SDS extract, the detergent-rich phase, and purified PIN-a and PIN-b. Polyclonal antibodies against PIN-a crossreacted strongly with PIN-a and weakly with PIN-b. The protein dilutions used in enzyme-linked immunosorbent assay (ELISA) (1:1,500) allowed the antibodies to cross-react only with PIN-a. Immunochemical Determination of PIN-a Content Sample preparation. To determine the puroindoline contents in different cultivars, 1 g of seed was ground in a mortar, and puroindolines were extracted by stirring the wholemeal flour at 4°C for 1 hr with 30 mL of Tris-HCl 50 mM, pH 7.8, 100 mM KCl, and 5 mM ethylenediaminetetraacetic acid (EDTA) (buffer A). After centrifugation at 10,000 × g, the supernatant was removed, and the pellet was extracted at 4°C for 1 hr with buffer A, containing 2% (v/v) Triton X-114. After centrifugation at 10,000 × g and 4°C for 30 min, phase partitioning was performed at 30°C as previously described (Blochet et al 1991). The lower detergent-rich phase was recovered, and its final volume was adjusted to 30 mL with fresh buffer A to constitute the stock puroindoline extract for ELISA.
ELISA procedure. ELISA was performed by immobilizing puroindolines or TX-114 extracts on 96-well microtiter plates. PIN-a was detected indirectly by a primary polyclonal anti-PIN-a antibody and a second anti-rabbit IgG from goat conjugated with horseradish peroxidase (1:3,000). Each well was coated with 100 µL of PIN-a standard or assay solution in the 5–160 ng/mL range. The plate was left overnight at 4°C. The plate was washed three times with a phosphate saline buffer (Na2HPO4, 8 mM; KH2PO4, 1.5 mM; KCl, 2.7 mM; NaCl, 140 mM) containing 0.05% (v/v) Tween 20 (v/v) (Tween-PBS). PBS (250 µL) containing 1% (v/w) bovine serum albumin was added and incubated for 1 hr at 37°C. The plate was washed three times with Tween-PBS, and 100 µL of purified anti-PIN-a immunoglobulins (dilution 1:2,000) was added. After incubation for 1 hr at 37°C and washing three times with Tween-PBS, the antibodies linked to PIN-a were revealed by goat anti-rabbit IgG conjugated with horseradish peroxidase (dilution 1:3,000 in PBS). After 1 hr at 37°C, wells were washed three times, and 100 µL of an o-phenylenediamine (OPD) and H2O2 solution (20 mg of OPD in 50 mL of 50 mM citrate buffer, pH 5.5, and 20 mL of H2O2) was added. The plate was incubated for 30 min, and the enzyme activity was stopped by addition of 25 µL of 2N H2SO4. Absorbance measurements were then performed at 490 and 630 nm. Preparation of Dough Liquor Wheat flour (50 g) and distilled water (32 mL) were mixed in a Brabender Farinograph for 5 min at 30°C. Doughs were ultracentrifuged at 100,000 × g for 75 min at 30°C in a Beckman ultra-
TABLE I Puroindoline-aa Content in 32 Wheat Cultivars as Determined by Enzyme-Linked Immunosorbent Assay (ELISA) Hardnessb Cultivar AB1 Quality (good) Aubaine Darius Florence A. Prinqual Camp Remy Capitole Chopin Renan Monopol Glenlea Goya Courtot Damier B2C1 Quality (medium) Goelent Talent Fidel Creneau Rafale Soissons Thesee C2D Quality (poor) H. de Bersee Promentin Vilmorin Apollo Arminda Champlain Ecrin Etoile de C. Andain Duck Fortress Vicking a b c
PIN-a
Sample No.
NIR
Class
%/Dry Basis × 103
%/Total Protein × 102
µg/Kernel
1 2 3 4 5 6 7 8 9 10 11 12 13
69 76 87 75 76 21 92 76 78 85 ndc 57 30
H H VH H H S VH H H H nd MH S
55 ± 0.4 59 ± 0.4 0 0 71 ± 1.7 50 ± 4.9 59 ± 4.6 53 ± 2.1 59 ± 2.7 0 59 ±1.6 71 ±1.8 74 ±1.4
44 ± 0.3 49 ± 3.6 0 0 54 ± 1.3 41 ± 4 49 ± 4 43 ± 1.7 49 ± 0.2 0 42 ± 1.1 62 ± 1.6 61 ± 1.2
27.76 ± 1.39 25.41 ± 1.84 0 0 27.62 ± 1.44 25.22 ± 5.07 23.14 ± 4.40 24.17 ± 5.93 26.02 ± 1.06 0 22.15 ± 4.69 29.06 ± 0.75 37.73 ± 3.52
14 15 16 17 18 19 20
82 34 33 74 22 60 62
H MS MS H S MH MH
63 ± 2.6 59 ± 1.8 77 ± 4.4 55 ± 4.3 60 ± 6.6 66 ± 1.7 70 ± 1.1
54 ± 2.2 46 ± 1.4 65 ± 3.7 45 ± 3.6 51 ± 5.6 61 ± 1.6 69.7 ± 10.3
26.14 ± 2.35 22.25 ± 1.28 30.39 ± 3.59 18.22 ± 1.88 20.95 ± 0.83 29.47 ± 5.90 30.78 ± 3.30
21 22 23 24 25 26 27 28 29 30 31 32
25 26 35 26 34 15 80 32 60 nd nd nd
S S MS S MS S H MS MH nd nd nd
78 ± 0.46 72 ± 1 69 ± 9.58 84 ± 12.4 83 ± 13.3 100 ± 1 0 107 ± 1.45 104 ± 0.72 88 ± 5.51 85 ± 6.15 85 ± 0.6
47 ± 0.3 63 ± 0.9 55.6 ± 7.8 67 ± 9.9 73 ± 12 85 ± 0.8 0 76 ± 1 91.5 ± 0.6 76.2 ± 4.8 67 ± 4.8 70 ± 0.0
28.81 ± 0.11 27.67 ± 0.65 29.90 ± 3.66 41.14 ± 5.56 35.47 ± 5.39 33.36 ± 0.47 0 49.14 ± 3.52 42.76 ± 2.12 44.34 ± 0.91 32.10 ± 4.07 30.72 ± 0.49
PIN-a, the major isoform isolated from wheat flour. Classed in five groups of grain hardness: soft (S), medium soft (MS), hard (H), and very hard (VH). NIR = near-infrared reflectance. Not determined. Vol. 75, No. 2, 1998
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TABLE II Relationship Between Properties of Breadmaking Quality and Hardness Classes to Puroindoline-a a Content PIN-a Sample No.
Propertiesb
Dry Basis
Protein
Kernel
C2D B2C1 AB1 MS MH S H VH
0.0774a c
0.6206a 0.5601ab 0.3782b 0.6320ab 0.7090a 0.5937ab 0.3399bc 0.2470c
32.03a 25.45ab 20.51b 33.43a 33.01a 30.69a 17.53ab 11.57b
9 7 12 5 4 7 10 2 a b c
0.0643ab 0.0459b 0.0790a 0.0777a 0.0740a 0.0415ab 0.0295b
PIN-a, the major isoform isolated from wheat flour. C2D, B2C1, and AB1 indicate poor, medium, and good breadmaking qualities, respectively. Grain hardness groups: soft (S), medium soft (MS), hard (H), and very hard (VH). Values followed by the same letter are not significantly different ( P = 0.05). TABLE III Linear Correlation Between Puroindoline-a Content and Grain Hardness Determined in 26 Wheat Cultivars PIN-aa
Grain hardness (n = 26) PIN-a/dry basis (n = 32) PIN-a/protein (n = 32) a
Dry Basis
Protein
Kernel
–0.573**
–0.557** 0.975**
–0.555** 0.955*** 0.938***
PIN-a, the major isoform isolated from wheat flour. Statistically significant at P = 0.01 (**) and P = 0.001 (***).
TABLE IV Effect of Puroindolines on Alveograph Indicesa of Selected Flours Flourb
W
Puroindoline-free FA 342 FAEC 270 EC 224 0.1% Puroindolines added FA 478 FAEC 250 EC 206
L
P
P/L
60.4 79.3 55.75
147 119.9 115.4
2.51 1.5 2.29
77.59 55.48 36.9
172 123.2 134.4
2.21 2.5 3.64
a
centrifuge equipped with a SW rotor. After centrifugation, the upper liquid phase corresponding to dough liquor was recovered and freeze-dried. Foaming Measurements Foaming properties of dough liquor were determined using conductivity measurements as previously described (Loisel et al 1993, Dubreil et al 1997). The conductivity was related to the volume of liquid sustained in the foam lamellae (Guillerme et al 1993). During these experiments, the dough liquor concentration was constant and equal to 8 mg/mL of freeze-dried material. The concentration used for puroindoline was 0.2 mg/mL. Egg white proteins were used at 2 mg/mL. All solutions were prepared in 10 mM sodium phosphate buffer at pH 7. Alveograph Measurements The flours from different cultivars (70% extraction rate) were obtained using a Chopin mill CD1. The rheological properties of the dough were measured using the Chopin alveograph test according to Approved Methods (AACC 1995). To limit puroindoline consumption, the 50-g micromixer was used instead of the 250-g standard mixer, giving only two replicates. Under these conditions, the standard deviations of alveograph indices were in the range of 5–8%. The dough parameters (strength [W], tenacity [P], extensibility [L], and tenacity-to-extensibility ratio [P/L]) were computed using the RCV4 Chopin apparatus. Baking Test Baking experiments were performed according to a modification of the French baking method (Godon and Sarrazin 1973). Wheat flour (200 g) was mixed in a Chopin kneading machine with 5 g of baker’s compressed yeast dispersed in 126 mL of deionized water. Total mixing time was 13 min (2 min at low speed [60 rpm] and 11 min at high speed [120 rpm]). After 6 min at high speed, 4.4 g of sodium chloride was added. After mixing, the dough was rounded into a ball and incubated for 45 min in a fermenting room at 28°C and 85% rh. Fermented dough was divided into three portions of 85 g each, which were mechanically molded. After a second fermentation for 90 min, loaves were 224
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W = dough strength (×10–4 J); L = extensibility (mm); P = tenacity (mm); P/L = tenacity-to-extensibility ratio. b FA = Florence Aurore; EC = Ecrin; FAEC = mixture of FA and EC.
baked at 270°C for 20 min in a Chopin oven. Bread volume was measured by rapeseed displacement. Good reproducibility of this French breadmaking test was obtained when temperature of the dough at the end of mixing was in the range 22–25°C (standard deviation for bread volumes was in the range 4–9%). Statistical Method Linear correlations, variance analysis, and other statistical tests were computed using SAS statistical software (SAS Institute, Cary, NC). RESULTS PIN-a Content, Breadmaking Quality, and Grain Hardness This immunochemical study was performed only on PIN-a because this was the main and the most surface-active puroindoline isoform with the highest affinities for wheat polar lipids (Husband et al 1995, Dubreil et al 1997). Also, we did not succeed in producing highly specific polyclonal antibodies against the minor PIN-b isoform (results not shown). Determination of PIN-a contents in 32 cultivars revealed the existence of four cultivars free of PIN-a: Florence Aurore (FA), Prinqual, Ecrin (EC), and Glenlea (Table I). It is noteworthy that PIN-a was lacking in good (Florence Aurore) as well as in poor (Ecrin) quality wheats. For the other cultivars, PIN-a concentration was in the range of 0.05–0.1% of dry material (Table I). Multiple range tests were produced by considering the three groups of cultivars according to a quality scale: group I (A to B1), group II (B2 to C1), and group III (C2 to D). Significant differences in the PIN-a content were discovered between group I and group III (Table II). Variance analysis discriminated hard and very hard wheats from soft, medium soft, and medium hard classes (Table II). It was noteworthy that the three lowest PIN-a contents were obtained for the very hard class, and that all cultivars free of PIN-a were hard wheats. General linear model was used to test the effect of the hardness classes on the variations in PIN-a contents.
Fig. 2. Bread baked from Florence Aurore (FA) flour with 0.1 and 0.2% egg white proteins (egg) and 0.1 and 0.2% puroindoline (puro).
Fig. 1. Bread baked from Florence Aurore flour without puroindoline (FA0) and with 0.05, 0.1, and 0.2% (w/w) puroindoline (puro) added.
Grain hardness was negatively related to PIN-a content as expressed on a dry basis, on a protein content basis, or per kernel. These three measurements were all significantly related (Table III). Effect of Puroindolines on Rheological Properties Both rheological and baking experiments were performed with the same crude puroindoline batch containing >95% puroindolines with ≈80% PIN-a and 20% PIN-b. Similarly, these experiments were performed on the same batch of good (FA), poor (EC), and medium (FAEC) quality flours. The alveograph data reported in Table IV show that addition of 0.1% puroindolines had a significant impact on the rheological properties of the tested wheat flours. The dough strength (W)
value of EC and FAEC was reduced from 8 and 7.4%, respectively. For FA, W showed an increase of ≈40% (from 342 to 478.10–4 J) in the presence of puroindolines. The extensibility (L) value was decreased from 33.8 and 30% for EC and FAEC, both supplemented in puroindolines. For FA, addition of 0.1% puroindoline to flour increased L from 60.4 to 77.6 mm. The tenacity (P) value (resistance to deformation) was increased from 2 to 16%, and the P/L ratio was also increased from 59 to 67% by addition of 0.1% puroindoline in EC and FAEC. The addition of 0.1% puroindoline to FA increased the P value by 17% and decreased the P/L ratio by 12%. Effect of Puroindolines on Grain Crumb and Bread Volume Puroindolines (0.05, 0.1, and 0.2% [w/w]) were added to FA flour. In 0.05% puroindoline, control and reconstituted bread crumbs were similar (Fig. 1). With 0.1 and 0.2% puroindoline, a fine crumb structure was obtained with an homogeneous distribution of tiny gas cells (Fig. 1). A decrease of bread volume (277 ± 20, 250 ± 10, and 230 ± 10 mL, respectively) was observed when puroindolines were added at 0, 0.1, and 0.2% levels. A comparative experiment was made with egg white proteins instead of puroindolines. At 0.1 and 0.2% egg white protein, grain crumb was expanded and gas cells were not uniformly sized (Fig. 2). No significant increase of bread volume (277 ± 20, 278 ± 17, and 290 ± 23 mL, respectively) occurred with the addition of 0, 0.1, and 0.2% egg white proteins. Baking experiments were also performed on poor and medium breadmaking flours by adding 0.1% puroindoline (Fig. 3). This Vol. 75, No. 2, 1998
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was a favorable compromise between availability of puroindoline from purification, the endogenous content in flour, and the minimal concentration for which effects are observed. For the three tested flours, puroindoline-reconstituted flours provided breads with fine crumb texture (Fig. 3). When flour with poor breadmaking quality (EC) was supplemented with puroindoline, a decrease of bread volume of 20% was observed (Fig. 4). When experiments were performed with the medium quality flour (FAEC), the presence of puroindoline induced an increase of bread volume of 20% (Fig. 4). In this case, differences between the crumb texture of standard bread (without puroindoline) and a bread sample fabricated with 0.1% puroindoline was less obvious than with poor and good quality
breadmaking flour (Fig. 3). However, gas cells of bread containing puroindolines appeared more uniformly sized. Effect of Puroindolines on Foaming Properties of Dough Liquors No foam was obtained from puroindoline-free dough liquors provided by ultracentrifigation of nonyeasted EC and FA doughs. The foaming assay used 1.6 mg of puroindoline for 64 mg of freeze-dried dough liquor: a mass ratio corresponding to 0.05% puroindoline on a dry weight flour basis. Foam obtained from this mixture was very stable, and the texture of foam was very fine (Fig. 5). Foaming properties of dough liquor obtained from EC and FA flours with PIN-a were compared. Drainage kinetics of these dough liquors were quite similar (Fig. 6). Foam obtained from dough liquor reconstituted with egg white proteins entrained two times less liquid than foam obtained from dough liquor reconstituted with PIN-a. Furthermore, after 5 min of drainage, dough liquor enriched with PIN-a still contained 36% of the liquid volume incorporated at the end of bubbling. Foam of dough liquor containing egg white proteins retained only 18% of its original liquid volume (Fig. 6). Dough liquor foams with egg white proteins were composed of large and heterogeneously sized bubbles (Fig. 5). DISCUSSION
Fig. 3. Bread baked from Florence Aurore (FA) flour without puroindoline (FA0), with 0.1% puroindoline (puro) added. FA flour was blended with Ecrin (EC) flour (50:50, w/w) without puroindoline (FAEC0) and with 0.1% puroindoline (puro) added. EC flour without puroindoline (EC0) and with 0.1% puroindoline (puro) added. 226
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A significant variability of PIN-a content was detected in samples of 32 wheat cultivars. These wheat cultivars classed in three breadmaking quality groups were also characterized by their hardness indices. Previous results have suggested that friabilins, puroindoline-like proteins isolated from the surface of starch granules, are markers of hardness (Greenwell and Schofield 1986, Jolly et al 1993, Morris et al 1994). Our results lead to a similar conclusion because variance analysis showed that hard and very hard wheats exhibited the lowest PIN-a contents. This significant relationship is partly due to the genetic linkage between PIN-a locus and Ha (hardness) locus on 5D chromosomes (Sourdille et al 1996). It was noteworthy that ≈75 % of these hard and very hard cultivars were found in the good quality breadmaking group. This is in agreement with the fact that hard wheats are generally used to make bread, while soft wheats are generally used to make biscuits, cookies, or other friable cereal baked products (Pomeranz and Williams 1990, Faridi et al 1994). Finally, this wheat sampling revealed that cultivars free of PIN-a are found in both good and poor breadmaking quality wheats. This means that PIN-a could play a specific role in breadmaking but it is not essential to the formation of bread. To explore this role through reconstitution experiments, we sampled two puroindoline-free cultivars with opposite breadmaking qualities, FA and EC. Their breadmaking quality is well related to their high molecular weight glutenin composition based on GluA1, GluB1, GluD1 content (Payne et al 1984) because FA is 2*, 7–9, 5–10 and EC is null, 17–18, 2–12. No medium quality cultivar was detected in our samples, and we therefore created one by producing a 1:1 (w/w) mixture of FA and EC flours (FAEC). Its medium quality was confirmed by baking experiments as well as by some alveograph characteristics (strength, tenacity). Considering the parent flours, the higher extensibility of the FAEC mixture is surprising. However, it is important to keep in mind that: 1) the mixture does not give rise to a sticky dough, and 2) the mixture is composed of glutenin subunits with different effects on extensibility and tenacity (Branlard and Dardevet 1985). Relatively low amounts of puroindoline can drastically affect the rheological properties of wheat doughs. For the three tested flours, puroindolines induced an increase of tenacity, which suggests an increase in protein-protein interactions. The mechanism by which puroindolines interfere with these protein-protein interactions should first affect puroindoline-lipid interactions. Previous
experiments have shown that lipids and lipoproteins have no effect on gluten viscoelasticity (Hargreaves et al 1995), so puroindolinelipid interactions are probably without major consequences on protein-protein interactions. However, considering that puroindoline is the main component of friabilin (Morris et al 1994), it is also possible that puroindoline-lipid-starch interactions control protein-protein interactions through gluten-starch aqueous phase separations (Eliasson and Larsson 1993). In contrast to tenacity, addition of puroindoline can give rise to opposite effects on strength and extensibility in FA and EC flours. This could be related to the high molecular weight glutenin composition. For example, most of the glutenin subunits of FA positively influence the strength (2*, 5, 9, and 10) while subunits 2–12 of EC negatively influence the strength of the resulting doughs. Opposite effects on extensibility are also observed for subunits 7–9 of FA and subunits 17–18 of EC (Branlard and Dardevet 1985). The effects of puroindoline on dough rheology are well related to the results of baking. It has been shown that the low bread volumes produced by poor quality flours are not caused by less air occluded during mixing but by more carbon dioxide being released during fermentation and the early stage of baking (He and Hoseney 1991). Gas retention is mainly driven by the growth of existing air bubbles by CO2 diffusion during fermentation and the early stages of baking and not by the increase in bubble number (Baker and Mize 1941). Gas retention and thermal gas expansion during baking influence the bread loaf volume and crumb structure. This process also requires that dough be extensible to prevent premature rupture of membranes between gas cells. Furthermore, the resistance to deformation (tenacity) of dough film between growing bubbles must be optimal: not too high to allow dough film expansion and not too low to prevent premature film rupture (Bloksma 1990a,b). EC flour gave a sticky dough with the lowest extensibility and tenacity in the three tested flours. Therefore, standard breads obtained from this flour gave rise to the lowest volume. When puroindolines were added to EC, a dramatic decrease of extensibility occurred that was not balanced by the slight increase of tenacity; the decrease of bread volume was 20%. For FA, the dough extensibility and tenacity values were high, and the bread had the highest volume among the three tested flours. However, the bread volume slightly decreased (–10%) in the presence of puroindolines, while extensibility and tenacity increased. In this case, the resistance to deformation probably became too high in the presence of puroindoline, and dough expansion was impaired. Finally, even if puroindoline dramatically decreased extensibility of the FAEC flour blend (–30%), it was still higher than the extensibility of EC flour (+50%). Tenacity, which increased only slightly
Fig. 4. Bread volume as function of puroindoline content and flour breadmaking quality. Concentration of puroindoline corresponded to 0 (white) or 0.1% (black), on a dry flour basis. FA = Florence Aurore (good breadmaking quality, AB1); FA flour was blended with Ecrin (EC) flour FAEC = (medium, B2C1); EC = (poor, C2D).
in the presence of puroindoline, was not too high to oppose a resistance to fermentation and oven rise; puroindoline induced a 20% increase of bread volume. To explore the physicochemical phenomena responsible for the drastic effect of low puroindoline contents in breadmaking, we studied the foaming properties of dough liquor. This liquid phase separated from the dough by ultracentrifugation plays a role in loaf volume and crumb texture (MacRitchie 1976). At first, it was noteworthy that no foam was obtained from dough liquor obtained from either poor- or high-quality puroindoline-free flours. When puroindoline was added to the dough liquor, a stable foam composed of fine gas cells could form. On the contrary, when egg white proteins were added to dough liquors, an unstable foam composed of large bubbles was formed. Therefore, an apparent relationship was highlighted between the structure of puroindoline foams and the crumb structure of corresponding puroindolinereconstituted breads. The absence of foaming was mainly due to the presence of nonpolar lipids forming a creamy layer at the top of the dough liquor after ultracentrifugation. When this lipid layer was removed or when flour was defatted, the foaming properties of dough liquor were restored (MacRitchie 1976, Dubreil unpublished data). The antifoaming properties of nonpolar lipids have been observed on dough liquors (MacRitchie 1977), and the prevention effect of puroindoline against lipid destabilization of protein foams has been previously described in beer (Clark et al 1994) and in egg white protein foams (Husband et al 1995). In this regard, egg white proteins, chosen as a model of food proteins with good foaming properties, were unable to produce stable dough liquor foams. It might be argued that the absence of foaming properties of the dough liquor was due to the absence of most
Fig. 5. Dough liquor foams at the end of bubbling, containing 0.2 mg/mL of puroindoline (A) and 2 mg/mL of egg white proteins (C). After 10 min of drainage with puroindoline (B) and egg white proteins (D). Vol. 75, No. 2, 1998
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polar lipids, which remained trapped in the gluten matrix (Marion et al 1987, Sahi 1994). However, it has been shown that polar wheat lipids could first decrease the foaming properties of dough liquor by competitive adsorption with soluble wheat proteins before they increase to form a polar lipid film at the gas-water interface (MacRitchie 1977, Gan et al 1995). Similarly, we have previously shown that polar wheat lipids destabilize egg white protein foams. On the contrary, puroindolines that interact strongly with polar wheat lipids can improve the foaming properties of polar wheat lipid-puroindoline mixtures (Dubreil et al 1997). Therefore, these preliminary experiments with dough liquor suggest that puroindolines could first prevent dough liquor foams from destabilization by nonpolar lipids and act as an endogenous defatting agent. The fine and regular bread grain crumb obtained in presence of puroindoline has already been described for bread made with defatted flour (MacRitchie 1981). Also, puroindoline could act synergistically with polar lipids to form stable lipoprotein foams. These results suggest that the lipid-to-puroindoline ratio could be, with the nonpolar-to-polar lipid ratio (Chung et al 1980, Bekes et al 1986, McCormack et al 1991), the most pertinent biochemical parameter for predicting the structure and texture of bread crumb. CONCLUSIONS Although reconstitution experiments do not necessarily reproduce the effect of endogenous puroindolines, the results reported in this article show unambiguously that low amounts of puroindolines can induce important changes in the structure of grain crumb and bread volumes. These puroindoline concentrations are equal to or even lower than those normally used for synthetic surfactants, so that puroindolines are, at least, promising as natural additives. The minimal content of puroindoline necessary to observe a significant change in breadmaking is quite compatible with the content of puroindoline-rich wheat cultivars. However, the inverse relationship between hardness and puroindoline content could hinder future breeding programs to improve the breadmaking quality of hard wheat through puroindolines. The effect of puroindolines on bread volume is closely related to its strong effect on dough rheology, while their effect on grain crumb is closely related to the foaming properties of these proteins. The latter effect is due to the strong interactions between puroindolines and wheat lipids, but the precise molecular mechanism is still unknown. More investigations will be necessary, especially on soft wheat, before considering puroindolines as important proteins in future breeding programs.
Fig. 6. Drainage kinetics for dough liquor foam obtained from Ecrin (EC) ( ) and Florence Aurore (FA) (l) flours with puroindoline (0.2 mg/mL)and from FA flour with 2 mg/mL of egg white proteins (∇).
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[Received April 18, 1997. Accepted December 17, 1997.]
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