J. Agric. Food Chem. 2006, 54, 2567−2574
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Proteolysis in Model Sourdough Fermentations TERESA ZOTTA,*,† PAOLO PIRAINO,† ANNAMARIA RICCIARDI,† PAUL L. H. MCSWEENEY,§ AND EUGENIO PARENTE† Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Universita` degli Studi della Basilicata, Campus di Macchia Romana, 85100 Potenza, Italy, and Department of Food Science, Food Technology and Nutrition, University College Cork, Cork, Ireland
Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker’s yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits. KEYWORDS: Wheat sourdoughs; proteolysis; lactic acid bacteria; yeasts
INTRODUCTION
Sourdough fermentation is a traditional process used in the baking industry to improve the quality and flavor of baked goods. The addition of sourdoughs in breadmaking affects in several different ways the final properties of the bread. During the fermentation process several metabolic activities take place, and each is responsible for the changes that occur in the characteristics of wheat flour as well as in aroma, taste, nutritive value, and shelf life of the bread. Lactic acid bacteria (LAB) and yeasts have been reported to be the predominant microorganisms in sourdoughs (1) and, consequently, the organoleptic and nutritional properties of sourdough breads depend on their metabolism and their cooperative activities. Acidification and antimicrobial compounds produced by the sourdough microflora contribute to the stability of products and play an important role in the regulation of the complex interactions within the starter microflora and between the starter and contaminant microflora (2). Exopolysaccharides (EPS) can replace hydrocolloids used as texturizing, antistaling, or prebiotic additives in bread production, improving the textural and nutritional properties of the product (3). Production of CO2 from heterofermentative LAB and yeasts influences the leavening process of the final dough, improving bread softening. Moreover, the interaction between LAB and yeasts may affect the synthesis of volatile compounds that contribute to the flavor and aroma of sourdough products (1). The hydrolysis of chelating agents such as phytic acid during * Corresponding author (telephone + 39 971 205552; fax + 39 971 205503; e-mail
[email protected]). † Universita ` degli Studi della Basilicata. § University College Cork.
sourdough fermentation can have positive nutritional implications by increasing mineral bioavailability (4). Dough properties and the nutritional value of bread are strongly influenced by protein content as well as the protein composition of the flour. The quality of cereal proteins may be improved by the fermentation process. The degradation of gluten proteins influences the rheology of wheat sourdoughs and, consequently, the texture of bread (5); additionally, modification of the gluten network improves the dough’s machinability (6). Free amino acids and small peptides released during fermentation are important for rapid microbial growth as well as fermentative activity of yeasts and lactate production by LAB and as precursors for flavor development of baked goods. The proteolytic/peptidolytic activity of LAB can contribute to hydrolysis of bitter peptides and liberation of bioactive peptides (7). Several proteins of wheat flour and products of their hydrolysis have biological activities that can affect human health when wheat or its byproducts are consumed as foods. Peptides generated from the digestion of the wheat prolamins, which affect the human intestinal mucosa, resulting in celiac disease or gluten-sensitive enteropathy (8), may be degraded during proteolysis. Besides, there are also indications that some mental disorders, such as schizophrenia and autism, could be related to a diet rich in wheat proteins containing peptides with opioidlike activities (9). Several theories about whether the proteolytic activity during sourdough fermentation originates from LAB and/or yeasts or from indigenous cereal enzymes have been proposed (10). Cereal flour, yeasts, and LAB contain proteases and peptidases that can contribute in different ways to the proteolytic events. Thiele et al. (5) concluded that proteolysis during sourdough
10.1021/jf052504s CCC: $33.50 © 2006 American Chemical Society Published on Web 03/03/2006
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Table 1. Dough Formulas for Preparation of Control Doughs and Sourdoughs
flour (g) water (mL) NaCl (g) acid solutionb (µL) penicillin Gc (µL) cycloheximidec (µL) cloramphenicolc (µL) LAB startera (mL) yeast starter (mL) phosphate bufferd (mL)
neutral control
acid control
neutral yeast
acid yeast
LAB
LAB, buffer
LAB, yeast
20 20 0.4 0 20 200 200 0 0 0
20 20 0.4 600 20 200 200 0 0 0
20 18 0.4 0 20 0 200 0 2 0
20 18 0.4 600 20 0 200 0 2 0
20 18 0.4 0 0 200 0 2 0 0
20 16 0.4 0 0 200 0 2 0 2
20 16 0.4 0 0 0 0 2 0 2
a Lb. plantarum DBPZ1015 or Lb. plantarum DBPZ1025 or Lb. pentosus DBPZ0984 or Lb. curvatus DBPZ1020 or W. cibaria DBPZ1006 or Leuc. mesenteroides DBPZ1005. b Mixture of 4 volumes of lactic acid (90%) and 1 volume of acetic acid (98%). c 1 g/L cycloheximide and cloramphenicol; 50000 IU/mL of penicillin G. d 1 M sodium phosphate buffer, pH 6.8.
fermentation and the rheological consequences of gluten degradation are mainly related to the pH-mediated activation of cereal enzymes; the indigenous proteases of flour, in fact, are able to degrade cereal prolamins under acid conditions (10). On the contrary, other studies have shown that LAB had a substantial role in the proteolysis during sourdough fermentation (6, 8). The aim of the present study was to investigate the changes that occur during sourdough fermentation in wheat flour proteins, the sources of proteolytic activities, and the factors that may be responsible for these events such as activity of indigenous cereal enzymes, pH conditions, and microbial metabolism. MATERIALS AND METHODS Strains and Culture Conditions. Two strains of Lactobacillus plantarum and one strain each of Lactobacillus pentosus, Lactobacillus curVatus, Leuconostoc mesenteroides, and Weissella cibaria were used in this study. All strains were isolated from wheat sourdoughs used to produce Cornetto di Matera bread (11) and were maintained as freezedried stocks in the Culture Collection of the Department of Biologia, Difesa e Biotecnologie Agro-forestali, Universita` della Basilicata, Potenza, Italy. Commercial active dry yeast was obtained from Lesaffre Yeast Corp., North America. LAB strains were propagated overnight in modified MRS medium (mMRS) containing 10 g/L maltose and 5 g/L each of glucose and fructose (5). Cells were harvested by centrifugation (12000g, 10 min, 4 °C), washed twice, and resuspended in 0.85% (w/v) NaCl to obtain a final concentration of 107 colonyforming units (cfu)/g of dough. The yeast was revitalized with 0.85% (w/v) NaCl and added immediately to the dough to obtain a final concentration of 106 cfu/g of dough. Sourdough Fermentations. Commercial breadmaking wheat flour with 12% protein content (w/w dry basis), 0.5% ash content (w/w dry basis), 13% moisture (w/w), and a Hagberg falling number value of 250 (Odlum Group, Dublin, Ireland) was used in all tests. Flour was not treated or fortified with additives. Doughs were prepared as described in Table 1, mixing water and flour with a sterile spatula for 3 min to obtain a dough yield (DY) of 200. The fermentations were carried out for 24 h at 30 °C. Two control fermentations, obtained by adding antibiotics to the dough mixtures, were carried out aseptically at pH 6.0 and 3.5, respectively. Samples were taken at 0, 6, and 24 h for the subsequent analysis. Cell Counts and pH Measurement. LAB and yeasts were enumerated at the end of fermentation by plate counts on mMRS and YPD (yeast extract, 10 g/L; peptone, 20 g/L; glucose, 20 g/L) agar media, respectively. The pH values were measured on each dough suspension (1 g of dough and 9 mL of distilled water) after 0, 6, and 24 h of
fermentation using a glass electrode (pH-meter 210, Radiometer, Copenhagen, Denmark). Extraction of Dough Samples. Wheat proteins and their fractions were extracted sequentially from dough samples using the following solvents: (1) 0.5 mol/L NaCl and 150 mmol/L sodium phosphate, pH 6.8 (albumins and globulins) (5); (2) 50% (v/v) 2-propanol in H2O (gliadins); (3) 1.5% (w/w) SDS, 1% (w/w) DTT, and 0.063 mol/L TrisHCl, pH 7.5 (glutenins) (12). Solvent (6 mL) was added to 2 g of dough, and each extraction step was carried out for 30 min at room temperature (albumins, globulins, and gliadins) or at 70 °C (glutenins), vortexing the suspension every 10 min. Supernatants were recovered after centrifugation for 10 min at 25000g and 4 °C (albumins, globulins, and gliadins) or 20 °C (glutenins). A washing step with deionized water of the pellet following the saline extraction was included to remove salt and residual organic acids. The extracts were not combined. The dialyzed (Medicell International Ltd., London, U.K.; dialysis tubing cutoff 3500 Da, against deionized water at 4 °C for 24 h) albumin-globulin fraction was freezedried and stored at room temperature until analysis by SDS-PAGE and RP-HPLC. The gliadin and glutenin extracts were stored at -80 °C until analysis by SDS-PAGE. Determination of Free Amino Acids. The concentration of free amino acids in the salt-soluble extracts of control doughs and sourdoughs was determined by using the trinitrobenzenesulfonic acid (TNBS) method (13). A calibration curve was prepared using leucine (Leu, Sigma) as standard (range ) 0.0-1.0 mmol/L of Leu), and results were expressed as milligrams of Leu per kilogram of dough. RP-HPLC Analysis of Dough Extracts. Salt-soluble extracts were analyzed by RP-HPLC. The freeze-dried samples were dissolved (10 mg/mL) in 0.1% (v/v) trifluoroacetic acid (TFA, sequential grade, Sigma) in deionized HPLC grade water (Milli-Q system, Waters Corp.) and centrifuged at 12000g for 5 min at room temperature. The supernatants were filtered through 0.45 µm cellulose acetate filters (Sartorius GmbH, Goettingen, Germany) and injected (150 µL) into a Varian System HPLC (Varian Associates Inc., Walnut Creek, CA) comprising an autosampler, a ProStar solvent delivery system with three pumps, and a ProStar programmable multiwavelength spectrophotometric detector, interfaced with a PC with a software package for system control and data acquisition (Varian Star Workstation 5). The column was an analytical 250 × 4 mm Nucleosil C8 (5 µm particle size, 300 Å pore size, operated at room temperature) with a 4.6 × 10 mm guard column (Capital HPLC Ltd., Broxburn, West Lothian, U.K.). Eluents used were A, 0.1% (v/v) TFA in deionized HPLC grade water, and B, 0.1% TFA in acetonitrile (HPLC grade, Lab-scan Ltd., Dublin, Ireland). The separation was achieved using the following gradient: 100% A for 5 min, 50% B (v/v) from 6 to 55 min, 50% B (v/v) for 6 min, 60% B (v/v) from 62 to 66 min, and 60% B (v/v) for 3 min; the flow rate was 0.75 mL/min. The column was washed with 95% B (v/v) for 5 min, followed by equilibration with 100% A for 5 min before the next injection. The proteins were detected by measuring the absorbance at 214 nm. Some samples were run three times to measure the reproducibility of the patterns in the above conditions. SDS-PAGE Analysis of Dough Extracts. All protein fractions were analyzed by SDS-PAGE using a Bio-Rad MiniProtean III apparatus (Bio-Rad Laboratories). Freeze-dried albumin-globulin extracts (∼3.0 mg) were dissolved in 0.5 mL of sample buffer (sample buffer 2×, Sigma), whereas 1 volume of gliadin and glutenin extracts were mixed with 3 or 4 volumes of sample buffer, respectively. Samples were heated at 95 °C for 5 min before the run. Electrophoresis was performed using separating (12% w/v T, 2.67% w/v C) and stacking (4% T, 2.67% C) gels (14). Gels were run at a constant current of 20 mA for 3 h using a Power Pack 3000 unit (Bio-Rad Laboratories). Staining was carried out according to the method of Pot et al. (15). Data Processing and Statistical Analysis. RP-HPLC chromatograms (height and retention time of peaks) were processed by a logistic weighting function as described by Piraino et al. (16). Peak height was expressed as percent of the total, and the peaks with heights of
