Introduction

Wheat germ, corresponding to 2–3% of the total weight of wheat kernel, is almost systematically removed during milling since it adversely affects the keeping and processing quality of the flour [1, 2]. Due to the high concentration of a-tocopherol, vitamins of group B, dietary fibre, polyunsaturated fats, minerals and phytochemicals, wheat germ is one of the most attractive and promising source of vegetable functional compounds [1, 2]. Wheat germ is also considered to be the major alternative source of plant proteins [3]. Most of the essential amino acids contained in the proteins of wheat germ are present at a level higher than that found in the reference egg proteins [3, 4]. The poor stability of wheat germ during shelf-life of wheat flour and related baked goods is the main concern for the limited use in bakery industries. The high lipase and lipoxygenase activities cause sensitivity to oxidation which leads to the release of free fatty acids and, consequently, to the appearance of rancidity in baked goods [5]. Lipases of wheat germ are thermo-stable and maintain more than 20% of the residual activity at 60–90 C for 1 h [5]. Lipases have an optimum of pH of ca. 8.0, but their activity markedly decreases under acid conditions [5]. Increasing efforts are being made to stabilize wheat germ towards oxidation [5]. Treatments by heat, microwave and extrusion cooking [6] or the addition of antioxidants [7] were considered. Despite their effectiveness, the above technological treatments are in some cases expensive, not completely resolving and they negatively affect the nutritional value of wheat germ. Besides, synthetic antioxidants are increasingly looked at with suspicious because of their potential risks for consumer health [7]. 

Recently, some studies have considered the potential of wheat germ in medical and cosmetic applications, and also the effect of processed wheat germ on the nutritional and sensory properties of cereal based foods [2]. Previously, two lactic acid bacteria, Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5, were isolated from wheat germ, selected based on the kinetics of acidification and used as starters for the manufacture of sourdough fermented wheat germ [2]. Sourdough fermentation stabilized and enhanced some nutritional properties of the wheat germ. Due to lactic acidification, the lipase activity of the sourdough fermented wheat germ was markedly lower than that found in the raw wheat germ. As shown by SPME/GC/MS analysis, a very low level of volatile compounds deriving from lipid oxidation was found in the freeze-dried sourdough fermented wheat germ during 40 days of storage [2]. This paper aimed at using the sourdough fermented wheat germ as an ingredient for the manufacture of white bread. The nutritional, texture and sensory characteristics of this bread were compared to those of white breads made with or without the use of raw (non fermented) wheat germ. Materials and methods Wheat germ and chemical characterization Six samples of wheat germ were supplied by the industry Tandoi Pellegrino (Corato, BA, Italy). Before use, samples were pooled as usually done at industry plant. The germ was separated from refined flour during milling of Triticum aestivum cv. Appulo. A degerminator and a set of rollermills were used (Bu¨hler AG, Uzwil, Switzerland). Moisture, ash, proteins and fat were determined according to the approved methods of the American Association of Cereal Chemists [8].

 Total titratable acidity (TTA) was determined on 10 g of wheat germ homogenized with 90 ml of distilled water and expressed as the amount (ml) of 0.1 M NaOH to get pH of 8.3. The values of pH were determined by a Foodtrode electrode (Hamilton, Bonaduz, Switzerland). Sourdough fermentation of wheat germ Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were previously isolated from raw wheat germ and selected based on the kinetics of acidification [2]. Lactobacilli were cultivated in modified MRS (mMRS, maltose and fresh yeast extract were added to MRS at 1 and 5%, respectively, and the final pH was 5.6) until the late exponential phase of growth was reached (ca. 10 h), washed twice in phosphate buffer, pH 7.0, 50 mM and re-suspended in tap water. Two hundred grams of pooled wheat germ, 115 ml of tap water and 5 ml of the cell suspension, containing both lactic acid bacteria (final cell density in the dough ca. 108 cfu/g), were used to produce 320 g dough (dough yield of 160) with a continuous highspeed mixer (609g, dough mixing time, 5 min). Sourdough fermentation was carried out at 30 C for 24 h. After fermentation, sourdough wheat germ was freeze-dried and used for bread making. Fermentations were carried out in triplicate. Serial dilution of freeze-dried sourdough fermented wheat germ were made and plated onto MRS (Oxoid LTD, Basingstoke, Hampshire, UK). Enumeration of lactic acid bacteria was carried out after incubation at 30 C for 48 h. Lipase activity As previously shown [9], wheat germ lipase has good solubility in water/salt-buffers. Water/salt-soluble extracts from raw wheat germ and sourdough fermented wheat germ were prepared according to the method originally described by Osborne and modified by Weiss et al. [10]. 

The concentration of proteins in the water/salt-soluble extracts was determined by the Bradford method [11]. Tributyrin as the substrate and the agar diffusion assay [12] were used to determine the lipase activity of the water/saltsoluble extracts. Agar plates contained 1% (wt/vol) of triglyceride, 0.02% (wt/vol) sodium azide and 50 mM phosphate buffer, pH 8.0. As reported by Kapranchikov et al. [5], this value of pH was the optimum for wheat germ endogenous lipase activity. Lipase activity was expressed as the minimum dilution of the enzyme preparation that failed to give a detectable zone of hydrolysis after 24 h of incubation at 30 C. Bread making The characteristics of the wheat flour (T. aestivum, cv Appulo) used for bread making were as follows: moisture, 14.2%; protein (N 9 5.70), 11.5%, of dry matter (d.m.); fat, 1.6% of d.m.; ash, 0.6% of d.m.; and total soluble carbohydrates, 1.5% of d.m. According to typical Italian bread making, three breads having dough yield of 160 were manufactured at the pilot plant of the Department of Plant Protection and Applied Microbiology. The formulas were as follows: (1) wheat flour bread (WFB) made with 250 g flour, 150 g tap water and 2% (wt/wt) of baker’s yeast; (2) raw wheat germ bread (RWGB) made with 240 g flour, 10 g raw wheat germ (RWG) (4%, wt/wt of wheat flour), 150 g tap water and 2% of baker’s yeast (wt/wt); and (3) sourdough fermented wheat germ bread (SFWGB) made with 240 g flour, 10 g of freeze dried SFWG (4%, wt/wt of wheat flour), 150 g tap water and 2% (wt/wt) of baker’s yeast. A continuous high-speed mixer (609g, dough mixing time 5 min) was used to prepare the doughs.

 Fermentation of doughs was allowed at 30 C for 2.5 h. Before baking, doughs were characterized for pH, titratable acidity, organic acids, free amino acids, total phenols, and phytase as well as antioxidant activities. The rheology properties of the doughs were determined by a Brabender Farinograph (mixer type S300H Brabender, Duisburg, Germany), according to the AACC method [8]. From the farinograph normal curve, three main parameters such as water absorption capacity, development time and level of dough softening were determined. All breads were baked at 220 C for 40 min (Combo 3, Zucchelli, Verona, Italy). Fermentations were carried out in triplicate and each bread was analysed twice. Breads were packed in polyethylene bags to maintain constant the moisture and stored at room temperature for 8 days. Determination of organic acids and free amino acids Water/salt-soluble extracts from fermented doughs were prepared as reported elsewhere. Organic acids were determined by high performance liquid chromatography (HPLC) using an A¨ KTA Purifier system (GE Healthcare) equipped with an Aminex HPX-87H column (ion exclusion, Biorad) and a UV detector operating at 210 nm. Elution was at 60 C, with a flow rate of 0.6 ml/min, using H2SO4 10 mM as mobile phase [5]. Total and individual free amino acids were determined by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm inner diameter) as described by Rizzello et al. [5]. Phytase activity Phytase activity from water/salt-soluble extracts was measured in terms of inorganic ortophosphate released from the phytic acid by phytase [13]. 

The reaction mixture, containing 150 ll of water extract and 600 ll of substrate (3 mM Na-phytate in 0.2 M Na-acetate, pH 4.0), was incubated at 45 C. The reaction was stopped by adding 750 ll of 5% trichloroacetic acid. The released inorganic phosphate was measured by adding 750 ll of colour reagent, prepared daily by mixing four volumes of 1.5% (wt/vol) ammonium molybdate in 5.5% (vol/vol) sulphuric acid solution and one volume of a 2.7% (wt/vol) ferrous sulphate solution. The absorbance was measured at 700 nm. One unit (U) of phytase activity was defined as the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay conditions. Total phenols and antioxidant activity Extracts from dough were prepared by weighing 5 g of sample and mixing with 50 ml of 80% methanol. The mixture was purged with nitrogen stream, mixed for 30 min and centrifuged at 6,0009g for 20 min. Extracts were transferred into culture tubes, purged with nitrogen stream and stored at ca. 4 C before analysis. Analysis of total phenols was done according to the method of Slinkard and Singleton [14]. Gallic acid was the standard. The reaction mixture contained 20 ll of extract, 100 ll of Folin-Ciocalteu reagent (Sigma Chemical Co.) and 1.58 ml of distilled water. After few min, 300 ll of saturated sodium carbonate solution was added to the reaction mixture. 

Incubation was allowed at 20 C for 2 h and the absorbance at 765 nm was determined. The concentration of total phenols was calculated as gallic acid equivalent. The radical cation (2,20 -azino-di-[3-ethylbenzthiazoline sulphonate]) (ABTS
?) scavenging capacity was measured using the Antioxidant Assay Kit CS0790 (Sigma Chemical Co.), following the manufacturer’s instruction. Trolox (6-hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the antioxidant standard. The scavenging activity was expressed as Trolox equivalent. In vitro protein digestibility The in vitro protein digestibility of breads was determined according to the methods of Dahlin and Lorenz [15], with some modifications. Fifty millilitres of bread suspensions, containing 6.25 mg of crude protein/ml, was allowed to rehydrate at 5 C for 60 min. After rehydration, the suspension was placed in a water bath at 37 C and the pH was set to 8.0, using 0.1 N NaOH and/or 0.1 N HCl. Lyophilized, crystallized trypsin (Sigma Chemical Co., Milan, Italy), at the concentration of 1.6 mg/ml, was maintained in an ice bath and the pH was adjusted to 8.00 with 0.1 N NaOH and/or 0.1 N HCl. Five milliliters of the enzyme solution was added to the protein suspension under stirring at 37 C. The activity of trypsin was 13,000 BAEE units/mg protein.A rapid decline in pH occurred immediately. The pH drop was recorded 15 s after enzyme addition and at 1-min intervals for 10 min. 

 The enzyme solution was freshly prepared before each test. The percent protein digestibility (Y) was calculated according to the following equation [15]: Y = 210.4-18.1x, where x is the change in pH after 10 min. Texture and image analyses Instrumental texture profile analysis (TPA) was performed with a TA.XT2i Texture Analyzer, using a 35-mm flat-end aluminium compression disc (probe P/35). The selected settings were as follows: test speed 1 mm/s, 30% deformation of the sample and one compression cycle. TPA was carried out [16] using Texture Export Exceed software. Specific volume, height, width, depth and area of loaves were measured by the TA.XT2i system. The following textural parameters were obtained by the texturometer software: hardness (maximum peak force); fracturability (the first significant peak force during the probe compression of the bread); and resilience (area during the withdrawal of the penetration, divided by the area of the first penetration). Triplicate measurements for breads from each storage time were made. The crumb grain of breads was evaluated after 24 h of storage using the image analysis technology. Images of the sliced breads were scanned full-scale using an Image Scanner (Amersham Pharmacia Biotech, Uppsala, Sweden), at 300 dots per inch and analysed in grey scale (0–255). Image analysis was performed using the UTHSCSA ImageTool program (Version 2.0, University of Texas Health Science Centre, San Antonio, Texas, available by anonymous FTP from ftp://maxrad6.uthscsa.edu). A threshold method was used for differentiating gas cells and non-cells [17]. Analysis was carried out on two subimages of 500 9 500 pixels (field of view) selected from within the bread slice.

 Two slices were analysed per treatment. The crumb cell features recovered were number, area, perimeter, elongation, roundness and gas cell to total area ratio. Colour measurement Colour was measured at three different positions of the bread surface using a Minolta CR-10 camera [18]. The L*a*b* colour space analysis method was used, where L* represents lightness (white–black) and a* and b* the chromaticity co-ordinates (red–green and yellow–blue, respectively). Result was reported in the form of a colour difference, dEab * , as follows: where dL, da, and db are the differences for L, a, and b values between sample and reference (a white ceramic plate having L = 93.4, a = -1.8, and b = 4.4). Sensory analysis Sensory analysis of breads was carried out by ten nontrained panellists according to the method described by Haglund et al. [19]. Elasticity, colour, acid taste, acid flavour, sweetness, dryness and taste were considered as sensory attributes using a scale from 0 to 10, with 10 the highest score. Salty taste, previously described as another wheat sourdough bread attribute, was also included [20]. Statistical Analysis Data were subjected to one-way ANOVA; pair-comparison of treatment means was achieved by Tukey’s procedure at P\0.05, using the statistical software, Statistica 7.0 for Windows. Results Raw and sourdough fermented wheat germ The averaged values of the six samples of raw wheat germ were the following: moisture 11.11 ± 0.37%, protein (N 9 5.70) 28.56 ± 0.88% of dry matter (d.m.); fat 7.99 ± 0.04% of d.m.; and ash 3.77 ± 0.007% of d.m.

 The pH was 6.34 ± 0.08 and TTA was 18.1 ± 0.23 ml of 0.1 M NaOH/10 g. After freeze-drying, sourdough fermented wheat germ had values of moisture of 11.09 ± 0.41%, pH 4.15 ± 0.05 and TTA 25.5 ± 0.11 ml of 0.1 M NaOH/10 g. It contained ca. 5.9 9 109 cfu/g of viable lactic acid bacteria. The water/salt-soluble extracts of raw wheat germ and sourdough fermented wheat germ were used to determine the lipase activity. The minimum concentration of the crude enzyme extract that failed to give a detectable zone of hydrolysis was, respectively, 52.7 ± 3.4 and 152.8 ± 1.9 lg/ml. Chemical and nutritional characterization The values of pH were 5.45 ± 0.08, 5.76 ± 0.05 and 4.86 ± 0.04 for WFB, RWGB and SFWGB, respectively (Table 1). TTA was 3.5 ± 0.11, 4.8 ± 0.09 and 6.4 ± 0.16 of 0.1 M NaOH/10 g for WFB, RWGB and SFWGB, respectively. Lactic and acetic acids were not detectable in WFB and RWGB. Due to the preliminary sourdough fermentation of wheat germ, SFWGB contained 5.28 ± 0.07 and 2.07 ± 0.05 mM of lactic and acetic acids, respectively. Consequently, SFWGB bread had the quotient of fermentation (QF, molar ratio between lactic and acetic acids) of 2.55. This value approached that usually found in sourdough baked goods. The concentration of total free amino acids of WFB, made without wheat germ, was 751 ± 48 mg/kg (Table 1). It increased to 1,356 ± 56 mg/kg for RWGB which used non-fermented wheat germ as an ingredient. The concentration of total free amino acids of SFWGB (1,686 ± 56 mg/kg) was significantly (P\0.05) higher than that found in WFB and RWGB. Almost all free amino acids were found at higher concentration in RWGB and SFWGB breads with respect to WFB (Fig. 1).