Effect of Toasting Time on the Browning of Sliced Bread

Journal of the Science of Food and Agriculture J Sci Food Agric 81:513±518 &online: 2001)

Effect of toasting time on the browning of sliced bread Antonio Ramı´rez-Jimeńez, Beleń Garcıá-Villanova and Eduardo Guerra-Hernańdez* Departamento de Nutricioń y Bromatologıá, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja, 18012 Granada, Spain

Abstract: Slices of wheat bread were toasted for different times until a distinct intensity of brown colour was reached. Two assays were carried out: prolonged toasting times 5±60min) and reduced toasting times 0.5±5min). The browning indicators furosine,available lysine,hydroxymethylfurfural HMF),colour and absorbance at 284 and 420nm) were determined. The precision of all indicators used was high CV<4%). No furosine or HMF was detected in the dough before baking. The furosine content increased until 7min 299mg per 100g protein) and then decreased to 2.9mg per 100g protein at 60minutes. For the ®rst toasting times 0.5,1 and 2min) the furosine content decreased slightly. Available lysine reached losses of 50% after 25min of heating. The toasting of bread increased HMF values from 12 to 2025mg kgÀ1 for the assay at prolonged times of heating and from 1.3 to 4.2mg kgÀ1 at reduced times 0.5±5min). The HMF content decreased 1000mg kgÀ1) when the sliced bread was toasted until it burnt. Colour DE,100 ÀL*) and absorbance at 284 and 420nm always increased. High 2 linear correlations r >0.860) were obtained between browning indicators and time A284/time, A420/time,100 ÀL*/time and HMF/time). # 2001 Society of Chemical Industry

Keywords: browning indicators; toasting; sliced bread

INTRODUCTION syl-lysine produced by reaction of e-amino groups of A pleasant ¯avour and colour are produced when lysine with glucose, lactose and maltose.5 Furosine slices of wheat bread are toasted, corresponding to the determination has been applied to cereal products to organoleptic characteristics of bread crust.1 The monitor the extent of non-enzymatic browning in chemical reactions involved in this process are pasta and bakery products6 and commercial baby essentially the Maillard reaction and caramelisation. cereals7 and to control baby cereal processing.8 Both depend on the type of reagent, temperature, Lysine is limiting in cereal proteins. This de®ciency water activity and pH. can be further aggravated by losses from browning Maillard reactions are favoured in systems with an reactions during processing. Available lysine can be intermediate moisture content, temperatures above utilised for metabolism and is distinguished from total 50°C and pH 4±7 &ie in the pH range of food), lysine, which includes damaged or bound lysine. It is producing changes in colour &melanoidins), ¯avour suggested that lysine is not used biologically unless the &aldehydes and ketones), functional properties and e-amino group is free.9 The method using 1-¯uoro- nutritional value &blocking or destruction of lysine).2 2,4-dinitrobenzene &FDNB) is the most widely used10 Caramelisation depends on direct degradation of and there is reason to believe that lysine that does not sugars and requires more drastic conditions &tempera- react with FDNB is not nutritionally available.11 3 tures >120°C, pH<3orpH>9 and low Aw). Available lysine has been measured to evaluate the The manufacture of bread involves baking a loaf at effect of heating on the protein quality of the following 200°C for 50minutes, then cutting the loaf into slices. cereal products: pasta,12 noodles,13 breads,14 cereal The sliced bread can be consumed fresh or toasted. In ¯akes15 and infant cereals.16 this process, therefore, the Maillard reaction and Hydroxymethylfurfural &HMF) is formed from the caramelisation may occur simultaneously.4 degradation of hexoses heated in acid solution, even in Evaluation of the early stages of the Maillard mild acid solution,3 and is also an intermediate reaction can be achieved by the determination of product in the Maillard reaction.17 HMF has been 18 furosine &e-N-&furoylmethyl)-L-lysine), an amino acid used to control the drying of pasta and baking of formed during acid hydrolysis of the Amadori com- cookies19 and in baby20 and breakfast21 cereals. pounds fructosyl-lysine, lactulosyl-lysine and maltulo- Brown pigments with low and high molecular

* Correspondence to: Eduardo Guerra-Hernańdez, Departamento de Nutricioń y Bromatologıá, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja, 18012 Granada, Spain E-mail: [email protected] (Received 28 July 2000; accepted 7 December 2000)

# 2001 Society of Chemical Industry. J Sci Food Agric 0022±5142/2001/$30.00 513 A RamõÂrez-JimeÂnez et al weights are formed in the advanced stages of the Sample preparation browning reactions and are assumed to be water- Commercial samples of sliced bread were made by a soluble up to a molecular size of 100kDa.22 The bread-making company. The bread formula and absorbance at 420nm is a classic browning index that conditions used by the company were: wheat ¯our is very useful in sugar and sugar±amino acid model &50kg), water &27kg), baker's yeast &2kg), NaCl systems. Moreover, A420 has been applied to the &1kg), previously fermented dough &5kg) and dough- kinetics of bread baking.23 The brown colour index in conditioning agents; fermentation at 30°C for 60min; solid products has been applied to follow the model- and baking at 200°C for 50min. The 700g loaves were ling of bread crust browning kinetics during baking,4 cut into 10mm thick slices weighing 25g each. The the toasting of wheat bread1 and the twin-screw slices were toasted in the laboratory in a Moulinex extrusion of corn and soy products.24 model T-90 toaster &Spain). Two assays were carried Little information has been found about these out: prolonged toasting timesÐwhole &crust and crumb) indicators in breads. Rychlik and Grosch1 studied bread slices were toasted for between 5and 60min; the ¯avour components of toasted wheat bread by a reduced toasting timesÐbread slices without crust were complex methodology. However, studies on toasted toasted for between 0.5and 5min. The toasted bread bread with simpler indicators to evaluate the process of slices of both assays were ground in a Wiley mill to pass toasting have not been reported. through a 40-mesh screen and stored in screw-capped This paper describes the behaviour of browning bottles at À40°C. Each assay &prolonged and reduced indicators during the process of toasting bread for toasting times) was performed on bread slices from different times in order to give industry some useful different loaves in duplicate samples. All determina- indicators for evaluating and controlling this process. tions were carried out in duplicate. In addition, the study shows the in¯uence of toasting time on the loss of lysine. Colour The colour of the toasted and untoasted bread samples was measured in a re¯ectance spectrophotometer MATERIALS AND METHODS using the CIE L*, a*, b* colour system, where L* is Apparatus lightness, a* is redness and b* is yellowness.20 The results are expressed as 100ÀL and the colour . Elrepho 2000 re¯ectance spectrophotometer &Data- * difference &DE) between the untoasted bread and the color SA, Spain). toasted samples was determined according to the . Perkin Elmer model 551S UV/VIS spectro- following equation:25 DE =&DL2 Da2 Db2)1/2, photometer &Norwalk, CT, USA). where DL is the brightness difference, Da is the redness . Konik model 500A liquid chromatograph, Konik difference and Db is the yellowness difference. The model 200 variable-wavelength UV detector samples were lyophilised prior to the analysis. &Barcelona, Spain) and Hewlett Packard model 3394A integrator &Avondale, PA, USA). . Perkin Elmer model 250 liquid chromatograph Hydroxymethylfurfural (HMF) HMF determination was performed by HPLC follow- &Norwalk, CT, USA) with Waters plus 717 auto- ing the GarcõÂa-Villanova et al method.21 A Konik sampler &Milford, MA, USA) and Perkin Elmer liquid chromatograph was used and the HMF was model 235diode array detector &Norwalk, CT, separated in a reverse phase C column &Spherisorb USA). Data were collected by a 1020 software data 18 S5ODS2, 250mm Â4mm id; Sugelabor, Spain). system &Perkin Elmer, Norwalk, CT, USA). HMF was extracted by shaking and centrifugation with deionised water, and the supernatants were Reagents clari®ed with Carrez solutions. Analytical reagent grade chemicals were used. . A standard stock solution containing 200mg lÀ1 A A 284 and 420 5-&hydroxymethyl) furfural &Merck, Darmstadt, The aqueous extracts obtained from HMF extrac- Germany) was used to prepare the working standard tion21 were measured at 284 and 420nm in a UV/VIS solutions &0.02±0.5mg l À1). spectrophotometer using as a reference material the . A standard stock solution containing 1.2mg ml À1 solution obtained from untoasted bread. The solutions

furosine &Neosystem Laboratoire, Strasbourg, measured at A284 had to be diluted with water before France) was used to prepare the working standard determination. solution. À1 . A standard stock solution containing 1.256mg l Furosine HCl 8.1 M DNP-lysine &Sigma Chemical Co) was Furosine determination was performed by the used to prepare the working standard solution &1ml method described by Guerra-HernaÂndez and Corzo.7 with water to 10ml). A Perkin Elmer HPLC was used and the furosine was . Lysine derivative reagent: 1-¯uoro-2-4-dinitro- obtained by hydrolysis with HCl, puri®ed with a

benzene &FDNB) solution &Sigma Chemical Co) Sep-pak C18 cartridge &Millipore) and separated by in 3% ethanol. ion pair reverse phase chromatography using a

514 J Sci Food Agric 81:513±518 &online: 2001) Browning of sliced bread

Table 1. Colour parameters at prolonged toasting times Table 2. Browning parameter correlations

Time min) a* b* L* 100ÀL* DE Assay at prolonged Assay at reduced toasting times toasting times 0 2.2 15.1 84.1 15.9Æ0.17 Ð 5 2.6 16.7 81.6 18.4Æ0.19 3.0Æ0.03 Correlations a r2)r2) 7 4.1 20.1 80.6 19.4Æ0.17 6.4Æ0.06 100ÀL /time 0.962 0.919 10 3.6 18.5 78.8 21.2Æ0.18 6.4Æ0.06 * DE/time 0.913 0.835 14 7.0 23.8 74.6 25.4Æ0.19 13.8Æ0.13 HMF/time 0.866 0.952 18 6.3 22.8 72.2 27.8Æ0.17 14.7Æ0.14 Furosine/time 0.712 0.610b 21 10.3 29.4 67.7 32.3Æ0.15 22.0Æ0.35 DNP-L/time 0.694 0.903 25 9.8 29.0 67.1 32.9Æ0.15 23.3Æ0.13 A /time 0.927 Ð 30 11.5 30.4 60.9 39.1Æ0.13 29.3Æ0.32 284 A /time 0.939 Ð 40 13.6 31.3 54.4 45.6Æ0.12 35.7Æ0.35 420 HMF/100ÀL 0.871 0.954 60 14.1 30.0 48.9 51.1Æ0.11 40.0Æ0.40 * Furosine/100ÀL* 0.834 0.530c n =4. DNP-L/100ÀL* 0.788 0.755b HMF/furosine 0.612 0.639b DNP-L/furosine 0.748 0.491c Spherisorb ODS2 5mm column &250mmÂ4.6mm DNP-L/HMF 0.515b 0.855 id; Phenomenex, Torrance, USA). A284/A420 0.967 Ð A284/HMF 0.964 Ð A /100ÀL 0.935 Ð Available lysine with FDNB (DNP-L) 420 * A /DE 0.901 Ð Available lysine was determined by Carpenter's 420 method10 as modi®ed by Booth.11 e-DNP lysine was a All correlations have a value P <0.01; b P <0.05; c P <0.1. determined by spectrophotometric measurement at 435nm after hydrolysis of the FNDB derivate. water-soluble compounds and has also been applied in 23 Moisture, protein and reducing sugar kinetic studies of baked dough. The reproducibility determinations of A420 was studied on sliced bread at two times of Moisture was determined by a gravimetric method toasting &5and 40min). The coef®cients of variation &AOAC method 925.10).26 Protein was determined by &CV) were 3.56% &n =7) and 4.04% &n =7) respec- the Kjeldahl method &AOAC method 920.87).26 tively. These values increased during heat treatment

Reducing sugars were determined by a titrimetric &Table 3). The linear correlation between A420 and method &AOAC method 939.03).26 time was r2 =0.939 &Table 2). The relation between

The results for the different parameters are ex- 100ÀL* &ground bread sample) and A420 &water- pressed on a dry matter basis. soluble compounds) was r2 =0.935&Table 2).

Statistical analysis Assay at reduced toasting times The Sigma package &Horus Hardware SA, Madrid, Table 4 shows the effects of toasting on the colour Spain) was applied to study parameter correlations. values. The colour increased most at 4 and 5min. The 100ÀL* value &13.9) at t =0 was lower than for the assay at prolonged toasting times, since this sample did RESULTS AND DISCUSSION not contain the crust. The linear correlation between 2 2 Colour DE and time was r =0.835, while r was 0.919 for the The reproducibility of the refractometer colour correlation 100ÀL*/time &Table 2). method was studied on bread &n =7). The coef®cients of variation &CV) were 2.92%, 2.28% and 0.30% for a*, b* and L* respectively. The colour parameters Table 3. Absorbance at prolonged toasting timesa considered by other authors were DE for bread4 and Time min) A420 A284 corn and soy extruded products24 and 100ÀL* for pasta.18 Fernandez-Artigas et al 20 applied both par- 5 0.010Æ0.0004 0.034Æ0.0002 ameters to processed baby cereals. 7 0.025Æ0.001 0.153Æ0.001 10 0.026Æ0.001 0.209Æ0.002 14 0.029Æ0.001 0.520Æ0.005 Assay at prolonged toasting times 18 0.032Æ0.001 0.682Æ0.007 Table 1 illustrates the effects of toasting on the colour 21 0.057Æ0.002 0.741Æ0.008 values. The browning index &DE) for toasted bread 25 0.069Æ0.003 1.323Æ0.02 increased with heat treatment time in comparison with 30 0.132Æ0.005 2.260Æ0.04 untoasted bread. The linear correlation between DE 40 0.140Æ0.006 2.910Æ0.06 and time was r2 =0.913 &Table 2). When 100ÀL* 60 0.198Æ0.008 3.328Æ0.07 values were considered, the linear correlation was n =4. 2 r =0.962 &Table 2). a The toasted samples were measured against an The absorbance at 420nm measures the colour of untoasted sample.

J Sci Food Agric 81:513±518 &online: 2001) 515 A RamõÂrez-JimeÂnez et al

À1 Table 4. Colour parameters at reduced toasting times times and 1.3mg kg HMF for the bread used in the assay at reduced toasting times. The different HMF Time min) a* b* L* 100ÀL* DE values are due to the presence of crust in the sample 0 0.65 13.40 86.15 13.85Æ0.07 Ð with higher content. 0.5 0.65 13.50 86.07 13.93Æ0.04 0.14Æ0.02 1 0.70 13.50 85.75 14.25Æ0.04 0.42Æ0.03 2 0.70 13.55 85.73 14.27Æ0.08 0.45Æ0.10 Assay at prolonged toasting times 3 0.75 13.55 85.56 14.44Æ0.08 0.62Æ0.09 The toasting of sliced bread &25g) for 10min more 4 1.15 14.65 85.22 14.78Æ0.13 1.64Æ0.15 than doubled the HMF content &26mg kgÀ1) &Table 5 1.40 15.65 84.57 15.43Æ0.17 2.88Æ0.04 5). The HMF content increased by approximately À1 n =4. 2000mg kg after toasting for 40min &Table 5). The linear correlation between HMF and time was r2 =0.866 &Table 2). After 14min, when the loss of The 100ÀL index showed a better correlation with * moisture is very slight, the values ranged from 78 to time than did DE. Therefore 100ÀL may be a more * 2024mg kgÀ1. Using an HMF value determined by suitable parameter in such studies. The absorbance at HPLC and by applying the coef®cient of molar 420nm is a simple measure for the browning index of absorptivity &ε=16830), it is possible to calculate a toast and could be useful for rapid control of the theoretical absorbance value at 284nm for the HMF toasting process. determined by HPLC. After 30min the contribution

of the HMF to the A284 value was 74%, at 40min it HMF was 97% and at 60min it was 94%. After 30min of The identity and purity of the chromatographic peak toasting, HMF is probably produced by caramelisa- were con®rmed by diode array detection. The preci- tion and is presumably not a result of the Maillard sion &n =7) for bread with 3.41mg kgÀ1 HMF was reaction. 1.57% &CV), increasing to 2.60% for high concentra- tions &176.1mg kgÀ1). No HMF was detected in the dough. This suggests that the increase in this par- Assay at reduced toasting times ameter during the heat-processing step may be a useful The HMF values increased with the heating from indicator. The baking of the bread loaf was performed 1.3mg kgÀ1 &t =0) to 4.2mg kgÀ1 &t =5min) &Table at 200°C for 50min and produced 11.8mg kgÀ1 HMF 6). Thus an increase in HMF content can still be for the bread used in the assay at prolonged toasting observed when the toasting times are reduced. The

Furosine DNP-L Time min) HMF mg kgÀ1) mg kgÀ1 mg per 100g protein a gkgÀ1 protein a Loss %) 0 11.8Æ0.23 242.7 222.7Æ4.0 17.5Æ0.3 Ð 5 14.8Æ0.28 290.1 266.1Æ4.7 13.7Æ0.2 21.7 7 20.7Æ0.31 326.4 299.4Æ4.9 13.6Æ0.5 22.3 10 26.0Æ0.30 289.6 265.7Æ4.4 13.5Æ0.3 22.9 14 78.1Æ0.78 201.6 185.0Æ3.1 11.4Æ0.5 34.9 18 168.4Æ1.59 144.0 132.1Æ2.2 11.5Æ0.4 34.3 21 195.3Æ1.75 86.4 79.3Æ1.0 10.4Æ0.3 40.6 25 408.7Æ3.74 36.0 33.0Æ0.7 8.9Æ0.2 49.1 30 1096.4Æ7.60 18.0 16.5Æ0.3 8.2Æ0.4 53.1 40 1853.7Æ10.1 7.2 6.6Æ0.1 8.3Æ0.5 52.6 Table 5. HMF, furosine and available lysine 60 2024.8Æ15.3 3.2 2.9Æ0.1 8.5Æ0.4 51.4 contents at prolonged toasting times a (expressed on a dry matter basis) NÂ5.70; n =4.

Furosine DNP-L Time min) HMF mg kgÀ1) mg kgÀ1 mg per 100g protein a gkgÀ1 protein a Loss %) 0 1.3Æ0.02 106.5 97.8Æ1.7 19.6Æ0.01 Ð 0.5 1.6Æ0.02 72.3 66.3Æ1.3 19.0Æ0.21 3.1 1 1.6Æ0.02 62.3 57.2Æ1.5 19.0Æ0.01 3.1 2 1.9Æ0.03 100.6 92.4Æ1.1 18.9Æ0.13 3.6 3 2.6Æ0.04 110.5 101.4Æ0.8 17.4Æ0.21 11.2 4 3.2Æ0.05 122.2 112.2Æ1.0 17.4Æ0.22 11.2 Table 6. HMF, furosine and available lysine 5 4.2Æ0.06 138.9 127.4Æ1.5 17.1Æ0.17 12.8 contents at reduced toasting times a (expressed on a dry matter basis) NÂ5.70; n =4.

516 J Sci Food Agric 81:513±518 &online: 2001) Browning of sliced bread linear correlation between HMF and time was Studies carried out by the authors8 on wheat ¯our r2 =0.952 &Table 2). toasted at 140°C showed an increase in furosine A study of the HMF content in commercial toasted content from 10.6 to 14.3mg per 100g protein. sliced breads27 determined values between 13 and During this heat treatment the moisture content fell 90mg kgÀ1, with a mean value of 30mg kgÀ1. Our from 13.2% to 1.7% after toasting. A study with two corresponding values were obtained at 10±14min of toasting temperatures showed furosine values of 15.4 toasting. Toasting times of 5±20min &frequently used and 11.1mg per 100g protein at 140 and 150°C for this type of bread) show a linear increase with a respectively. The results obtained from the two studies shallow slope. A steeper slope can be observed &¯our and toasted bread) are basically similar. Early between 20 and 40min. The HMF value at 60min stages of the Maillard reaction are not favoured when was slightly higher than at 40min. Heat treatment the water content is very low, even if there are until the product was nearly burnt reduced the HMF precursors. The furosine degradation was linear until value to 1337mg kgÀ1. 25min of heating &r2 =0.994). A high correlation between HMF and 100ÀL* &colour index) was found in the study at reduced Available lysine with FDNB toasting times &r2 =0.954). A good correlation Available lysine was determined in untoasted bread to &r2 =0.871) was also found during the assay at assess the heat damage produced in the toasting of prolonged toasting times. Colour index and HMF sliced bread. The losses of lysine during heating were are adequate indicators to control the toasting time. determined with respect to sliced bread before toasting Studies carried out on the toasting of single and &Tables 5and 6). mixed wheat-based and rice-based ¯ours gave values of 1±5mg kg À1 HMF.20 In commercial breakfast Assay at prolonged toasting times cereals21 the HMF values ranged from 4 to 193mg The contents of available lysine decreased from kgÀ1. Acquistucci and Bassotti28 and Resmini et al 18 17.5gkg À1 protein &t =0) to 8.5gkg À1 protein found 7.0 and 0.45mg kg À1 HMF respectively during &t =60min) &Table 5). From 25min the losses of pasta drying. lysine were 50%, and subsequently no increase was

The reproducibility of the A284 value was studied observed. The correlation between available lysine and with seven samples of sliced bread at two times of time was r2 =0.694 &Table 2). toasting &5and 40min). The coef®cients of variation &CV) were 0.68% and 2.10% respectively. The Assay at reduced toasting times absorbance of water-soluble compounds at 284nm Available lysine contents decreased at reduced times of &Table 3) showed a linear increase with toasting time heating from 19.6 to 17.1gkgÀ1 protein. The losses of 2 &r =0.927). The correlation between A284 and HMF lysine ranged from 3.1% to 12.8% at 5min of heating was 0.964 &Table 2). A284 is a rapid and sensitive &Table 6). parameter and could be an adequate browning index Losses of lysine of around 40% have been found in for such samples. bread toasted for 20min which exhibited good organoleptic quality. Furosine Considerable heat damage has been reported in Assay at prolonged toasting times heated cereal products such as pasta, breads, breakfast The furosine content in baked bread &t =0) was cereals and biscuits.5 Lightly toasted extruded wheat 223mg per 100g protein &Table 5). Toasting the breakfast cereals showed no decrease in lysine, while sliced bread produced an increased furosine value after darkly toasted ones suffered a 50% loss of available 5and 7min of treatment. Furosine levels fell after lysine.29 During the industrial production of wheat- 10min. The reducing sugars were determined in the based and rice-based baby cereals, available lysine was samples, and the content was between 4.72% for affected by the toasting and drying processes. Con- baked bread &t =0) and 1.68% for toasted sliced bread siderable reduction was observed as a result of the &t =60min). The reducing sugar content when the roller-drying process.16 furosine value began to decrease was 3.5%. The highest loss of moisture was reached at 10±14min of heat treatment. Furosine levels began to decrease CONCLUSIONS when the moisture content fell below 10%. The linear Studies of the relationship between browning indica- correlation between furosine and time was r2 =0.712 tors and times of prolonged toasting &Table 2) showed

&Table 2). high correlations of A420, A284, 100ÀL* and HMF with time. In studies at reduced toasting times, Assay at reduced toasting times available lysine also showed a high correlation with The furosine value at t =0 was 97.8mg per 100g time. HMF is the best indicator of browning at usual protein &Table 6). Heating for 0.5and 1min produced toasting times. Absorbance measurement is a simple a decrease in furosine values, but from 2min the and rapid method and therefore could be a useful furosine content increased. The correlation between technique for rapid control of the industrial process. furosine and time showed an r2 value of 0.610. When moisture loss during heating is greatest, the

J Sci Food Agric 81:513±518 &online: 2001) 517 A RamõÂrez-JimeÂnez et al

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