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Effect of sodium selenite addition and sponge dough fermentation on selenomethionine generation during production of yeast-leavened breads
Journal of Cereal Science 58 (2013) 164e169
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Effect of sodium selenite addition and sponge dough fermentation on selenomethionine generation during production of yeast-leavened breads Marco A. Lazo-Vélez, Víctor A. Gutiérrez-Díaz, Alicia Ramírez-Medrano, Sergio O. Serna-Saldívar* Centro de Biotecnología-FEMSA, Escuela de Biotecnología y Alimentos, Tecnológico de Monterrey, Monterrey, N.L., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2012 Received in revised form 6 March 2013 Accepted 25 March 2013
The effect of sodium selenite addition and fermentation times on production of selenomethionine (SeM) during sponge bread production was evaluated. Doughs were supplemented with sodium selenite (Na2SeO3) and fermented with yeast, avoiding addition of sulfur salts. The effect of Na2SeO3 on yeast activity was evaluated using a pressurometer. Results showed that there were not statistical differences (p < 0.05) in CO2 production and dough pH at all the sodium selenite concentrations tested. HPLCfluorescence data showed that SeM production was higher with the increase of fermentation times, while less significant effects were observed due to changes in Na2SeO3 concentration. Two slices of Seenriched bread can provide about 200 mg SeM, the dose recommended to prevent cancer and oxidative stress. The physical features (water absorption, bread weight, bread volume, color, density, oven spring, etc.) and organoleptic evaluations for the enriched loaves were evaluated. In all these parameters, the experimental enriched breads had practically identical attributes compared to the control. The breads rich in SeM have potential to be used as functional foods because this amino acid is synthesized into higher quantities of glutathione peroxidase, an enzyme considered as one of the most protective mechanisms against oxidative stress and preventive of cancer and chronic diseases. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Selenium-enriched bread Dry instant yeast Selenomethionine Sponge dough
1. Introduction Cereal-based foods are the most important for mankind because they provide from 30 to 60% of the intake and are considered as the major suppliers of carbohydrates and calories. The consumption of cereals is comparatively higher in developing countries around the globe. Epidemiological studies clearly show that whole cereals have a protective effect against chronic diseases that are responsible for most of the deaths in the western world (Poutanen, 2009). Among the wide array of cereal-based products, bread is still considered as the main staple food for most of the inhabitants in the world. The production of bread is considered as one of the oldest biotechnological processes developed (Fleet, 2006).
Abbreviations: ANOVA, analysis of variance; DM, dry matter; HPLC, high pressure liquid chromatography; SeM, selenomethionine. * Corresponding author. Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico C.P. 64849, Monterrey, N.L., Mexico. Tel.: þ52 81 8358 1400; fax: þ52 81 8328 4262. E-mail address: [email protected] (S.O. Serna-Saldívar). 0733-5210/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2013.03.019
Undoubtedly, the nutrients and nutraceuticals associated with bread can impact the health status of consumers. White bread is considered as an excellent source of digestible energy and intermediate quality protein whereas whole grain and variety breads are additionally rich in dietary fiber and phenolics. The bread also contains important amounts of some micronutrients intrinsically associated with wheat and yeast and purposely added to refined flours (Fe, Zn, thiamin, riboflavin, niacin and folic acid) (WHO et al., 2009). It is documented that flours and bread vary in the concentration of the trace mineral selenium according to the growing location of the wheat and the flour extraction rate. Selenium (Se) is considered an essential micronutrient because it is required for the synthesis of important antioxidant proteins such as cytosolic glutathione peroxidase (Combs et al., 2011). This enzyme protects mammalian systems from oxidative stress and has been related to prevention of chronic diseases and cancer (Beckett and Arthur, 2005). The bioavailability of Se is higher when associated to selenoproteins (Katsuhiko et al., 2000). These proteins are synthesized from selenocysteine (SeC), obtained after the reversible methylation of SeM stored in different tissues. The SeM is considered the main source of organic selenium in foods and the moiety with the highest bioavailability.
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An analysis of diets in the U.S. by Schubert et al. (1987) revealed that five foods, including white bread, contributed about 50% of the total selenium intake, and that 80% of the total dietary Se was provided by only 22 core food items (Schubert et al., 1987). The supplementation of Se salts to yeast cultures enhances the conversion of inorganic Se into organic sources. The amount synthesized will vary according to the yeast food composition especially in terms of the Se/S ratio (Ouerdane and Zoltán, 2008; Mapelli et al., 2012). Bakery yeast (Saccharomyces cerevisiae) is capable of metabolizing up to 3000 mg Se/g converting approximately 90% of the selenium into L-SeM and leaving traces of the inorganic form (California Environmental Protection Agency, 2010). Therefore, yeast-leavened bakery products could be an ideal carrier for the production and supplementation of organic selenium (Ouerdane and Zoltán, 2008). The advantage of bread is that without an extensive modification of the process, it can yield significant amounts of organic selenium and be mass produced. The Se-enriched breads may contain a high Se concentration: the 100 g wheat roll prepared with selenium-enriched yeast contained 50 mg Se in the form of SeM (Stabnikova et al., 2008), compared to 3e6 mg/100 g commonly seen in regular counterparts (Bryszewska et al., 2005; Ramiro-Anaya, 2011). However, the uncontrolled addition of Se may modify the sensory characteristics and overall acceptability of breads. The feasibility of producing SeM enriched breads has not been properly addressed in terms of manufacturing procedures and bread quality. This research was undertaken in order to investigate the effects of sodium selenite addition as yeast food in order to obtain SeM enriched breads manufactured from sponge doughs fermented with dry instant yeast (S. cerevisiae). 2. Materials and methods 2.1. Sulfur-free yeast food preparation The sulfur-free yeast food was prepared according to the procedure described by Anghileri and Márquez (1965) with minor modifications. Briefly, a first solution was prepared by mixing 3.5 g di-ammonium phosphate, 0.1 g magnesium chloride, 0.8 g zinc acetate, 0.2 mg iron(III) chloride, 50 mg copper (II) chloride (D.E.Q Monterrey, N.L., Mexico), 1.0 g monosodium citrate (Sigmae Aldrich, Toluca Edo. Mexico, Mexico) and 0.8 L distilled water (Lab. Monterrey, Monterrey, N.L., Mexico). This pre-solution was treated at 121 C at 15 psi in an autoclave (Sterilizer SE-510, Yamato, Japan) for 15 min. The sterilized solution was cooled and stored aseptically. The second solution was prepared by diluting 2.5 g monohydrate-Lasparagine, 10 mg biotin, 0.5 mg calcium pantothenate, 10 mg inositol, 6 mg thiamine, 1 mg pyridoxine, 20 g glucose (Sigmae Aldrich, Toluca Edo. Mexico, Mexico), and 0.20 L distilled water. The two solutions were blended in a laminar flow hood (Labconco, Model 36212, Kansas City, MO, USA). The resulting yeast food solution was passed through 0.2 mm sterile filters, adjusted to pH 4.5e 5.0 (phosphoric acid 0.5 M) and stored at 4 C. The selenite salts were added to the sterile solution utilizing a stock solution of Na2SeO3 (SigmaeAldrich, Toluca Edo. Mexico, Mexico) to a concentration of 100 mg/mL. 2.2. Determination of the effects of sodium selenite concentration on yeast gassing power and dough pH The effects of different concentrations of Na2SeO3 on the CO2 gassing power of commercial dry instant yeast was tested in a pressurometer according to AACC method 22-11.01 (AACC, 2000) and Serna-Saldívar (2012) with slight modifications. Before the test, the flour was treated with ultraviolet light for 15 min with the aim
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of inactivating wild yeast and the associated microbial load. The sanitized flour was immediately packaged in sealed plastic bags and refrigerated at 4 C. Briefly, 35.15 g (14% moisture basis) of refined wheat flour and 0.23 g of dry yeast were first blended and allowed to rest for 5 min. Then, the flour-yeast blend was hydrated with 15.07 mL of sulfurfree yeast food and 9.47 mL distilled water in which different amounts of Na2SeO3 were dosed (1.9, 3.8 and 7.6 mg Se/g flour). The resulting blend was placed in the pressurometer, weighed and mixed with a spatula until attaining a soft and smooth slurry. The pressurometer was immediately sealed and tightened with a wrench and placed in a water bath (Hot-Saker 7746-12110, Bellco Glass Inc., Vineland, NY, USA) set at 28.5 2 C for 4.5 h. The gas in the pressurometer was released after the first 5 min. The pressure (mm Hg) generated by the gas produced by the yeast was measured for 90 min with a frequency of 10 min. The slurry pH was registered after three degassing steps of the pressurometer every 90 min during 4.5 h fermentation. The pH was measured with a potentiometer (SevenGo-2B, Mettler-Toledo, Schwerzenbach, Switzerland) equipped with a special dough probe (SEREX01072, SensoreXÒ, Garden Grove, CA, USA) 2.3. Bread making preparation The optimized sponge bread making AACC method 10-11.01 (AACC, 2000) was utilized with slight variations in formulation. The breads were manufactured in order to obtain commercial loaves for sensory and chemical analyses. The basic loaf formulation included 100 g (14% moisture basis) bread flour (10.7% protein and 0.4% ash), 5 g refined cane sugar (Avance, Avance Comercial de Monterrey, Monterrey, NL, Mexico), 4 g vegetable shortening (Inca, Unilever de Mexico S.A de C.V., Tultitlán, Edo. de Mexico, Mexico), 2 g refined iodinated salt (La Fina, Sales del Istmo, Coatzacoalcos, Veracruz, Mexico), 2 g dry yeast (Saccharomyces cereviceae) (AztecaÒ Levadura, Iztapalapa, Mexico, D.F., Mexico), 2 g dry milk (Nestle de Mexico, Mexico, D.F., Mexico), 0.2 g diastastic malt (SudChemic de Mexico, Puebla, Pue., Mexico) and 0.5 g lecithin (Proveedores de Ingeniería Alimentaria S.A. de C.V. Monterrey, N.L., Mexico). Bake absorption, mixing time, proof height, loaf height, oven spring, loaf volume, loaf apparent density and crumb grain texture were determined. Sponges were fermented for 8, 16 or 24 h with different Na2SeO3 concentrations: 0 (control), 4.33, 7.83 and 23.8 mg Se/g as Na2SeO3. The sponge stage consisted of mixing by hand 80 g of refined bread flour, 3 g instant dry yeast and 48 mL of the yeast food solution that contained the different Na2SeO3 concentrations. The resulting sponge dough was placed in a plastic container in a fermentation cabinet (National Manufacturing Co., Lincoln, NE, USA) set at 27 2 C for the predetermined fermentation time. Resulting sponges were mixed with the dough stage ingredients: 120 g bread flour, 10 g sugar, 8 g shortening, 4 g of dry whole milk, 4 g salt, 0.4 g diastatic malt, 0.4 g lecithin, 1 g dry yeast instant and 72 mL water for 2 min at low speed in a Hobart mixer equipped with the hook attachment. The speed was switched to medium until attaining optimum dough development. Optimum mix times were subjectively determined by observing dough properties or gluten development (film formation, gloss and dough stickiness). Resulting doughs were weighed and cut into two identical parts before placing them in a fermentation cabinet set at 27 2 C and 85% relative humidity. After 30 min resting, doughs were punched thru 0.56 cm roll spacing and dough sheets folded in half and in half again and returned to the fermentation cabinet for two additional 15 min fermentation periods. Doughs were punched through 0.95 cm roll spacing in preparation for molding and panning in 7 cm height metal pans that had the following dimensions on the base and top, respectively: 33 and 35 cm long and
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10.5 and 14.5 cm wide. Baking pans were previously greased with vegetable shortening on the bottom and sides. Panned doughs were proofed for 45 min before baking for 25 min in an oven (Electrolux EOG Gas single oven X 601) set at 220 C. The height of proof doughs and breads were measured at the center of the loaf in order to calculate oven spring. In addition, loaves were weighed and volumes determined by rapeseed displacement (National Manufacturing Co., Lincoln, NE, USA). Finally, breads were packed in sealed polyethylene bags and stored at room temperature. 2.4. Determination of bread crumb color Crumb color was objectively measured with a colorimeter (CR 300, Minolta, Japan) standardized with a white reference plate (L ¼ 92.90, a ¼ 1.05, b ¼ 0.82). Bread crumb color parameters measured were L*, a*, b*. 2.5. Baking commercial procedure Se-enriched white breads were manufactured in a pilot test using sponge doughs fermented for 16 h with a concentration of 3.5 mg Se/kg flour as Na2SeO3. Two slices or servings (72 g) of this enriched bread contained 200 mg of total Se. 2.6. Sensory analyses of the breads Twenty one untrained panelists evaluated the sensory characteristics of breads using triangle testing that were conducted with one-day old control and selenized breads. Briefly, each panelist was given simultaneously three coded samples (two samples equal and one different) along with a ballot. When all panel members completed the trial, their ballots were marked as correct when they identified the different bread sample or as incorrect when they failed to identify the odd sample. Using the binomial test from oneend, the total number of panelists with correct answers with the total number of the observations was compared (Watts et al., 1995). 2.7. Detection and quantification of SeM by HPLC-fluorescence 2.7.1. Sample preparation The bread obtained from different experiments was sliced and dried for 3 h in an oven (Electrolux EOG 601 X Gassingle oven) at a temperature of 50 C. All dehydrated samples were reduced to a fine powder in a cutter mill (Standard Mod N03, Philadelphia, PA, USA) equipped with a 1 mm sieve. The final product was transferred into labeled polyethylene bags with airtight seal and frozen at 20 C. 2.7.2. In vitro gastrointestinal digestion method Bread samples were subjected to the in vitro digestion method previously described by Pasini et al. (2001). Briefly, 60 mg of ground sample were suspended in 4 mL of 0.2 N HCl (pH 2.0) and 0.05 mg/ mL of porcine pepsin P-7000 (SigmaeAldrich, Toluca Edo. Mexico, Mexico) to emulate gastric protein hydrolysis. The final concentration of protein was measured at 1.87 mg/mL with a ratio of enzyme/protein of 1:37 (w/w). The control samples were treated the same way but without pepsin. The digestion was carried out for 1 h at a temperature of 37 C in a shaking water bath (Hot-Shaker 7746-12110, Bellco Glass Inc., Vineland, NJ, USA) adjusted to a stirring speed of 28 rpm. The stock solution was prepared 50/50 (v/ v) with boric acid (DEQ, Monterrey, N.L., Mexico), previously heated to a concentration 1.0 M and a 0.5N NaOH solution (JT Baker, Xalostoc, Tlaxcala, Mexico). This stock solution was adjusted to pH 7.8 with HCl (5 N) and subsequently treated with 0.25 mg/mL pancreatin (SigmaeAldrich, Toluca, Edo. Mexico, Mexico). An aliquot of 1.15 mL of the pancreatin solution was added to the
pepsin-hydrolyzed bread solution. The resulting pH was recorded at 7.5, with a ratio pancreatin/protein of 1:17 (w/w). The biocatalytic reaction was allowed to proceed at 37 C in a shaking water bath (Hot-Shaker 7746-12110, Bellco Glass Inc., Vineland, NJ USA) and stopped after 120 min by inactivation in a hot water bath (80 C) for two min. After standing for 1 h, the samples were centrifuged for 10 min at 10000g and 4 C (SL16R, Thermo Scientific, Germany). The supernatant and pellet were separated and then analyzed for SeM. If SeM was not detected in the pellet, the supernatant was sonicated (Branson Ultrasonic Corporation, Danbury, CT, USA) for 10 min. The sonicated sample was filtered through a 0.45 mm sterile filter (Corning, NY, USA) into 1.5 mL vials, which were immediately stored at 20 C. 2.7.3. Selenomethionine analysis The SeM was analyzed after the in vitro gastrointestinal digestion method described above, according to the Waters AccQTag Amino Acid Analysis MethodÔ (Millipore Corporation; Milford, MA, USA). SeM was derivatized with the Waters AccQFluor reagent and separated by reversed-phase high performance liquid chromatography in an AccQTag column (Waters Nova-PakÔ C18 4 mm, 3.9 150 mm column). A Waters HPLC system equipped with a 1525 Binary pump and Multi Fluorescence detector (Waters 2475) was used. The external calibration standard containing SeM (Sigmae Aldrich, Toluca Edo. Mexico, Mexico) was prepared according to the instruction manual of Waters (Millipore Corporation, 1993). 3. Statistical analysis The data for breads are presented as means standard deviations of determinations. The difference of SeM content for each loaf was analyzed by one-way analysis of variance (ANOVA). Multiple comparisons of means were performed using Tukey HSD tests at a level of significance of P < 0.05. Data obtained from the pressurometer were analyzed using ANOVA procedures and correlated using Pearson correlations. All computations were made by the statistical software’s Minitab (version 15) and JPM (version 10.2). 4. Results and discussion 4.1. Effect of sodium selenite on carbon dioxide generation by yeast The CO2 generation capacity of yeast was registered at different fermentation times (Fig. 1). A highly significant correlation among all experimental treatments and the control (all >r ¼ 0.99) was observed. The gas volumes generated by the control fermented for
Fig. 1. Effect of different concentrations of sodium selenite on carbon dioxide generation by dry instant yeast (S. cerevisiae). Evaluated in a pressurometer at a temperature of 28 2 C for 4.5 h (n ¼ 3; error bars indicate standard deviation). Pearson correlation: r ¼ 1 [1.90 mg Se/g ], r ¼ 0.99 [3.80 mg Se/g ], r ¼ 1 [7.60 mg Se/g ]1.
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4.5 h was 360.1 mL whereas, for treatments supplemented with the three increasing Na2SeO3 concentrations, were 344.1, 320.2 and 339.7 mL, respectively. These data clearly indicate that the three tested Na2SeO3 concentrations did not significantly affect final CO2 production (p < 0.05). Stabnikova et al. (2008) reported that yeast growing in cultures containing 2e5 mg Se/mL as NaHSeO3 did not differ from the control treatment. 4.2. Effect of sodium selenite on dough pH The comparison of the control dough pH after 4.5 h fermentation at 28.5 2 C in the pressurometer with doughs containing 1.9, 3.8 and 7.6 mg Se/g flour (Fig. 2) indicated a highly significant correlation for the first two concentrations (r ¼ 0.97, 0.93) and significant for the highest concentration (p < 0.05 and r ¼ 0.76). The addition of the different concentrations of Na2SeO3 to the slurry in the pressurometer did not significantly affect dough pH (p > 0.05). As expected, the pH values decreased significantly (p < 0.05) throughout the different fermentation times. The pH values in the pressurometer were within the expected range for the optimum yeast activity and CO2 generation (4e5.8) (Fig. 2). Likewise, the sponge doughs fermented for 8, 16 and 24 h with Na2SeO3 concentrations of 4.33, 7.84 and 23.8 mg Se/g flour had pH with strong correlations with respect to the control sponge dough (r ¼ 0.95; 0.99 y 0.98) (data not shown). When sponge dough pH values were compared, the different selenite supplemented doughs tended to have slightly significant different pH as fermentation proceeded. However, the pH values were not significantly different (p < 0.05) from the control tested at 24 h fermentation. The sponge dough lowest pH observed after 24 h fermentation was 5.1 for the control and 5.21 for the experimental treatment supplemented with 23.8 mg Se/g flour) (data not shown). Under these conditions, it is reported that gluten retains the maximum amount of gas (Calveras, 2004; Cauvain and Young, 1998; Romano et al., 2006). It is also known that the maximum selenite incorporation into the yeast cells occur at the pH values (4.5e5) achieved (McDermott et al., 2010). Interestingly, the dough pH decreased to <4.3 in sponge doughs fermented for 16 or 24 h with selenite concentrations > of 7.82 mg Se/g. (data not published). These doughs acquired a pink-reddish coloration and the bread crumb retained these colored dots after baking. This is due to the formation of elemental selenium owing to reduction reactions at the acidic pH values (Combs and Combs, 1996). This phenomenon was also observed in yeast biomass
Fig. 2. Effect of selenium in the form of sodium selenite concentrations on pH values of doughs fermented with dry instant yeast (S. cerevisiae). Evaluated in the pressurometer at a temperature of 28 2 C for 4.5 h (n ¼ 3; error bars indicate standard deviation). Pearson correlation: r ¼ 0.97 [1.90 mg Se/g ], r ¼ 0.93 [3.80 mg Se/g ], r ¼ 0.76 [7.60 mg Se/g ] 1.
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cultured with concentrations higher than 10 mg Se/mL (Stabnikova et al., 2008). Alzate et al. (2007) described a similar color reduction mechanism when producing yogurt containing 50 mg Se/g and higher. The yogurt fermented with Lactobacillus suffered a reduction of the Se (IV) to Se0, which produced a reddish coloration. 4.3. Properties of breads The addition of the different concentrations of Na2SeO3 to sponge doughs did not significantly affect bread color (Table 1). The comparison of the control dough proof height produced from sponges fermented for 8 or 16 h were not significantly different from all experimental treatments. Nevertheless, the sponges fermented for 24 h with Na2SeO3 concentrations of 7.84 and 23.8 mg Se/g flour produced doughs with proof heights significantly lower compared to the control counterpart. On the other hand, the comparison of the apparent densities of experimental breads containing 23.8 mg Se/g flour obtained from sponges fermented for 24 h were significantly higher compared to the control bread. The observed differences in the last two parameters may be due to a lower yeast activity during proofing and the first stage of baking. The oven spring of Se enriched breads manufactured from sponge doughs fermented for 24 h was negatively affected, especially in those loaves supplemented with the highest concentration of sodium selenite. The bread volume was slightly lower in breads supplemented with 23.8 mg Se/g in all the sponge dough fermented for different times (737e754 cm3) (Table 1). Breads supplemented with 7.84 mg Se/g flour and manufactured from sponge doughs fermented for 16 or 24 h also had slightly different volumes (750e 790 cm3). The rest of the experimental and control breads developed similar bread volumes (820e860 cm3) (p > 0.05). For pup loaves produced from 100 g of 14% moisture based flours, the optimum bread volumes were between 850 and 925 cm3 (SernaSaldívar and Abril, 2011). Garvin et al. (2011) reported volumes between 750 and 850 cm3 for breads produced from patent flour rich in Se (0.53e7.56 mg Se/g flour). The bread volumes obtained herein are similar to the ones reported by Garvin et al. (2011) and are considered adequate. The bread volumes can be improved by the use of oxidizing agents such as ascorbic acid or potassium bromate, a higher protein flour and/or vital gluten (Serna-Saldivar and Abril, 2011, 2012). The addition of 0.5% more instant dry yeast (not Se-enriched) during the dough stage mixing helped to prevent low bread volume in loaves prepared with less than 4 mg Se/g flour. 4.4. Selenium-methionine content of breads determined by HPLCfluorescence Preliminary studies indicated that the peak and elution time for a SeM standard was 32.80 0.02 min (Fig. 3). The SeM did not coelute with any other amino acid present in the standard solution. The identification of SeM was reconfirmed by the use of an internal standard containing additional known quantities of this specific amino acid. Breads manufactured from sponge doughs supplemented with different concentrations of Na2SeO3 (4.33e23.8 mg Se/g) contained between 120 and 250 mg Se/100 g bread (DM.) as SeM (Fig. 4). Statistical analysis indicated that the concentration in bread was mainly affected by fermentation time and to a lesser extent by the original Na2SeO3 concentration; the SeM concentration significantly increased (p < 0.05) with prolonged sponge dough fermentation times. On the other hand, the different sodium selenite concentrations tested did not significantly affect SeM concentration. As expected, all experimental breads contained significantly higher concentrations of SeM when compared to the
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Table 1 Effects of different concentrations of Se in the form of Na2SeO3 and fermentation times in the physical characteristics of breads prepared with a sponge dough procedure. Concentration [Se as SeO3Na2]
Proof height (cm)
Loaf height (cm)
Oven springa (cm)
Bread weight (g)
Bread volume (cm3)
Apparent density (g/cm3)
Color crumb (dE*ab)
Fermentation time ¼ 8 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.17 7.30 7.23 7.27
0.1A 0.1A 0.2A 0.2A
9.27 9.27 8.90 8.97
0.12A 0.06A 0.20A 0.15A
1.29 1.27 1.23 1.23
0.03A 0.01A 0.02A 0.05A
148.00 151.33 148.67 152.00
0.00A 4.62A 6.11A 2.00A
860.0 820.0 800.0 741.7
17.32A 17.32A 43.59A,B 24.66B
0.17 0.18 0.19 0.21
0.003A 0.008A,B 0.015A,B 0.005B
1.94 1.39 1.73 1.40
0.45A 0.15A 0.50A 0.21A
Fermentation time ¼ 16 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.20 7.33 7.13 7.20
0.1A 0.2A 0.1A 0.1A
9.33 9.43 8.97 8.67
0.15A,B 0.12A 0.21A,C 0.06C
1.30 1.29 1.26 1.20
0.02A 0.02A 0.03A,B 0.02B
148.00 148.67 149.33 149.33
2.00A 1.15A 2.31A 2.31A
846.7 821.7 788.3 753.3
15.28A 10.41A,B 54.85A,B 5.77B
0.17 0.18 0.18 0.19
0.010A 0.004A,B 0.002A,B 0.004B
2.60 1.71 2.32 1.60
0.27A 0.45A 0.33A 0.76A
Fermentation time ¼ 24 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.53 7.93 7.40 7.23
0.1A 0.1A,B 0.3B 0.1B
9.53 9.43 8.83 8.10
0.12A 0.25A 0.29B 0.1C
1.27 1.19 1.20 1.12
0.01A 0.02A,B 0.07A,B 0.01B
141.67 148.00 147.33 145.67
2.89A 2.00A 3.06A 2.52A
820.0 850.0 750.0 736.7
20.00A 20.00A 10.00B 11.55B
0.17 0.17 0.19 0.19
0.002A 0.003A 0.003B 0.004B
2.13 1.76 2.09 1.46
0.07A 0.24A 0.77A 0.15A
Means standard deviation with the levels not connected by same letter are significantly different at p < 0.05. a Oven spring ¼ bread height proof height.
control or unsupplemented bread (36.71 0.78 mg Se/100g bread). The concentration of Na2SeO3 was not relevant in terms of the final amount of SeM in breads produced with 2% yeast. However, if more SeM concentration is desired, the best way to achieve it is through the supplementation of additional amounts of yeast especially during the sponge dough stage. The bread that contained the highest SeM concentration was the one manufactured from a sponge dough supplemented with 23.8 mg Se as Na2SeO3/g and fermented for 24 h. The bread contained 5.6 and 1.7 times more SeM concentrations compared to the control (37.59 mg Se/100g bread) and counterparts produced from sponge doughs fermented for 8 h (126.24 mg Se/g bread). Also, breads produced from sponge doughs fermented for 8e16 h and 16e24 h incremented SeM by a factor of one for each of these periods. Most of the commercial organic selenium is obtained from yeast (S. cerevisiae) cultured in a medium rich in inorganic selenium. It is reported that the reactor selenized yeast production yields from 500 to 2124 mg Se/g DM (Nagodawithana and Gutmanis, 1985; Ponce de León et al., 2002). Stabnikova et al. (2008) produced enriched selenized breads with 55 mg of Se as SeM from yeast cultures previously supplemented with inorganic sodium hydroxyselenite. A theoretical comparison of selenized proteins generated in a yeast bio-fermentation reactor or bread doughs indicate that
Fig. 3. Identification and elution time of selenomethionine in a chromatograph obtained after HPLC-Fluorescence analysis of enzyme hydrolyzed bread samples. Analyzed with injection volume of 10 mL, wavelength of 250 nm emission, 395 nm excitation and linear gradient. TYR: Tyrosine; VAL: Valine; MET: Methionine; SeM: Selenomethionine; LYS: Lysine; ILE: Isoleucine; L: Leucine.
these two contrasting processes generate equivalent amounts of SeM. These earlier studies that produced breads with selenized yeast cultured for 72 h with 5 mg Se as hydroselenite/mL yielded an enriched yeast with 154 mg Se/g dry biomass; the breads manufactured with 1.5% of the Se enriched yeast contained 55 mg Se as SeM/100 g bread (Stabnikova et al., 2008). A comparison of the SeM concentrations of SeM of the different bread systems indicated that the bread produced with the lowest sodium selenite (4.33 mg Se/g) concentration fermented for 24 h contained z200 mg Se/100 g fresh bread. Therefore, this particular bread system contained approximately 4 times more SeM compared to the breads produced by Stabnikova et al. (2008). The commercial fermentation processes aimed towards the production of selenized yeast generally cultured for 24e72 h (Nagodawithana and Gutmanis, 1985) was equivalent to the 24 h sponge dough process described herein. The advantages of sponge doughs are that they require fewer controls and are performed in a solid state. Furthermore, the food grade dough can be dried in order to produce an enriched protein selenized product. Moreover, the amounts of selenized proteins generated during sourdough
Fig. 4. Effect of yeast (S. cerevisiae) fermentation time and concentration of sodium selenite on the production of selenomethionine in different loaves of bread. SeM biosynthesis was favored by the increase in fermentation times, with less significant changes in concentration of sodium selenite. Letters comparing selenite concentration means denote significant differences (at p < 0.05). Within each fermentation time cluster.
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fermentation can be higher because these types of breads are produced using higher fermentation times. Thus, the yeast in the sponge dough was very efficient in the transformation of sodium selenite into SeM especially when fermented for more than 16 h. 4.5. Sensory evaluation The triangle tests clearly indicated that the SeM enriched breads had similar color, texture, flavor and overall acceptability compared to the unsupplemented control bread (p < 0.005). 5. Conclusions This research clearly showed that it was feasible to produce breads with high content of SeM by preparing sponge doughs fermented for less than 16 h with dry instant yeast and supplemented with less than 23.8 mg Se as SeO3Na2/g flour. The resulting enriched breads were practically identical to regular counterparts and consumers were not capable of perceiving sensory differences. The consumption of two slices of selenium-enriched bread (36 g for slice) produced from sponge doughs fermented for 24 h with only 4.33 mg Se as SeO3Na2/g flour can provide approximately 200 mg of SeM, a dose which has proven to have chemopreventive effects in epidemiological studies (California Environmental Protection Agency, 2010). Acknowledgments Authors acknowledge Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico, Secretaría Nacional de Estudios Superiores, Ciencia, Tecnología e Innovación (SENESCyT), Ecuador and the Nutrigenomic Research Chair of Tecnológico de Monterrey which provided research and support funds for graduate students. References AACC, 2000. Approved methods of the American association of cereal chemists. In: Methods 10-11.01 and 22-11.01, eleventh ed. AACC International, St. Paul. Alzate, A., Cañas, B., Pérez-Munguía, S., Hernández-Mendoza, H., Pérez-Conde, C., Gutiérrez, A.M., Cámara, C., 2007. Evaluation of the inorganic selenium biotransformation in selenium-enriched yogurt by HPLC-ICP-MS. Journal of Agricultural and Food Chemistry 55, 9776e9783. Anghileri, L.J., Marquez, R.G., 1965. Preparación biosintética de selenometionina y selenocisteina, marcados con 75Se. Su incorporación y distribución en ratones. Reporte Técnico NP-15594. Comisión Nacional de Energía Atómica, Buenos Aires. Beckett, G.J., Arthur, J.R., 2005. Selenium and endocrine systems (review). Journal of Endocrinology 184, 455e465. Bryszewska, M.A., Ambroziak, W., Diowksz, A., Baxter, M.J., Langford, N.J., Lewis, D.J., 2005. Changes in the chemical form of selenium observed during the manufacture of a selenium-enriched sourdough bread for use in a human nutrition study. Food Additives & Contaminants 22, 135e140. California Environmental Protection Agency, 2010. Public Health Goal for Selenium in Drinking Water. Pesticide and Environmental Toxicology Section, Office of Environmental Health Hazard Assessment, Berkely.
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Calveras, J., 2004. Nuevo tratado de panificación y bollería, second ed. Ediciones Mundi-Prensa, Madrid. Cauvain, S.P., Young, L.S., 1998. Fabricación de Pan, second ed. Editorial ACRIBIA S.A, Zaragoza. Combs, G.F., Jackson, M.I., Watts, J.C., Johnson, L.K., Zeng, H., Idso, J., Schomburg, L., et al., 2011. Differential responses to selenomethionine supplementation by sex and genotype in healthy adults. The British Journal of Nutrition 107, 1514e1525. Combs Jr., G.F., Combs, S.B., 1996. The Role of Selenium in Nutrition, first ed. Academic Press, INC, Orlando. Fleet, G., 2006. The commercial and community significance of yeasts in food and beverage production. In: Querol, A., Fleet, G. (Eds.), 2006. Yeasts in Food and Beverages, vol. 2. Springer, Berlin, pp. 1e11. Garvin, D.F., Harelan, G., Gregoire, B.R., Finley, J.W., 2011. Impact of wheat grain selenium content variation on milling and bread baking. Cereal Chemistry 88, 195e200. Katsuhiko, N., Tomofuni, O., Tatsuya, H., 2000. Metabolism of selenoaminoacids and contribution of selenium methylation to their toxicity. Journal of Health Source 46, 418e421. Mapelli, V., Hillestrøm, P.R., Patil, K., Larsen, E.H., Olsson, L., 2012. The interplay between sulphur and selenium metabolism influences the intracellular redox balance in Saccharomyces cerevisiae. FEMS Yeast Research 12, 20e32. McDermott, J.R., Barry, R.P., Liu, Z., 2010. Jen1p: a high affinity selenite transporter in yeast. Molecular Biology of the Cell 21, 3934e3941. Millipore Corporation, 1993. Waters AccQ-tag Chemistry Package. Instruction Manual. Millipore Corporation, Melford. Nagodawithana, T., Gutmanis, F., 1985. Method for the production of selenium yeast, Patent US 4530846. Ouerdane, L., Zoltán, M., 2008. Production and characterization of fully Selenomethionine-labeled Saccharomyces. Journal of Agriculture and Food Chemistry 56, 11792e11799. Pasini, G., Simonato, B., Giannattasio, M., Peruffo, A.D., Curioni, A., 2001. Modifications of wheat flour proteins during in vitro digestion of bread dough, crumb, and crust: an electrophoretic and immunological study. Journal of Agricultural and Food Chemistry 49, 2254e2261. Ponce de León, C.A., Bayoán, M.M., Paquin, C., 2002. Selenium incorporation into Saccharomyces cerevisiae cells: a study of different incorporation methods. Journal of Applied Microbiology 92, 602e610. Poutanen, K., 2009. Cereal foods in diet and health. In: Shewry, P.R., Ward, J.L. (Eds.), Analysis of Bioactive Components in Small Grain Cereals. American Association of Cereal Chemists, St. Paul, pp. 34e71. Ramiro-Anaya, M., 2011. Premio Nacional en Ciencia y Tecnología de Alimentos. Available on line at: http://www.pncta.com.mx/pages/pncta_investigaciones_ 04h.asp?page¼04e4#up (accessed on 08.11.2011). Romano, P., Capece, A., Jespersen, L., 2006. Taxonomic and ecological diversity of food and beverage yeasts. In: Querol, A., Fleet, G. (Eds.), 2006. Yeasts in Food and Beverages, vol. 2. Springer, Berlin, pp. 13e53. Schubert, A., Holden, J., Wolf, W., 1987. Selenium content of a core group of foods based on a critical evaluation of published analytical data. Journal of American Diet Association 87, 285e299. Serna-Saldivar, S.O., 2012. Cereal Grains: Laboratory Reference and Procedures Manual. CRC Press, Boca Raton. Serna-Saldivar, S.R., Abril, R., 2011. Production and nutraceutical properties of breads fortified with DHA-and Omega-3-containing oils. In: Preedy, V.R., Ross Watson, R., Patel, V.B. (Eds.), Flour and Breads, and Their Fortification in Health and Disease Prevention, firth ed. Academic Press, London, pp. 313e323. Stabnikova, O., Ivanov, V., Larionova, I., Stabnikov, V., Bryszewska, M.A., LewisLebenson, J., 2008. Ukrainian dietary bakery product with selenium enriched yeast. LWT e Food Science and Technology 41, 890e895. Watts, B.M., Ylimaki, G.L., Jeffery, L.E., Ellías, L.G., 1995. Métodos sensoriales básicos para la evaluación de alimentos, first ed. International Development Research Centre, Ottawa. WHO, FAO, UNICEF, GAIN, MI, FFI, 2009. Recommendations on Wheat and Maize Flour Fortification. Meeting report: interim consensus statement. World Health Organization, Geneva. Available on line at: http://www.who.int/nutrition/ publications/micro-nutrients/wheat_maize_fort.pdf (accessed on 05.11.2012).
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Effect of sodium selenite addition and sponge dough fermentation on selenomethionine generation during production of yeast-leavened breads Marco A. Lazo-Vélez, Víctor A. Gutiérrez-Díaz, Alicia Ramírez-Medrano, Sergio O. Serna-Saldívar* Centro de Biotecnología-FEMSA, Escuela de Biotecnología y Alimentos, Tecnológico de Monterrey, Monterrey, N.L., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2012 Received in revised form 6 March 2013 Accepted 25 March 2013
The effect of sodium selenite addition and fermentation times on production of selenomethionine (SeM) during sponge bread production was evaluated. Doughs were supplemented with sodium selenite (Na2SeO3) and fermented with yeast, avoiding addition of sulfur salts. The effect of Na2SeO3 on yeast activity was evaluated using a pressurometer. Results showed that there were not statistical differences (p < 0.05) in CO2 production and dough pH at all the sodium selenite concentrations tested. HPLCfluorescence data showed that SeM production was higher with the increase of fermentation times, while less significant effects were observed due to changes in Na2SeO3 concentration. Two slices of Seenriched bread can provide about 200 mg SeM, the dose recommended to prevent cancer and oxidative stress. The physical features (water absorption, bread weight, bread volume, color, density, oven spring, etc.) and organoleptic evaluations for the enriched loaves were evaluated. In all these parameters, the experimental enriched breads had practically identical attributes compared to the control. The breads rich in SeM have potential to be used as functional foods because this amino acid is synthesized into higher quantities of glutathione peroxidase, an enzyme considered as one of the most protective mechanisms against oxidative stress and preventive of cancer and chronic diseases. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Selenium-enriched bread Dry instant yeast Selenomethionine Sponge dough
1. Introduction Cereal-based foods are the most important for mankind because they provide from 30 to 60% of the intake and are considered as the major suppliers of carbohydrates and calories. The consumption of cereals is comparatively higher in developing countries around the globe. Epidemiological studies clearly show that whole cereals have a protective effect against chronic diseases that are responsible for most of the deaths in the western world (Poutanen, 2009). Among the wide array of cereal-based products, bread is still considered as the main staple food for most of the inhabitants in the world. The production of bread is considered as one of the oldest biotechnological processes developed (Fleet, 2006).
Abbreviations: ANOVA, analysis of variance; DM, dry matter; HPLC, high pressure liquid chromatography; SeM, selenomethionine. * Corresponding author. Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico C.P. 64849, Monterrey, N.L., Mexico. Tel.: þ52 81 8358 1400; fax: þ52 81 8328 4262. E-mail address: [email protected] (S.O. Serna-Saldívar). 0733-5210/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2013.03.019
Undoubtedly, the nutrients and nutraceuticals associated with bread can impact the health status of consumers. White bread is considered as an excellent source of digestible energy and intermediate quality protein whereas whole grain and variety breads are additionally rich in dietary fiber and phenolics. The bread also contains important amounts of some micronutrients intrinsically associated with wheat and yeast and purposely added to refined flours (Fe, Zn, thiamin, riboflavin, niacin and folic acid) (WHO et al., 2009). It is documented that flours and bread vary in the concentration of the trace mineral selenium according to the growing location of the wheat and the flour extraction rate. Selenium (Se) is considered an essential micronutrient because it is required for the synthesis of important antioxidant proteins such as cytosolic glutathione peroxidase (Combs et al., 2011). This enzyme protects mammalian systems from oxidative stress and has been related to prevention of chronic diseases and cancer (Beckett and Arthur, 2005). The bioavailability of Se is higher when associated to selenoproteins (Katsuhiko et al., 2000). These proteins are synthesized from selenocysteine (SeC), obtained after the reversible methylation of SeM stored in different tissues. The SeM is considered the main source of organic selenium in foods and the moiety with the highest bioavailability.
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An analysis of diets in the U.S. by Schubert et al. (1987) revealed that five foods, including white bread, contributed about 50% of the total selenium intake, and that 80% of the total dietary Se was provided by only 22 core food items (Schubert et al., 1987). The supplementation of Se salts to yeast cultures enhances the conversion of inorganic Se into organic sources. The amount synthesized will vary according to the yeast food composition especially in terms of the Se/S ratio (Ouerdane and Zoltán, 2008; Mapelli et al., 2012). Bakery yeast (Saccharomyces cerevisiae) is capable of metabolizing up to 3000 mg Se/g converting approximately 90% of the selenium into L-SeM and leaving traces of the inorganic form (California Environmental Protection Agency, 2010). Therefore, yeast-leavened bakery products could be an ideal carrier for the production and supplementation of organic selenium (Ouerdane and Zoltán, 2008). The advantage of bread is that without an extensive modification of the process, it can yield significant amounts of organic selenium and be mass produced. The Se-enriched breads may contain a high Se concentration: the 100 g wheat roll prepared with selenium-enriched yeast contained 50 mg Se in the form of SeM (Stabnikova et al., 2008), compared to 3e6 mg/100 g commonly seen in regular counterparts (Bryszewska et al., 2005; Ramiro-Anaya, 2011). However, the uncontrolled addition of Se may modify the sensory characteristics and overall acceptability of breads. The feasibility of producing SeM enriched breads has not been properly addressed in terms of manufacturing procedures and bread quality. This research was undertaken in order to investigate the effects of sodium selenite addition as yeast food in order to obtain SeM enriched breads manufactured from sponge doughs fermented with dry instant yeast (S. cerevisiae). 2. Materials and methods 2.1. Sulfur-free yeast food preparation The sulfur-free yeast food was prepared according to the procedure described by Anghileri and Márquez (1965) with minor modifications. Briefly, a first solution was prepared by mixing 3.5 g di-ammonium phosphate, 0.1 g magnesium chloride, 0.8 g zinc acetate, 0.2 mg iron(III) chloride, 50 mg copper (II) chloride (D.E.Q Monterrey, N.L., Mexico), 1.0 g monosodium citrate (Sigmae Aldrich, Toluca Edo. Mexico, Mexico) and 0.8 L distilled water (Lab. Monterrey, Monterrey, N.L., Mexico). This pre-solution was treated at 121 C at 15 psi in an autoclave (Sterilizer SE-510, Yamato, Japan) for 15 min. The sterilized solution was cooled and stored aseptically. The second solution was prepared by diluting 2.5 g monohydrate-Lasparagine, 10 mg biotin, 0.5 mg calcium pantothenate, 10 mg inositol, 6 mg thiamine, 1 mg pyridoxine, 20 g glucose (Sigmae Aldrich, Toluca Edo. Mexico, Mexico), and 0.20 L distilled water. The two solutions were blended in a laminar flow hood (Labconco, Model 36212, Kansas City, MO, USA). The resulting yeast food solution was passed through 0.2 mm sterile filters, adjusted to pH 4.5e 5.0 (phosphoric acid 0.5 M) and stored at 4 C. The selenite salts were added to the sterile solution utilizing a stock solution of Na2SeO3 (SigmaeAldrich, Toluca Edo. Mexico, Mexico) to a concentration of 100 mg/mL. 2.2. Determination of the effects of sodium selenite concentration on yeast gassing power and dough pH The effects of different concentrations of Na2SeO3 on the CO2 gassing power of commercial dry instant yeast was tested in a pressurometer according to AACC method 22-11.01 (AACC, 2000) and Serna-Saldívar (2012) with slight modifications. Before the test, the flour was treated with ultraviolet light for 15 min with the aim
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of inactivating wild yeast and the associated microbial load. The sanitized flour was immediately packaged in sealed plastic bags and refrigerated at 4 C. Briefly, 35.15 g (14% moisture basis) of refined wheat flour and 0.23 g of dry yeast were first blended and allowed to rest for 5 min. Then, the flour-yeast blend was hydrated with 15.07 mL of sulfurfree yeast food and 9.47 mL distilled water in which different amounts of Na2SeO3 were dosed (1.9, 3.8 and 7.6 mg Se/g flour). The resulting blend was placed in the pressurometer, weighed and mixed with a spatula until attaining a soft and smooth slurry. The pressurometer was immediately sealed and tightened with a wrench and placed in a water bath (Hot-Saker 7746-12110, Bellco Glass Inc., Vineland, NY, USA) set at 28.5 2 C for 4.5 h. The gas in the pressurometer was released after the first 5 min. The pressure (mm Hg) generated by the gas produced by the yeast was measured for 90 min with a frequency of 10 min. The slurry pH was registered after three degassing steps of the pressurometer every 90 min during 4.5 h fermentation. The pH was measured with a potentiometer (SevenGo-2B, Mettler-Toledo, Schwerzenbach, Switzerland) equipped with a special dough probe (SEREX01072, SensoreXÒ, Garden Grove, CA, USA) 2.3. Bread making preparation The optimized sponge bread making AACC method 10-11.01 (AACC, 2000) was utilized with slight variations in formulation. The breads were manufactured in order to obtain commercial loaves for sensory and chemical analyses. The basic loaf formulation included 100 g (14% moisture basis) bread flour (10.7% protein and 0.4% ash), 5 g refined cane sugar (Avance, Avance Comercial de Monterrey, Monterrey, NL, Mexico), 4 g vegetable shortening (Inca, Unilever de Mexico S.A de C.V., Tultitlán, Edo. de Mexico, Mexico), 2 g refined iodinated salt (La Fina, Sales del Istmo, Coatzacoalcos, Veracruz, Mexico), 2 g dry yeast (Saccharomyces cereviceae) (AztecaÒ Levadura, Iztapalapa, Mexico, D.F., Mexico), 2 g dry milk (Nestle de Mexico, Mexico, D.F., Mexico), 0.2 g diastastic malt (SudChemic de Mexico, Puebla, Pue., Mexico) and 0.5 g lecithin (Proveedores de Ingeniería Alimentaria S.A. de C.V. Monterrey, N.L., Mexico). Bake absorption, mixing time, proof height, loaf height, oven spring, loaf volume, loaf apparent density and crumb grain texture were determined. Sponges were fermented for 8, 16 or 24 h with different Na2SeO3 concentrations: 0 (control), 4.33, 7.83 and 23.8 mg Se/g as Na2SeO3. The sponge stage consisted of mixing by hand 80 g of refined bread flour, 3 g instant dry yeast and 48 mL of the yeast food solution that contained the different Na2SeO3 concentrations. The resulting sponge dough was placed in a plastic container in a fermentation cabinet (National Manufacturing Co., Lincoln, NE, USA) set at 27 2 C for the predetermined fermentation time. Resulting sponges were mixed with the dough stage ingredients: 120 g bread flour, 10 g sugar, 8 g shortening, 4 g of dry whole milk, 4 g salt, 0.4 g diastatic malt, 0.4 g lecithin, 1 g dry yeast instant and 72 mL water for 2 min at low speed in a Hobart mixer equipped with the hook attachment. The speed was switched to medium until attaining optimum dough development. Optimum mix times were subjectively determined by observing dough properties or gluten development (film formation, gloss and dough stickiness). Resulting doughs were weighed and cut into two identical parts before placing them in a fermentation cabinet set at 27 2 C and 85% relative humidity. After 30 min resting, doughs were punched thru 0.56 cm roll spacing and dough sheets folded in half and in half again and returned to the fermentation cabinet for two additional 15 min fermentation periods. Doughs were punched through 0.95 cm roll spacing in preparation for molding and panning in 7 cm height metal pans that had the following dimensions on the base and top, respectively: 33 and 35 cm long and
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10.5 and 14.5 cm wide. Baking pans were previously greased with vegetable shortening on the bottom and sides. Panned doughs were proofed for 45 min before baking for 25 min in an oven (Electrolux EOG Gas single oven X 601) set at 220 C. The height of proof doughs and breads were measured at the center of the loaf in order to calculate oven spring. In addition, loaves were weighed and volumes determined by rapeseed displacement (National Manufacturing Co., Lincoln, NE, USA). Finally, breads were packed in sealed polyethylene bags and stored at room temperature. 2.4. Determination of bread crumb color Crumb color was objectively measured with a colorimeter (CR 300, Minolta, Japan) standardized with a white reference plate (L ¼ 92.90, a ¼ 1.05, b ¼ 0.82). Bread crumb color parameters measured were L*, a*, b*. 2.5. Baking commercial procedure Se-enriched white breads were manufactured in a pilot test using sponge doughs fermented for 16 h with a concentration of 3.5 mg Se/kg flour as Na2SeO3. Two slices or servings (72 g) of this enriched bread contained 200 mg of total Se. 2.6. Sensory analyses of the breads Twenty one untrained panelists evaluated the sensory characteristics of breads using triangle testing that were conducted with one-day old control and selenized breads. Briefly, each panelist was given simultaneously three coded samples (two samples equal and one different) along with a ballot. When all panel members completed the trial, their ballots were marked as correct when they identified the different bread sample or as incorrect when they failed to identify the odd sample. Using the binomial test from oneend, the total number of panelists with correct answers with the total number of the observations was compared (Watts et al., 1995). 2.7. Detection and quantification of SeM by HPLC-fluorescence 2.7.1. Sample preparation The bread obtained from different experiments was sliced and dried for 3 h in an oven (Electrolux EOG 601 X Gassingle oven) at a temperature of 50 C. All dehydrated samples were reduced to a fine powder in a cutter mill (Standard Mod N03, Philadelphia, PA, USA) equipped with a 1 mm sieve. The final product was transferred into labeled polyethylene bags with airtight seal and frozen at 20 C. 2.7.2. In vitro gastrointestinal digestion method Bread samples were subjected to the in vitro digestion method previously described by Pasini et al. (2001). Briefly, 60 mg of ground sample were suspended in 4 mL of 0.2 N HCl (pH 2.0) and 0.05 mg/ mL of porcine pepsin P-7000 (SigmaeAldrich, Toluca Edo. Mexico, Mexico) to emulate gastric protein hydrolysis. The final concentration of protein was measured at 1.87 mg/mL with a ratio of enzyme/protein of 1:37 (w/w). The control samples were treated the same way but without pepsin. The digestion was carried out for 1 h at a temperature of 37 C in a shaking water bath (Hot-Shaker 7746-12110, Bellco Glass Inc., Vineland, NJ, USA) adjusted to a stirring speed of 28 rpm. The stock solution was prepared 50/50 (v/ v) with boric acid (DEQ, Monterrey, N.L., Mexico), previously heated to a concentration 1.0 M and a 0.5N NaOH solution (JT Baker, Xalostoc, Tlaxcala, Mexico). This stock solution was adjusted to pH 7.8 with HCl (5 N) and subsequently treated with 0.25 mg/mL pancreatin (SigmaeAldrich, Toluca, Edo. Mexico, Mexico). An aliquot of 1.15 mL of the pancreatin solution was added to the
pepsin-hydrolyzed bread solution. The resulting pH was recorded at 7.5, with a ratio pancreatin/protein of 1:17 (w/w). The biocatalytic reaction was allowed to proceed at 37 C in a shaking water bath (Hot-Shaker 7746-12110, Bellco Glass Inc., Vineland, NJ USA) and stopped after 120 min by inactivation in a hot water bath (80 C) for two min. After standing for 1 h, the samples were centrifuged for 10 min at 10000g and 4 C (SL16R, Thermo Scientific, Germany). The supernatant and pellet were separated and then analyzed for SeM. If SeM was not detected in the pellet, the supernatant was sonicated (Branson Ultrasonic Corporation, Danbury, CT, USA) for 10 min. The sonicated sample was filtered through a 0.45 mm sterile filter (Corning, NY, USA) into 1.5 mL vials, which were immediately stored at 20 C. 2.7.3. Selenomethionine analysis The SeM was analyzed after the in vitro gastrointestinal digestion method described above, according to the Waters AccQTag Amino Acid Analysis MethodÔ (Millipore Corporation; Milford, MA, USA). SeM was derivatized with the Waters AccQFluor reagent and separated by reversed-phase high performance liquid chromatography in an AccQTag column (Waters Nova-PakÔ C18 4 mm, 3.9 150 mm column). A Waters HPLC system equipped with a 1525 Binary pump and Multi Fluorescence detector (Waters 2475) was used. The external calibration standard containing SeM (Sigmae Aldrich, Toluca Edo. Mexico, Mexico) was prepared according to the instruction manual of Waters (Millipore Corporation, 1993). 3. Statistical analysis The data for breads are presented as means standard deviations of determinations. The difference of SeM content for each loaf was analyzed by one-way analysis of variance (ANOVA). Multiple comparisons of means were performed using Tukey HSD tests at a level of significance of P < 0.05. Data obtained from the pressurometer were analyzed using ANOVA procedures and correlated using Pearson correlations. All computations were made by the statistical software’s Minitab (version 15) and JPM (version 10.2). 4. Results and discussion 4.1. Effect of sodium selenite on carbon dioxide generation by yeast The CO2 generation capacity of yeast was registered at different fermentation times (Fig. 1). A highly significant correlation among all experimental treatments and the control (all >r ¼ 0.99) was observed. The gas volumes generated by the control fermented for
Fig. 1. Effect of different concentrations of sodium selenite on carbon dioxide generation by dry instant yeast (S. cerevisiae). Evaluated in a pressurometer at a temperature of 28 2 C for 4.5 h (n ¼ 3; error bars indicate standard deviation). Pearson correlation: r ¼ 1 [1.90 mg Se/g ], r ¼ 0.99 [3.80 mg Se/g ], r ¼ 1 [7.60 mg Se/g ]1.
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4.5 h was 360.1 mL whereas, for treatments supplemented with the three increasing Na2SeO3 concentrations, were 344.1, 320.2 and 339.7 mL, respectively. These data clearly indicate that the three tested Na2SeO3 concentrations did not significantly affect final CO2 production (p < 0.05). Stabnikova et al. (2008) reported that yeast growing in cultures containing 2e5 mg Se/mL as NaHSeO3 did not differ from the control treatment. 4.2. Effect of sodium selenite on dough pH The comparison of the control dough pH after 4.5 h fermentation at 28.5 2 C in the pressurometer with doughs containing 1.9, 3.8 and 7.6 mg Se/g flour (Fig. 2) indicated a highly significant correlation for the first two concentrations (r ¼ 0.97, 0.93) and significant for the highest concentration (p < 0.05 and r ¼ 0.76). The addition of the different concentrations of Na2SeO3 to the slurry in the pressurometer did not significantly affect dough pH (p > 0.05). As expected, the pH values decreased significantly (p < 0.05) throughout the different fermentation times. The pH values in the pressurometer were within the expected range for the optimum yeast activity and CO2 generation (4e5.8) (Fig. 2). Likewise, the sponge doughs fermented for 8, 16 and 24 h with Na2SeO3 concentrations of 4.33, 7.84 and 23.8 mg Se/g flour had pH with strong correlations with respect to the control sponge dough (r ¼ 0.95; 0.99 y 0.98) (data not shown). When sponge dough pH values were compared, the different selenite supplemented doughs tended to have slightly significant different pH as fermentation proceeded. However, the pH values were not significantly different (p < 0.05) from the control tested at 24 h fermentation. The sponge dough lowest pH observed after 24 h fermentation was 5.1 for the control and 5.21 for the experimental treatment supplemented with 23.8 mg Se/g flour) (data not shown). Under these conditions, it is reported that gluten retains the maximum amount of gas (Calveras, 2004; Cauvain and Young, 1998; Romano et al., 2006). It is also known that the maximum selenite incorporation into the yeast cells occur at the pH values (4.5e5) achieved (McDermott et al., 2010). Interestingly, the dough pH decreased to <4.3 in sponge doughs fermented for 16 or 24 h with selenite concentrations > of 7.82 mg Se/g. (data not published). These doughs acquired a pink-reddish coloration and the bread crumb retained these colored dots after baking. This is due to the formation of elemental selenium owing to reduction reactions at the acidic pH values (Combs and Combs, 1996). This phenomenon was also observed in yeast biomass
Fig. 2. Effect of selenium in the form of sodium selenite concentrations on pH values of doughs fermented with dry instant yeast (S. cerevisiae). Evaluated in the pressurometer at a temperature of 28 2 C for 4.5 h (n ¼ 3; error bars indicate standard deviation). Pearson correlation: r ¼ 0.97 [1.90 mg Se/g ], r ¼ 0.93 [3.80 mg Se/g ], r ¼ 0.76 [7.60 mg Se/g ] 1.
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cultured with concentrations higher than 10 mg Se/mL (Stabnikova et al., 2008). Alzate et al. (2007) described a similar color reduction mechanism when producing yogurt containing 50 mg Se/g and higher. The yogurt fermented with Lactobacillus suffered a reduction of the Se (IV) to Se0, which produced a reddish coloration. 4.3. Properties of breads The addition of the different concentrations of Na2SeO3 to sponge doughs did not significantly affect bread color (Table 1). The comparison of the control dough proof height produced from sponges fermented for 8 or 16 h were not significantly different from all experimental treatments. Nevertheless, the sponges fermented for 24 h with Na2SeO3 concentrations of 7.84 and 23.8 mg Se/g flour produced doughs with proof heights significantly lower compared to the control counterpart. On the other hand, the comparison of the apparent densities of experimental breads containing 23.8 mg Se/g flour obtained from sponges fermented for 24 h were significantly higher compared to the control bread. The observed differences in the last two parameters may be due to a lower yeast activity during proofing and the first stage of baking. The oven spring of Se enriched breads manufactured from sponge doughs fermented for 24 h was negatively affected, especially in those loaves supplemented with the highest concentration of sodium selenite. The bread volume was slightly lower in breads supplemented with 23.8 mg Se/g in all the sponge dough fermented for different times (737e754 cm3) (Table 1). Breads supplemented with 7.84 mg Se/g flour and manufactured from sponge doughs fermented for 16 or 24 h also had slightly different volumes (750e 790 cm3). The rest of the experimental and control breads developed similar bread volumes (820e860 cm3) (p > 0.05). For pup loaves produced from 100 g of 14% moisture based flours, the optimum bread volumes were between 850 and 925 cm3 (SernaSaldívar and Abril, 2011). Garvin et al. (2011) reported volumes between 750 and 850 cm3 for breads produced from patent flour rich in Se (0.53e7.56 mg Se/g flour). The bread volumes obtained herein are similar to the ones reported by Garvin et al. (2011) and are considered adequate. The bread volumes can be improved by the use of oxidizing agents such as ascorbic acid or potassium bromate, a higher protein flour and/or vital gluten (Serna-Saldivar and Abril, 2011, 2012). The addition of 0.5% more instant dry yeast (not Se-enriched) during the dough stage mixing helped to prevent low bread volume in loaves prepared with less than 4 mg Se/g flour. 4.4. Selenium-methionine content of breads determined by HPLCfluorescence Preliminary studies indicated that the peak and elution time for a SeM standard was 32.80 0.02 min (Fig. 3). The SeM did not coelute with any other amino acid present in the standard solution. The identification of SeM was reconfirmed by the use of an internal standard containing additional known quantities of this specific amino acid. Breads manufactured from sponge doughs supplemented with different concentrations of Na2SeO3 (4.33e23.8 mg Se/g) contained between 120 and 250 mg Se/100 g bread (DM.) as SeM (Fig. 4). Statistical analysis indicated that the concentration in bread was mainly affected by fermentation time and to a lesser extent by the original Na2SeO3 concentration; the SeM concentration significantly increased (p < 0.05) with prolonged sponge dough fermentation times. On the other hand, the different sodium selenite concentrations tested did not significantly affect SeM concentration. As expected, all experimental breads contained significantly higher concentrations of SeM when compared to the
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Table 1 Effects of different concentrations of Se in the form of Na2SeO3 and fermentation times in the physical characteristics of breads prepared with a sponge dough procedure. Concentration [Se as SeO3Na2]
Proof height (cm)
Loaf height (cm)
Oven springa (cm)
Bread weight (g)
Bread volume (cm3)
Apparent density (g/cm3)
Color crumb (dE*ab)
Fermentation time ¼ 8 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.17 7.30 7.23 7.27
0.1A 0.1A 0.2A 0.2A
9.27 9.27 8.90 8.97
0.12A 0.06A 0.20A 0.15A
1.29 1.27 1.23 1.23
0.03A 0.01A 0.02A 0.05A
148.00 151.33 148.67 152.00
0.00A 4.62A 6.11A 2.00A
860.0 820.0 800.0 741.7
17.32A 17.32A 43.59A,B 24.66B
0.17 0.18 0.19 0.21
0.003A 0.008A,B 0.015A,B 0.005B
1.94 1.39 1.73 1.40
0.45A 0.15A 0.50A 0.21A
Fermentation time ¼ 16 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.20 7.33 7.13 7.20
0.1A 0.2A 0.1A 0.1A
9.33 9.43 8.97 8.67
0.15A,B 0.12A 0.21A,C 0.06C
1.30 1.29 1.26 1.20
0.02A 0.02A 0.03A,B 0.02B
148.00 148.67 149.33 149.33
2.00A 1.15A 2.31A 2.31A
846.7 821.7 788.3 753.3
15.28A 10.41A,B 54.85A,B 5.77B
0.17 0.18 0.18 0.19
0.010A 0.004A,B 0.002A,B 0.004B
2.60 1.71 2.32 1.60
0.27A 0.45A 0.33A 0.76A
Fermentation time ¼ 24 h Control 04.33 mg/g 07.83 mg/g 23.80 mg/g
7.53 7.93 7.40 7.23
0.1A 0.1A,B 0.3B 0.1B
9.53 9.43 8.83 8.10
0.12A 0.25A 0.29B 0.1C
1.27 1.19 1.20 1.12
0.01A 0.02A,B 0.07A,B 0.01B
141.67 148.00 147.33 145.67
2.89A 2.00A 3.06A 2.52A
820.0 850.0 750.0 736.7
20.00A 20.00A 10.00B 11.55B
0.17 0.17 0.19 0.19
0.002A 0.003A 0.003B 0.004B
2.13 1.76 2.09 1.46
0.07A 0.24A 0.77A 0.15A
Means standard deviation with the levels not connected by same letter are significantly different at p < 0.05. a Oven spring ¼ bread height proof height.
control or unsupplemented bread (36.71 0.78 mg Se/100g bread). The concentration of Na2SeO3 was not relevant in terms of the final amount of SeM in breads produced with 2% yeast. However, if more SeM concentration is desired, the best way to achieve it is through the supplementation of additional amounts of yeast especially during the sponge dough stage. The bread that contained the highest SeM concentration was the one manufactured from a sponge dough supplemented with 23.8 mg Se as Na2SeO3/g and fermented for 24 h. The bread contained 5.6 and 1.7 times more SeM concentrations compared to the control (37.59 mg Se/100g bread) and counterparts produced from sponge doughs fermented for 8 h (126.24 mg Se/g bread). Also, breads produced from sponge doughs fermented for 8e16 h and 16e24 h incremented SeM by a factor of one for each of these periods. Most of the commercial organic selenium is obtained from yeast (S. cerevisiae) cultured in a medium rich in inorganic selenium. It is reported that the reactor selenized yeast production yields from 500 to 2124 mg Se/g DM (Nagodawithana and Gutmanis, 1985; Ponce de León et al., 2002). Stabnikova et al. (2008) produced enriched selenized breads with 55 mg of Se as SeM from yeast cultures previously supplemented with inorganic sodium hydroxyselenite. A theoretical comparison of selenized proteins generated in a yeast bio-fermentation reactor or bread doughs indicate that
Fig. 3. Identification and elution time of selenomethionine in a chromatograph obtained after HPLC-Fluorescence analysis of enzyme hydrolyzed bread samples. Analyzed with injection volume of 10 mL, wavelength of 250 nm emission, 395 nm excitation and linear gradient. TYR: Tyrosine; VAL: Valine; MET: Methionine; SeM: Selenomethionine; LYS: Lysine; ILE: Isoleucine; L: Leucine.
these two contrasting processes generate equivalent amounts of SeM. These earlier studies that produced breads with selenized yeast cultured for 72 h with 5 mg Se as hydroselenite/mL yielded an enriched yeast with 154 mg Se/g dry biomass; the breads manufactured with 1.5% of the Se enriched yeast contained 55 mg Se as SeM/100 g bread (Stabnikova et al., 2008). A comparison of the SeM concentrations of SeM of the different bread systems indicated that the bread produced with the lowest sodium selenite (4.33 mg Se/g) concentration fermented for 24 h contained z200 mg Se/100 g fresh bread. Therefore, this particular bread system contained approximately 4 times more SeM compared to the breads produced by Stabnikova et al. (2008). The commercial fermentation processes aimed towards the production of selenized yeast generally cultured for 24e72 h (Nagodawithana and Gutmanis, 1985) was equivalent to the 24 h sponge dough process described herein. The advantages of sponge doughs are that they require fewer controls and are performed in a solid state. Furthermore, the food grade dough can be dried in order to produce an enriched protein selenized product. Moreover, the amounts of selenized proteins generated during sourdough
Fig. 4. Effect of yeast (S. cerevisiae) fermentation time and concentration of sodium selenite on the production of selenomethionine in different loaves of bread. SeM biosynthesis was favored by the increase in fermentation times, with less significant changes in concentration of sodium selenite. Letters comparing selenite concentration means denote significant differences (at p < 0.05). Within each fermentation time cluster.
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fermentation can be higher because these types of breads are produced using higher fermentation times. Thus, the yeast in the sponge dough was very efficient in the transformation of sodium selenite into SeM especially when fermented for more than 16 h. 4.5. Sensory evaluation The triangle tests clearly indicated that the SeM enriched breads had similar color, texture, flavor and overall acceptability compared to the unsupplemented control bread (p < 0.005). 5. Conclusions This research clearly showed that it was feasible to produce breads with high content of SeM by preparing sponge doughs fermented for less than 16 h with dry instant yeast and supplemented with less than 23.8 mg Se as SeO3Na2/g flour. The resulting enriched breads were practically identical to regular counterparts and consumers were not capable of perceiving sensory differences. The consumption of two slices of selenium-enriched bread (36 g for slice) produced from sponge doughs fermented for 24 h with only 4.33 mg Se as SeO3Na2/g flour can provide approximately 200 mg of SeM, a dose which has proven to have chemopreventive effects in epidemiological studies (California Environmental Protection Agency, 2010). Acknowledgments Authors acknowledge Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico, Secretaría Nacional de Estudios Superiores, Ciencia, Tecnología e Innovación (SENESCyT), Ecuador and the Nutrigenomic Research Chair of Tecnológico de Monterrey which provided research and support funds for graduate students. References AACC, 2000. Approved methods of the American association of cereal chemists. In: Methods 10-11.01 and 22-11.01, eleventh ed. AACC International, St. Paul. Alzate, A., Cañas, B., Pérez-Munguía, S., Hernández-Mendoza, H., Pérez-Conde, C., Gutiérrez, A.M., Cámara, C., 2007. Evaluation of the inorganic selenium biotransformation in selenium-enriched yogurt by HPLC-ICP-MS. Journal of Agricultural and Food Chemistry 55, 9776e9783. Anghileri, L.J., Marquez, R.G., 1965. Preparación biosintética de selenometionina y selenocisteina, marcados con 75Se. Su incorporación y distribución en ratones. Reporte Técnico NP-15594. Comisión Nacional de Energía Atómica, Buenos Aires. Beckett, G.J., Arthur, J.R., 2005. Selenium and endocrine systems (review). Journal of Endocrinology 184, 455e465. Bryszewska, M.A., Ambroziak, W., Diowksz, A., Baxter, M.J., Langford, N.J., Lewis, D.J., 2005. Changes in the chemical form of selenium observed during the manufacture of a selenium-enriched sourdough bread for use in a human nutrition study. Food Additives & Contaminants 22, 135e140. California Environmental Protection Agency, 2010. Public Health Goal for Selenium in Drinking Water. Pesticide and Environmental Toxicology Section, Office of Environmental Health Hazard Assessment, Berkely.
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