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The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by treating the nixtamalized masa with commercial baking yeast
Accepted Manuscript The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by treating the nixtamalized masa with commercial baking yeast E.J. Vernon-Carter, J. Alvarez-Ramirez, L.A. Bello-Perez, A. Garcia-Hernandez, S. Garcia-Diaz, C. Roldan-Cruz PII:
S0733-5210(18)30228-5
DOI:
10.1016/j.jcs.2018.07.001
Reference:
YJCRS 2595
To appear in:
Journal of Cereal Science
Received Date: 19 March 2018 Revised Date:
29 June 2018
Accepted Date: 2 July 2018
Please cite this article as: Vernon-Carter, E.J., Alvarez-Ramirez, J., Bello-Perez, L.A., GarciaHernandez, A., Garcia-Diaz, S., Roldan-Cruz, C., The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by treating the nixtamalized masa with commercial baking yeast, Journal of Cereal Science (2018), doi: 10.1016/j.jcs.2018.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by
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treating the nixtamalized masa with commercial baking yeast
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E.J. Vernon-Cartera, J. Alvarez-Ramireza,*, L.A. Bello-Perezb, A. Garcia-Hernandeza, S. Garcia-
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Diazc, C. Roldan-Cruzc
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Iztapalapa. A.P. 55-534, CDMX, 09340 México.
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CEPROBI, Instituto Politécnico Nacional, Yautepec, Morelos, México.
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Departamento de Biotecnología. Universidad Autónoma Metropolitana-Iztapalapa. A.P. 55-534,
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CDMX, 09340 México.
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* Corresponding Author. Email: [email protected]. Fax/phone: +52-55-58044650
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Departamento de Ingeniería de Procesos e Hidráulica. Universidad Autónoma Metropolitana-
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Abstract
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Baking yeast exhibiting amylolytic activity was used for treating masa made from nixtamalized
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maize flour (NMF). Baking yeast (0.25, 0.50 and 1.0 g˖100 g-1 NMF) was added to the basic masa
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recipe (40 NMF:60 water mass ratio). Two masa controls without yeast addition were prepared:
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CM1 used as such, and CM2 subjected to a mild incubation treatment (2 h, 38 °C). Tortillas were
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made (350 °C, 1.0 min) with the yeast-treated masa. Baking yeast reduced total sugars, apparent
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amylose and viscoelasticity of the masa. Tortillas made with treated masa exhibited significant
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lower hardness than tortillas made with CM1 and CM2, and this effect was more pronounced in the
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tortillas stored for 4 days. Tortillas freshly made from yeast-treated masa displayed reduced RDS
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and SDS, but increased RS fractions. When stored for 4 days, they showed reduction in RDS, but an
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increase in SDS and RS fractions (∼30%) with respect to tortillas made with CM1 and CM2.
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Hardness of the yeast-treated tortillas was significantly lower and remained practically without
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change during storage, while the untreated tortillas hardened significantly. Treatment with baking
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yeast induces beneficial health and textural effects in tortillas.
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Keywords: Nixtamalized maize masa; baking yeast; amylolytic activity; tortilla; starch
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digestibility.
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1. Introduction
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Maize tortillas are an important source of daily calories (~50-70%) and proteins (~50%) for urban
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and rural populations in Mexico, the consumption estimated at 90 kg by inhabitant per year. The
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production of maize tortillas requires of a process known as nixtamalization. In the traditional
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nixtamalization process, maize grains are cooked in a solution of water with lime, followed by
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overnight steeping, washing and grinding to obtain a wet dough (masa) from which tortillas are
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formed (Peña-Reyes et al., 2017). Due to economic and technological benefits, nowadays most of
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the nixtamalization process is carried out at industrial level, and the use of instantaneous
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nixtamalized maize flour (NMF) has increasingly displaced traditional maize grain processing.
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Nixtamalized maize has several benefits over unprocessed grain: it is more easily ground, its
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nutritional value is increased, flavor and aroma are improved, and mycotoxins are reduced (Garcia
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and Heredia, 2006). The masa produced from nixtamalized maize grains is more cohesive and
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adhesive, which allow the formation of a sheet and thus, favor its cutting and shaping as round disks
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or another shape (Arámbula-Villa et al., 2017). Besides, the nutritional, textural and sensory
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properties of tortillas are enhanced (Hernandez-Uribe et al., 2007).
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The nutritional aspects of maize tortillas have been studied to some extent. Bressani (1990)
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reported that lime-based nixtamalization improves bioavailability of calcium, nicotinic acid and
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overall protein quality. In contrast, Saldana and Brown (1984) found that traditional maize tortillas
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had lower protein, thiamin, riboflavin and niacin contents than white enriched bread. Pappa et al.
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(2010) reported that the protein quality of maize tortillas was lower than that of raw maize. The
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bioavailability of carbohydrate constituents has been also considered. Carbohydrates are the main
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fraction of maize tortilla, accounting for up 70% of the dry matter. Of these, starch is major
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constituent. Rendon-Villalobos et al. (2002) reported that available starch decreased, while resistant
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starch increased slightly with storage time. Sayago-Ayerdi et al. (2005) reported that the observed
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increase of resistant starch during storage can be explained as due to retrogradation effects. Rendon-
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Villalobos et al. (2006) pointed out that, in general, maize tortillas added with hydrocolloids had
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lower resistant starch fraction than their traditional counterparts. Hernandez-Uribe et al. (2007)
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studied tortillas made from white and blue maize varieties, finding that the latter exhibited lower
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predicted glycemic index than the former.
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The maize tortilla per capita consumption in Mexico has decreased about 30 % in the last two
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decades. Research results (Denova-Gutierrez et al., 2010) have associated this decline with the
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perception by consumers that tortillas are responsible for weight gain and metabolic syndrome
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related diseases. Since maize tortillas are still a major staple in Mexican dietary pattern, an ongoing
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research topic is to study maize varieties, technologies and processing conditions that may help to 3
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produce tortillas with reduced glycemic index. Santiago-Ramos et al. (2015) reported that tortillas
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made from maize flour obtained by using alternative nixtamalization treatments based on calcium
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salts had lower predicted glycemic index than tortillas made with commercial flour obtained using
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traditional lime-based nixtamalization. Camelo-Mendez et al. (2017) reported negative correlations
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between phenolic content and starch digestion for tortillas made from blue maize varieties.
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The in vivo digestion of starch involves amylase-mediated enzymatic hydrolysis of starch
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chains. Amylases digest amylose into maltose subunits and amylopectin into dextrins. Both maltose
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and dextrins are digested by maltase, resulting in the formation of glucose monomers. Resistant
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starch fraction limits the formation rate of maltose and dextrins, and hence the absorption of glucose
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units in small intestine. In contrast, small-length starch chains might contribute to faster hydrolysis
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rate, leading to increased glycemic index (Butterworth et al., 2011). However, lime-based
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nixtamalization of maize grains induces breaks in starch chains, which could lead to destabilization
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of semi-crystalline structures and favor continued amylolysis (Lobato-Calleros et al., 2015). A
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possible strategy to reduce starch digestibility of maize tortillas is to deplete the masa of sugars and
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short chains that are susceptible of rapid digestion.
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The aim of this study was (i) to explore the effect of adding baking yeast with amylolytic activity
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to masa obtained from nixtamalized maize flour on viscoelasticity, total sugars and apparent
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amylose content; and (ii) to produce tortillas from the yeast-treated masa, evaluating their hardness
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and in vitro starch digestibility, when freshly made and after four days of storage.
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2. Materials and methods
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2.1. Materials
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Maize starch (MS; S4126; amylose: 25.3%; moisture <15%) was obtained from Sigma-Aldrich
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(Toluca, Mexico). Nixtamalized maize flour (NMF) was purchased from Maseca S.A. de C.V.
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(Monterrey, Mexico; batch I02882) with reported composition per 100 g: 68 g starch, 1 g sugars, 8
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g proteins, 4.5 g lipids, 7 g dietary fiber and 11.5 g moisture. The particle size of the maize flour
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was estimated with a Mastersizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire, UK),
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with the help of a Scirocco dry disperser unit used for dispersing the powders at a feed pressure of 2
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bars and a feed rate of 40%, with monomodal distribution centered at 143.2 µm. Instant dry yeast
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was obtained from (Safmex S.A. de C.V., Toluca, Mexico). All reagents used were analytical grade
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obtained from (Sigma-Aldrich, Toluca, Mexico). Distilled water was used in all experiments.
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2.2. Amylolytic activity of the yeast strain
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The amylolytic activity of the commercial baking yeast (S. cerevisae strain) was tested by
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subjecting native starch granules to the metabolic action of the baking yeast strain. To this end,
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eight Erlenmeyer flasks (250 mL) containing native maize starch (3 g˖100 g-1) dispersions were
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inoculated with the yeast strain (3 g˖100 g-1). The flasks were gently stirred in a shaking water bath
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(200 rpm) at 38 ºC for up to 24 h. The maize starch samples were withdrawn at different sampling
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times, and centrifuged (6,000×g) for 10 min. The precipitates were oven-dried at 30 oC until
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constant weight was achieved (∼24 h), put into desiccators with a silica-gel bed at the bottom as
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adsorbent, until required for analysis.
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Polarized light microscopy was used to examine the morphology of the starch granules subjected to
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the metabolic action of the yeast strain. Samples were placed on viewing slides, upon which cover
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slips were gently placed. Olympus BX45 microscope (Olympus Optical Co., Tokyo, Japan) was
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used and images were captured with Carl Zeiss AxioCamERc 5s camera. Selected micrographs at
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100× were shown.
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2.4. Preparation of dough and tortilla variations
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The preparation of masa consisted of mixing NMF with water in a 40:60 mass ratio. Given the lack
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of guidelines in the literature, we decided to use typical yeast levels employed for different wheat
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breads preparation. The idea was to emulate the fermentation process of wheat dough. In this way,
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masa variations were prepared by adding baking yeast in 0.25, 0.50 and 1.0 g˖100 g-1 NMF in the
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basic masa recipe. To this end, the NMF (0.4 kg) was put into the mixing bowl of a Costway Tilt-
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head stand mixer (ON, Canada), and aqueous yeast dispersion was added in small portions to
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prevent grumps, and continuously mixed and kneaded with the equipment dough hook at speed
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level 3 for 5 min. The masa variations were individually packed into 20×30 cm poly-ethylene bags
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to reduce moisture loss and placed in a SL Shel Lab oven (Sheldon Manufacturing, OR, USA) for 2
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h at 38 °C to promote the activity of the yeast strain. Spherical portions of 30±0.01 g of masa were
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formed, placed between two plastic sheets in a manual wooden tortilla press, and pressed for
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obtaining masa sheet circles with a 14±0.2 cm diameter and 2.0±0.02 mm thickness. The masa
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circles were placed on a hotplate (C-MAG HS 10, IKA Industrie-und Kraftfahrzeugausrüstung
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GmbH, Königswinter, Germany) at 350 °C for 1 minute, then turned and placed for further 30
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seconds and flipped again for 15 seconds. Tortillas were left to cool down for 5 min, and a portion
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of them was used immediately for analyses, while the rest were individually packed into poly-
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ethylene bags and stored at 4 °C for up to 4 days. Stored tortillas were withdrawn from the plastic
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bags, and allowed to achieve the temperature at which specific analyses were carried out (Rendon-
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Villalobos et al., 2002). For simplicity in notation, masa and tortillas were tagged as Mx and Tx,
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where the symbol “x” denotes the amount of added yeast. In addition, two controls for masa and
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tortilla were considered to contrast the results: a) The first control consisted of forming the basic
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masa recipe without yeast and without subjecting it to the incubation step at 38 oC (CM1), and to
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form tortillas (CT1). b) The second control consisted of forming the basic masa recipe without
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yeast, but subjecting it to the heating process (2 h, 38 °C) (CM2), and to form tortillas (CT2).
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161 2.5. Rheological properties of masa variations
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Oscillatory measurements of masa variations were performed with a Physica MCR 300 rheometer
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(Physica Messtechnik GmbH, Stuttgart, Germany), and cone and plate geometry was selected, with
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a rotating cone 50 mm in diameter, cone angle of 2°, and a gap of 0.05 mm. Each sample (~5.0 g)
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was placed in the measuring system, and left to stabilize for 5 min at 25 °C for structure recovery.
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To prevent dehydration, mineral oil was used to cover the edges between geometry and masa.
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Temperature maintenance was maintained with a Physica TEK 150P temperature control system.
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Amplitude sweeps were carried out at 1 Hz and a strain range of 0.01-100%.
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2.6. Moisture content of masa and tortillas
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The moisture content of masa and tortilla variations was obtained by the AOAC-2000 vacuum oven
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drying method 930.15. All samples were vacuum oven dried at 60 °C for 24 hours.
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2.7. ATR-FTIR of masa and tortilla variations
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Masa and tortilla variations were characterized by ATR-FTIR. The spectra were recorded on a
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Perkin Elmer spectrophotometer (Spectrum 100-Perkin Elmer, Waltham, MA, USA) equipped with
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a crystal diamond universal ATR sampling accessory. Each spectrum represented an average of four
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scans. All spectra were deconvoluted using Gaussian and Lorentzian functions. In this case, the
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assumed line shape was Lorentzian with a half-width of 15 cm-1. The resolution enhancement factor
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was chosen as 1.5.
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2.8. Tortilla hardness
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Hardness of tortilla variations was measured with a Brookfield CT3-4500 texturometer (AMETEK,
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Middleborough, MA, USA) equipped with a spherical probe TA18 (1.27 cm diameter). A whole
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piece of each tortilla variation was placed on the texturometer plate with a square pierced hole (2.54 6
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cm diameter). Samples were compressed (1 cycle) at a rate of 1 mm·sec-1, until 50% penetration
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was achieved.
189 2.9. Apparent amylose content
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The method proposed by Zhu et al. (2008) was followed, with slight modifications. Masa samples
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(500.0±0.1 mg) were transferred into an l00 mL volumetric flask, and 1 mL of ethanol was added.
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The samples were dispersed in 10 mL of KOH (1N) for 1 h for complete dissolution. The volume
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was adjusted to 100 mL in a volumetric flask. Then 2 mL of the diluted solution were taken and 50
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mL of distilled water and 100 µL of alcoholic phenolphthalein solution (1% w/v) were added.
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Hydrochloric acid (0.1N) was added dropwise until a slight pink color was obtained. Afterwards 2
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mL of iodine solution (2.0 g of potassium iodide and 0.2 g of iodine in 100 mL of distilled water)
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was added to the neutralized solution and then adjusted to 100 mL in a volumetric flask. The
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solution was allowed to rest for 30 min to fully color development. The apparent amylose content
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was calculated from the ratio of absorbance measurements at 620/510 nm. Scans were performed by
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UV-Visible Spectrophotometer (Spectronic Genesys 2, Thermo Electron Corporation, Madison,
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WI, USA).
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203 2.10. Total sugars
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Total sugar determination of the masa variations was done using the phenol-sulfuric method
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(Dubois et al., 1956) with some modifications. Samples of masa variations (0.5 g) were dispersed in
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25 mL of water, an aliquot of 0.625 mL was taken and put into screw cap tubes (13×100 mm) and
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0.625 mL of a phenol solution (5 % w/w) were added. The mixture was vortex-stirred and placed in
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an ice bath ice for 5 min. Then, 2.5 mL of concentrated sulfuric acid was carefully added. The tubes
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were then vortex-stirred again for 5 s and incubated for 5 min at boiling temperature. The tubes
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were allowed to cool down to room temperature before reading the absorbance at 490 nm in UV-
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Visible Spectrophotometer (Spectronic Genesys 2, Thermo Electron Corporation, Madison, WI,
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USA). A glucose calibration curve was used as standard.
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2.11. In vitro starch digestibility
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About 100 mg of each tortilla variation were treated with a 2M KOH to disperse all starch fractions,
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and then hydrolyzed by incubation with α-amylase (A-3176, 10-30 units.mg-1 solid, Sigma
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Chemical Co. St Louis, MO, USA) and amyloglucosidase (A-7420, 14 units.mg-1, Sigma Chemical
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Co. St Louis, MO, USA). Resistant starch (RS) was measured using the AACC-2000 method 32-40.
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The tortilla samples were incubated with pancreatic α-amylase and amiloglucosidase during 16 h to 7
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hydrolyze the non-RS. The reaction was stopped by the addition of ethanol. Afterwards, the
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samples were centrifuged to collect the RS precipitate. The pellet was then treated with 2M KOH
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and hydrolyzed by incubation with AMG. For these determinations, the TS and RS kit analysis
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from Megazyme International Ireland Ltd. (Bray Business Park, Bray, Co., Wicklow, Ireland) was
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used. Available starch was calculated as the difference between TS and RS. The in vitro digestion
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of the tortilla variations was performed using the Englyst et al. (1992) method. Starch classification
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was based on the rate of hydrolysis, RDS (digested within 20 min) and SDS (digested between 20-
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120 min). Values were adjusted at the available starch content previously determined.
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Data were analyzed using a one-way analysis of variance (ANOVA) and a Tukey’s test for a
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statistical significance of p<0.05, using the Statgraphics 7 software (Statistical Graphics Corp.
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Manugistics Inc., Cambridge, MA). The data were reported as means ± S.D. All experiments were
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done in triplicate.
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235 3. Results and discussion
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3.1. Amylolytic activity
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The S. cerevisiae strain should display amylolytic activity for guaranteeing the reduction of amylose
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chain fractions in the masa variations bulk. The kinetics exhibited a sigmoidal-like behavior with
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lag phase lasting about 2 h (Figure 1). In this phase, the hydrolysis was relatively slow since starch
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granules are compactly packed and relatively free of water. For longer times, water hydrates the
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starch granules, facilitating the action of the amylolytic enzymes produced by S. cerevisiae. The
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micrograph corresponding to hydrolysis time of ∼3.5 h, shows that the starch granule morphology
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was characterized for displaying a central line known as the hilum or Maltese cross, where the
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beginning of a fracture can be discerned. In the micrograph corresponding to hydrolysis time of ∼ 6
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h, and fractures in the central region or hilum of the starch granule resulted evident, which cause the
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Maltese cross to deform. As hydrolysis proceeded to ∼ 8 h, the starch grain was broken into several
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fragments (the Maltese cross pattern disappeared), and at longer hydrolysis times (∼12 h) the starch
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fragments were disrupted into smaller entities, which are more susceptible for amylolytic attack.
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Overall, the results exhibited in Figure 1 show that the baking yeast used for masa treatment had
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strong amylolytic activity.
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3.2. Moisture and total starch content of masa
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Non-significant different moisture contents were exhibited among the masa variations after thermal
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incubation. Moisture content varied between from 69.8±0.6 g·100 g-1 for M0.25 to 70.6±0.7 g·100
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g-1 for CM1. This was expected as the masa variations were put into poly-ethylene bags during
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incubation. The total starch of masa after incubation was also quantified. The starch contents of
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CM1 (68.4±0.6 g·100 g d.b.) and CM2 (68.3±0.5 g·100 g d.b.) and were non-significantly different
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between themselves, but significantly different from those of the masa variations containing yeast.
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The starch content for M0.25, M0.5 and M1.0 was of 66.3±0.5, 64.1±0.5 and 63.6±0.6 g·100 g d.b.,
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respectively, and the reduction of the total starch content was ascribed to the amylolytic activity
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induced by the yeast addition.
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NMF contains about 1.0 g total sugars 100 g-1. These sugars are used by the S. cerevisiae strain
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producing carbon dioxide and water via invertase enzyme. The two masa controls, CM1 and CM2,
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contained non-significantly different total sugar content (Figure 2.a). In contrast, the masa
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variations containing S. cerevisiae showed a significant decrease in total sugars with respect to both
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controls, and the reduction in total sugar contents was significantly higher the higher was the S.
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cerevisiae concentration used. On the other hand, Figure 2.b shows that CM1 and CM2 had a non-
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significantly different amylose content of about 32-34%. Upon nixtamalization the native maize
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starch granules (which have an amylose content of about 25%), undergo partial gelatinization which
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mainly takes place at the periphery of the grain, where fractionation of starch chains, including
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debranching of amylopectin molecules, can occur. In turn, debranching increases the content of
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apparent amylose (Lobato-Calleros et al., 2015). The masa variations showed a significant lower
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amylose content with respect to CM1 and CM2. These results pinpoint that the amylolytic activity
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of S. cerevisiae decomposed starch chains into simple glucose units via glucoamylase enzymes.
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Gelatinized starch chains dispersed in the masa variations bulk are more susceptible to enzymatic
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attack, producing in this way an important reduction in the apparent amylose content. The amylose
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content was of about 28% for M0.25, 25% for M0.50 and 24% for M1.0. In subsequent reactions,
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the produced sugars are used by gasification reactions catalyzed by invertase enzymes. The overall
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effect was reflected as important reductions of total sugar and apparent amylose contents. The
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reduction in amylose content in the masa variations with respect to CM1 and CM2 ranged between
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12.5-29.4, depending on the baking yeast concentration used in masa variations.
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3.4. Viscoelasticity of masa
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The viscoelastic properties are important for assessing the malleability of masa, which ultimately
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affects its lamination, cutting and shaping, and has an impact on tortilla making and quality.
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Figure 3.a exhibits the storage (G’) and loss (G’’) moduli for CM1 and CM2, as well as for the
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yeast-treated masa variations M0.25, M0.5, and M1.0. For all cases, the storage modulus
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displayed a monotonous decreasing trend, with a power-law decay γ
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γ > 10% . This indicates that masa microstructure underwent rearrangements that reduced the
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capacity of mechanical energy storage. G’ was lower for the masa variations incorporating yeast
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than for CM1 and CM2, meaning that the storage modulus was reduced by the action of the yeast
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treatment. Figure 3.b presents the behavior of G’ at γ = 1.0 % for the different masa variations.
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CM1 and CM2 exhibited similar values, suggesting that the 2 h thermal treatment did not induce
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appreciable changes in the masa microstructure. However, the storage modulus decreased
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continuously with the amount of added yeast. It is apparent that yeast activity modified the
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components of the masa, possibly the fraction of leached amylose dispersed in the continuous
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phase of masa variations. On the other hand, the loss modulus in Figure 2.a (open symbols)
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exhibited an interesting behavior reflected by a non-monotonous pattern. In fact, the loss modulus
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decreased for small strain values ( γ < 0.3 %). However, increasing loss modulus was observed
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for strain values in the range from 1.0% to 10%. This behavior could be attributed to the
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aggregation of particles dispersed in the masa, producing a jamming-like pattern (Brown and
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Jaeger, 2014). For higher strain values, the loss modulus decreased continuously with power-law
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behavior γ
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suggesting that the additional thermal treatment induced poor rearrangement of the starch fraction
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dispersed in the masa. In contrast, the addition of yeast produced a significant reduction in the
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loss modulus, being of about 35% for M1.0. The cause of such reduction can be attributed to
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hydrolysis of amylose present in the periphery of starch granules induced possibly by the
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amylolytic activity of the baking yeast colonies, reflected as a significant reduction of amylose
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(Figure 2.a).
for strain values
. CM2 exhibited significant higher loss modulus than its CM1 counterpart,
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3.5. FTIR of masa
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The FTIR spectra for the masa controls and the yeast-treated masa variations showed similar
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characteristic bands (Figure 4.a). For all cases, a pronounced broad band was displayed at 3000-
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3700 cm-1, resulting from the overlapping combination of -OH stretching (~3500 cm-1) linked to
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hydration effects, and NH stretching (3300 cm-1) attributed to Amide A groups of proteins (Kong 10
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and Yu, 2007). These peaks were unchanged by yeast treatment. The peak at ~2930 cm-1 can be
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related to CH stretching and can be attributed to free fatty acids and amylose-fatty acid inclusion
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complexes (Flores-Morales et al., 2012). The formation of these complexes was an expected result
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since nixtamalized maize flour contains ~4-5 % lipids, which can form amylose-lipid inclusion
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complexes during the nixtamalization process. The broad band at 1700-1600 cm-1 with a large peak
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at ~1640 cm-1 can be ascribed to C=O stretching and is linked to the Amide I group of maize
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proteins (Flores-Morales et al., 2012). The bands at 1540, 1418, 1364 and 1242 cm-1 can be related
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to conjugated carbonyl and carboxyl groups and C-O vibrations. The broad band at ~1080-950 cm-1
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reflects C-O, C-C and C-H stretching and C-O-H bending vibrations, with starch corresponding to
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the major fraction in maize flour. This wide band has been considered as a fingerprint of starch
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structure (van Soest et al., 1995), finding that the band close to 1022 cm−1 increased for more
332
amorphous samples, while the bands at close to 995 and 1047 cm−1 were more prominent in more
333
crystalline samples. The ratio 1047/1022 was proposed as indicator of ordered structures in starch
334
(van Soest et al., 1995). The band at 995 cm−1is mainly due to COH bending vibrations, and is
335
sensitive to water-starch interactions, including hydrogen bonding (van Soest et al., 1995). Figure
336
4.b illustrates the deconvoluted FTIR spectra in the starch fingerprint region, showing three main
337
peaks at 1044, 1021 and 995 cm-1. The first two peaks are close to the nominal peaks 1047 and 1022
338
cm-1, a shift that could be caused by the limited amount of water in masa formulations. To be in line
339
with the terminology by van Soest et al. (1995), the ratio 1047/1022 was estimated by taking the
340
closest peaks to 1047 and 1022 cm-1. CM1 and CM2 exhibited similar ratio (Figure 4.c), indicating
341
that the heating of masa for 2 h at 38 oC (CM2) did not affect the short-range ordering of starch
342
chains. However, the ratio 1047/1022 increased with the yeast treatment, suggesting that yeast
343
activity reduced the amorphous starch components dispersed in the tortilla bulk. The increase of the
344
ratio 1047/1022 was higher as the amount of yeast added to masa was increased.
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3.6. Weight and thickness of tortillas
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The weight of tortillas was 27.2±0.5 g for CT1, and 26.9±0.4 g for CT2. On the other hand, tortillas
348
added with yeast showed similar weight, with variations from 27.1±0.4 g for T0.25 to 27.3±0.5 g
349
for T1.0. This means that the addition of yeast did not have a significant effect on the weight of
350
fresh tortillas. Similar effects were observed for tortilla thickness as the parameter varied from
351
1.82±0.2 mm for CT1 to 1.84±0.2 mm for T1.0.
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3.7. Moisture content and hardness of tortillas
356
The moisture content of tortillas is an important parameter since it has determinant effects in the
357
staling phenomenon. Figure 5.a shows that the moisture content of all the tortilla variations
358
decreased significantly between 2-3 % over the 4 days of storage. In general terms, the moisture
359
content of the tortilla variations was from lower to higher during storage as follows: CT1 < CT2 <
360
T0.25 < T0.5 < T1.0. These results indicate that the thermal treatment (2 h, 38 °C) to which CM2
361
was subjected induced a higher water retention respect to CT1, which was obtained from non-
362
thermally treated masa (CM1). The yeast-treated masa variations produced tortillas with improved
363
water retention with respect to the non-yeast treated controls CT1 and CT2. The higher the yeast
364
concentration the higher the moisture content of the tortilla variations. T1.0 exhibited an increase of
365
about 10% moisture content respect that of CT1, throughout storage time. A possible explanation
366
for the improved moisture content exhibited by the yeast-treated tortillas is that the cell wall of
367
yeast (S. cerevisae) represents about 50-65% of β-glucan, a branched polysaccharide that has a high
368
water holding capacity (Borchani et al., 2016), and to the increased yeast activity which produced
369
an increase in cells population in the masa bulk. Similar effect was reported for wheat bread added
370
with different hydrocolloids (sodium alginate, xanthan, κ-carrageenan and hydroxypropyl-
371
methycellulose). In fact, breads containing hydrocolloids showed lower loss of moisture content
372
(Clubbs et al et al., 2008).
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Loss of water is commonly accompanied by an increase in tortillas hardness. Fresh tortillas CT1
374
and CT2 exhibited the highest values of hardness (Figure 5.b), and became increasingly harder with
375
storage time. In contrast, the yeast-treated tortillas exhibited significantly lower hardness values
376
than the control tortillas throughout the storage time. The higher the yeast content, the lower was
377
the tortilla variation hardness.
378
significant hardness change with storage time, suggesting that yeast cells and in situ produced
379
polysaccharides contributed to practically maintain without change the tortillas hardness values.
380
Roman-Brito et al. (2007) reported that the addition of xanthan gum to maize tortillas decreased
381
hardness and retarded staling effects.
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3.8. FTIR of tortillas
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Storage of tortillas is accompanied by retrogradation effects, which are originated by loss of
385
moisture content (Figure 5.a), increased hardness (Figure 5.b) and an increase in the organization of
386
the structure. In fact, Flores-Morales et al. (2012) detected that the band close to 1047 cm-1
387
increased with tortilla staling. As done for masa, deconvolution of tortilla variations was carried out
388
to detect changes in the ratio R1047/1022. The results are presented in Figure 5.c and show that the 12
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ratio R1047/1022 was larger for the yeast-treated tortillas than for the non-yeast treated tortillas. This
390
result is in line with results in Figure 4.c for masa variations. Amorphous structures are more
391
susceptible to enzymatic attack by baking yeast, leaving more ordered molecular structures (double
392
helical crystalline states) that are identified as resistant starch (Tester et al., 2004). Storage
393
promoted the formation of ordered molecular structures as reflected by the increased values of the
394
ratio R1047/1022. Besides, the rate of increase of such ratio was higher for tortillas made with masa
395
treated with baking yeast. In principle, the increased value of R1047/1022 accompanied with the
396
reduction of moisture content should lead to tortillas with increased hardness (i.e., staling).
397
However, this effect was not observed since the yeast-treated tortillas exhibited lower hardness
398
values than the non-yeast treated tortilla controls. The metabolic activity of the baking yeast should
399
play an important role in the modification of the tortilla properties. As mentioned above, increased
400
yeast cell population and the production of exo-polysaccharides acting as in situ hydrocolloid could
401
be behind the hindering of the staling effects in treated tortillas.
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402 3.9. Digestibility
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TC1 exhibited about 79.21% RDS, 5.77% SDS and 15.23% RS (Figure 6.a), and these contents are
405
in line with those results reported by Bello-Perez et al. (2014) for tortillas made with commercial
406
nixtamalized flour. The heat treatment (38 oC by 2 h) did not affect the in vitro digestibility
407
fractions of TC2, which presented non-significant different fractions values similar to TC1.
408
However, the baking yeast treatment had an important effect on the in vitro digestibility. In fact, the
409
RDS fraction decreased and the RS fraction increased with the baking yeast content. Specifically,
410
the starch fractions for treated tortilla T1.0 were 70.75% RDS, 2.48% SDS and 27.52% RS.
411
Following the results described above, the increase of the RS fraction and the decrease of the RDS
412
fraction was likely induced by the metabolic activity of the baking yeast. These results are in line
413
with the decreases in total sugars and apparent amylose reported above. It can be postulated that
414
baking yeast consumes, via amylolytic activity, short-length starch chains that otherwise are easily
415
accessible by enzymatic activity. Storage time can modify the digestibility of tortillas by the effect
416
of starch retrogradation and moisture loss and re-distribution (Rendon-Villalobos et al., 2006;
417
Bello-Perez et al., 2014). Figure 6.b displays the starch fractions for the tortilla variations after four
418
days of storage time. The starch fractions for TC1 were 56.91% RDS, 19.02% SDS and 29.07% RS,
419
results that are similar to those reported by Bello-Perez et al. (2014) for tortillas made with
420
commercial nixtamalized maize flour. After four days of storage, T1.0 exhibited significant
421
increases of SDS and RS at the expenses of significant decreases in RDS. In fact, tortilla made with
422
masa treated with baking yeast exhibited 42.23% RDS, 23.54% SDS and 34.23% RS.
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Reorganization of starch molecules within complex semi-crystalline structures are linked to the
424
increase of the SDS and RS fractions in tortillas made with treated masa. Since the metabolic
425
activity of baking yeast is prompted to reduce the fraction of easily accessible starch chains (e.g.,
426
short-length chains), reorganized starch molecules in storage become conformed by preferably by
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long-length chains, offering increased resistance for amylolytic action.
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428 4. Conclusions
430
The treatment of nixtamalized maize masa with baking yeast altered the in vitro digestibility of
431
freshly made and stored (4 days) tortillas with respect to that exhibited by the non-yeast treated
432
controls. Yeast-treated fresh tortillas exhibited lower RDS and SDS, but higher RS fractions than
433
the untreated controls. In the yeast-treated tortillas stored for four days, the beneficial changes on in
434
vitro digestibility were more pronounced, as the RDS fraction decreased, but the SDS and RS
435
fractions increased significantly with respect to that of the controls. The effect can be explained
436
from the amylolytic activity of the baking yeast, leading to reduced contents of total sugars and
437
apparent amylose. In all cases the hardness of the yeast-treated tortillas was lower than for the
438
untreated tortilla controls, and this was attributed to the increased yeast population and in situ
439
polysaccharides formation that acted as hydrocolloids.
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440 References
442
Arámbula-Villa, G., Saavedra-Rosiles, G., Rendón-Villalobos, R., Rodríguez-González, F.,
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Figueroa-Cárdenas, J.D., Méndez-Albores, J.A., 2017. Structural, thermal and rheological
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properties of nixtamalized maize masa obtained from varying the concentration of calcium
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Bressani, R., 1990. Chemistry, technology, and nutritive value of maize tortillas. Food Rev. Int. 62, 225-264. Butterworth, P.J., Warren, F.J., Ellis, P.R., 2011. Human α-amylase and starch digestion: An interesting marriage. Starch/Stärke 63, 395-405. Camelo-Méndez, G.A., Agama-Acevedo, E., Tovar, J., Bello-Pérez, L.A., 2017. Functional study of
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Clubbs, E.A., Vittadini, E., Shellhammer, T.H., Vodovotz, Y., 2008. Effects of storage on the
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changes by FT-IR, Raman, and CP/MAS 13C NMR spectroscopy on retrograded starch of
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maize tortillas. Carbohydr. Polym. 87, 61-68.
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Hernández‐Uribe, J. P., Agama‐Acevedo, E., Islas‐Hernández, J. J., Tovar, J., Bello‐Pérez, L. A.
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Kong, J., Yu, S., 2007. Fourier transform infrared spectroscopic analysis of protein secondary
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Lobato-Calleros, C., Hernandez-Jaimes, C., Chavez-Esquivel, G., Meraz, M., Sosa, E., Lara, V.H.,
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Alvarez-ramirez, J., Vernon-Carter, E.J., 2015. Effect of lime concentration on gelatinized
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maize starch dispersions properties. Food Chem. 172, 353-360.
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Pappa, M.R., de Palomo, P. P., Bressani, R., 2010. Effect of lime and wood ash on the
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nixtamalization of maize and tortilla chemical and nutritional characteristics. Plant Foods
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Hum. Nutr. 65, 130-135.
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Peña-Reyes, R.A., Ramírez-Romero, G.A., Fernández-Perrino, F.J., Cruz-Guerrero, A.E., 2017.
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Effect of nixtamalization processing temperature on maize hydration and the textural
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properties of masa and tortillas. J. Food Process. Pres. 41, e13136. Rendon-Villalobos, R., Bello-Pérez, L.A., Osorio-Díaz, P., Tovar, J., Paredes-López, O., 2002.
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Effect of storage time on in vitro digestibility and resistant starch content of nixtamal, masa,
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and tortilla. Cereal Chem.79, 340-344.
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Rendón-Villalobos, R., Agama-Acevedo, E., Islas-Hernández, J.J., Sánchez-Muñoz, J., Bello-Perez,
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L.A., 2006. In vitro starch bioavailability of corn tortillas with hydrocolloids. Food Chem.
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97, 631-636.
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Román-Brito, J.A., Agama-Acevedo, E., Méndez-Montealvo, G., Bello-Pérez, L.A., 2007. Textural studies of stored corn tortillas with added xanthan gum. Cereal Chem. 84, 502-505. Saldana, G., Brown, H.E., 1984. Nutritional composition of corn and flour tortillas. J. Food Sci. 49, 1202-1203.
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Santiago-Ramos, D., Figueroa-Cárdenas, J.D.D., Véles-Medina, J.J., Mariscal-Moreno, R.M.,
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Reynoso-Camacho, R., Ramos-Gómez, M., Gaytán-Martínez, M., Morales-Sánchez, E.,
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2015. Resistant starch formation in tortillas from an ecological nixtamalization
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process. Cereal Chem. 92, 185-192.
Sáyago-Ayerdi, S.G., Tovar, J., Osorio-Diaz, P., Paredes-López, O., Bello-Pérez, L.A., 2005. In
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vitro starch digestibility and predicted glycemic index of corn tortilla, black beans, and
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tortilla-bean mixture: effect of cold storage. J. Agric. Food Chem. 53, 1281-1285.
510 511
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Tester, R.F., Karkalas, J., Xin, Q., 2004. Starch-composition, fine structure and architecture. J. Cereal Sci. 39, 151-165.
van Soest, J.J.G., Tournois, H., de Wit, D., Vliegenthart, J.F.G., 1995. Short-range structure in
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(partially) crystalline potato starch determined with attenuated total reflectance Fourier-
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transform IR spectroscopy. Carbohydr. Res. 279, 201-214.
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Zhu, T., Jackson, D. S., Wehling, R. L., & Geera, B. (2008). Comparison of amylose determination
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methods and the development of a dual wavelength iodine binding technique. Cereal
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Chem. 85, 51-58.
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Figure 1. Hydrolysis advance of native maize starch subjected to the metabolic action of
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the yeast strain. The inset images illustrate the morphology of the starch granules for
11
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different hydrolysis times.
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c
0.6 0.5 CM1
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a
d
M0.5
M1.0
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c
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CM1
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Figure 2. (a) Total sugars and (b) apparent amylose contained in the different masa
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variations. Small letters above the bars denote significant statistical differences (p < 0.05).
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(a)
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G' and G'' (Pa)
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4
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1
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a
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b
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5x10
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Strain (%)
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Figure 3. (a) Viscoelasticity moduli of the different masa variations. Closed and open
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circles correspond to storage (G’) and loss (G’’) moduli, respectively. Squares – MC1,
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circles – MC2, upper triangle – M0.25, sided triangle – M0.5 and star – M1.0. (b) Storage
28
modulus for strain 1.0% and the five different masa variations. (b) Loss modulus for strain
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1.0% and the five different masa variations. Small letters above bars indicate significant
30
different values (p < 0.05).
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- 931
-1155 - 1081
- 1242
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- 1418 - 1364
Amide I
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- 1540
Starch
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Absorbance (a.u.)
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1600 1400 1200 1000 -1
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Wavenumber (cm )
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Wavenumber (cm )
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c cd
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CM1
CM2
M0.25
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M1.0
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Figure 4. (a) FTIR spectra of masa formulations (solid – CM1, dash – CM2, dot – M0.25,
38
dash-dot – M0.5 and short dash – M1.0). (b) Example of deconvolution of the starch
39
fingerprint region. (c) Ratio R1047/1022 for the different masa variations. Letter above bars
40
indicate statistical significant differences (p < 0.05).
41 4
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(a)
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2.0 1.8 1.6 1.4
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0.24 0.22
0
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Time (days)
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Figure 5. (a) Evolution of the moisture content with storage time for tortilla variations. (b)
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Hardness of tortilla variations as function of storage time. (c) FTIR ratio R1047/1022 as
50
function of storage time. For the three panels, the legends are as follows: Squares – TC1,
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circles – TC2, upper triangle – T0.25, sided triangle – T0.5 and star – T1.0.
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Figure 6. Starch digestibility fractions for the different tortilla variations: (a) Fresh tortillas,
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and (b) tortillas stored for 4 days. Unfilled bar – RDS; Oblique-fill bar – SDS; Vertical-fill
63
bar – RS.
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Highlights Baking yeast treatment of maize masa reduced sugars and apparent amylose content
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Tortillas made with treated masa exhibited reduced contents of RDS and increased content of RS Baking yeast treatment led to tortillas with reduced hardness
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S0733-5210(18)30228-5
DOI:
10.1016/j.jcs.2018.07.001
Reference:
YJCRS 2595
To appear in:
Journal of Cereal Science
Received Date: 19 March 2018 Revised Date:
29 June 2018
Accepted Date: 2 July 2018
Please cite this article as: Vernon-Carter, E.J., Alvarez-Ramirez, J., Bello-Perez, L.A., GarciaHernandez, A., Garcia-Diaz, S., Roldan-Cruz, C., The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by treating the nixtamalized masa with commercial baking yeast, Journal of Cereal Science (2018), doi: 10.1016/j.jcs.2018.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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5 (Corrected Manuscript No. YJCRS_2018_220)
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The in vitro digestibility of starch fractions in maize tortilla can be rendered healthier by
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treating the nixtamalized masa with commercial baking yeast
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E.J. Vernon-Cartera, J. Alvarez-Ramireza,*, L.A. Bello-Perezb, A. Garcia-Hernandeza, S. Garcia-
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Diazc, C. Roldan-Cruzc
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a
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Iztapalapa. A.P. 55-534, CDMX, 09340 México.
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b
CEPROBI, Instituto Politécnico Nacional, Yautepec, Morelos, México.
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c
Departamento de Biotecnología. Universidad Autónoma Metropolitana-Iztapalapa. A.P. 55-534,
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CDMX, 09340 México.
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* Corresponding Author. Email: [email protected]. Fax/phone: +52-55-58044650
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Departamento de Ingeniería de Procesos e Hidráulica. Universidad Autónoma Metropolitana-
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Abstract
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Baking yeast exhibiting amylolytic activity was used for treating masa made from nixtamalized
35
maize flour (NMF). Baking yeast (0.25, 0.50 and 1.0 g˖100 g-1 NMF) was added to the basic masa
36
recipe (40 NMF:60 water mass ratio). Two masa controls without yeast addition were prepared:
37
CM1 used as such, and CM2 subjected to a mild incubation treatment (2 h, 38 °C). Tortillas were
38
made (350 °C, 1.0 min) with the yeast-treated masa. Baking yeast reduced total sugars, apparent
39
amylose and viscoelasticity of the masa. Tortillas made with treated masa exhibited significant
40
lower hardness than tortillas made with CM1 and CM2, and this effect was more pronounced in the
41
tortillas stored for 4 days. Tortillas freshly made from yeast-treated masa displayed reduced RDS
42
and SDS, but increased RS fractions. When stored for 4 days, they showed reduction in RDS, but an
43
increase in SDS and RS fractions (∼30%) with respect to tortillas made with CM1 and CM2.
44
Hardness of the yeast-treated tortillas was significantly lower and remained practically without
45
change during storage, while the untreated tortillas hardened significantly. Treatment with baking
46
yeast induces beneficial health and textural effects in tortillas.
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Keywords: Nixtamalized maize masa; baking yeast; amylolytic activity; tortilla; starch
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digestibility.
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1. Introduction
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Maize tortillas are an important source of daily calories (~50-70%) and proteins (~50%) for urban
53
and rural populations in Mexico, the consumption estimated at 90 kg by inhabitant per year. The
54
production of maize tortillas requires of a process known as nixtamalization. In the traditional
55
nixtamalization process, maize grains are cooked in a solution of water with lime, followed by
56
overnight steeping, washing and grinding to obtain a wet dough (masa) from which tortillas are
57
formed (Peña-Reyes et al., 2017). Due to economic and technological benefits, nowadays most of
58
the nixtamalization process is carried out at industrial level, and the use of instantaneous
59
nixtamalized maize flour (NMF) has increasingly displaced traditional maize grain processing.
60
Nixtamalized maize has several benefits over unprocessed grain: it is more easily ground, its
61
nutritional value is increased, flavor and aroma are improved, and mycotoxins are reduced (Garcia
62
and Heredia, 2006). The masa produced from nixtamalized maize grains is more cohesive and
63
adhesive, which allow the formation of a sheet and thus, favor its cutting and shaping as round disks
64
or another shape (Arámbula-Villa et al., 2017). Besides, the nutritional, textural and sensory
65
properties of tortillas are enhanced (Hernandez-Uribe et al., 2007).
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The nutritional aspects of maize tortillas have been studied to some extent. Bressani (1990)
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reported that lime-based nixtamalization improves bioavailability of calcium, nicotinic acid and
68
overall protein quality. In contrast, Saldana and Brown (1984) found that traditional maize tortillas
69
had lower protein, thiamin, riboflavin and niacin contents than white enriched bread. Pappa et al.
70
(2010) reported that the protein quality of maize tortillas was lower than that of raw maize. The
71
bioavailability of carbohydrate constituents has been also considered. Carbohydrates are the main
72
fraction of maize tortilla, accounting for up 70% of the dry matter. Of these, starch is major
73
constituent. Rendon-Villalobos et al. (2002) reported that available starch decreased, while resistant
74
starch increased slightly with storage time. Sayago-Ayerdi et al. (2005) reported that the observed
75
increase of resistant starch during storage can be explained as due to retrogradation effects. Rendon-
76
Villalobos et al. (2006) pointed out that, in general, maize tortillas added with hydrocolloids had
77
lower resistant starch fraction than their traditional counterparts. Hernandez-Uribe et al. (2007)
78
studied tortillas made from white and blue maize varieties, finding that the latter exhibited lower
79
predicted glycemic index than the former.
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The maize tortilla per capita consumption in Mexico has decreased about 30 % in the last two
81
decades. Research results (Denova-Gutierrez et al., 2010) have associated this decline with the
82
perception by consumers that tortillas are responsible for weight gain and metabolic syndrome
83
related diseases. Since maize tortillas are still a major staple in Mexican dietary pattern, an ongoing
84
research topic is to study maize varieties, technologies and processing conditions that may help to 3
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produce tortillas with reduced glycemic index. Santiago-Ramos et al. (2015) reported that tortillas
86
made from maize flour obtained by using alternative nixtamalization treatments based on calcium
87
salts had lower predicted glycemic index than tortillas made with commercial flour obtained using
88
traditional lime-based nixtamalization. Camelo-Mendez et al. (2017) reported negative correlations
89
between phenolic content and starch digestion for tortillas made from blue maize varieties.
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The in vivo digestion of starch involves amylase-mediated enzymatic hydrolysis of starch
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chains. Amylases digest amylose into maltose subunits and amylopectin into dextrins. Both maltose
92
and dextrins are digested by maltase, resulting in the formation of glucose monomers. Resistant
93
starch fraction limits the formation rate of maltose and dextrins, and hence the absorption of glucose
94
units in small intestine. In contrast, small-length starch chains might contribute to faster hydrolysis
95
rate, leading to increased glycemic index (Butterworth et al., 2011). However, lime-based
96
nixtamalization of maize grains induces breaks in starch chains, which could lead to destabilization
97
of semi-crystalline structures and favor continued amylolysis (Lobato-Calleros et al., 2015). A
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possible strategy to reduce starch digestibility of maize tortillas is to deplete the masa of sugars and
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short chains that are susceptible of rapid digestion.
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The aim of this study was (i) to explore the effect of adding baking yeast with amylolytic activity
101
to masa obtained from nixtamalized maize flour on viscoelasticity, total sugars and apparent
102
amylose content; and (ii) to produce tortillas from the yeast-treated masa, evaluating their hardness
103
and in vitro starch digestibility, when freshly made and after four days of storage.
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2.1. Materials
107
Maize starch (MS; S4126; amylose: 25.3%; moisture <15%) was obtained from Sigma-Aldrich
108
(Toluca, Mexico). Nixtamalized maize flour (NMF) was purchased from Maseca S.A. de C.V.
109
(Monterrey, Mexico; batch I02882) with reported composition per 100 g: 68 g starch, 1 g sugars, 8
110
g proteins, 4.5 g lipids, 7 g dietary fiber and 11.5 g moisture. The particle size of the maize flour
111
was estimated with a Mastersizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire, UK),
112
with the help of a Scirocco dry disperser unit used for dispersing the powders at a feed pressure of 2
113
bars and a feed rate of 40%, with monomodal distribution centered at 143.2 µm. Instant dry yeast
114
was obtained from (Safmex S.A. de C.V., Toluca, Mexico). All reagents used were analytical grade
115
obtained from (Sigma-Aldrich, Toluca, Mexico). Distilled water was used in all experiments.
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2.2. Amylolytic activity of the yeast strain
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The amylolytic activity of the commercial baking yeast (S. cerevisae strain) was tested by
121
subjecting native starch granules to the metabolic action of the baking yeast strain. To this end,
122
eight Erlenmeyer flasks (250 mL) containing native maize starch (3 g˖100 g-1) dispersions were
123
inoculated with the yeast strain (3 g˖100 g-1). The flasks were gently stirred in a shaking water bath
124
(200 rpm) at 38 ºC for up to 24 h. The maize starch samples were withdrawn at different sampling
125
times, and centrifuged (6,000×g) for 10 min. The precipitates were oven-dried at 30 oC until
126
constant weight was achieved (∼24 h), put into desiccators with a silica-gel bed at the bottom as
127
adsorbent, until required for analysis.
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Polarized light microscopy was used to examine the morphology of the starch granules subjected to
131
the metabolic action of the yeast strain. Samples were placed on viewing slides, upon which cover
132
slips were gently placed. Olympus BX45 microscope (Olympus Optical Co., Tokyo, Japan) was
133
used and images were captured with Carl Zeiss AxioCamERc 5s camera. Selected micrographs at
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100× were shown.
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2.4. Preparation of dough and tortilla variations
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The preparation of masa consisted of mixing NMF with water in a 40:60 mass ratio. Given the lack
138
of guidelines in the literature, we decided to use typical yeast levels employed for different wheat
139
breads preparation. The idea was to emulate the fermentation process of wheat dough. In this way,
140
masa variations were prepared by adding baking yeast in 0.25, 0.50 and 1.0 g˖100 g-1 NMF in the
141
basic masa recipe. To this end, the NMF (0.4 kg) was put into the mixing bowl of a Costway Tilt-
142
head stand mixer (ON, Canada), and aqueous yeast dispersion was added in small portions to
143
prevent grumps, and continuously mixed and kneaded with the equipment dough hook at speed
144
level 3 for 5 min. The masa variations were individually packed into 20×30 cm poly-ethylene bags
145
to reduce moisture loss and placed in a SL Shel Lab oven (Sheldon Manufacturing, OR, USA) for 2
146
h at 38 °C to promote the activity of the yeast strain. Spherical portions of 30±0.01 g of masa were
147
formed, placed between two plastic sheets in a manual wooden tortilla press, and pressed for
148
obtaining masa sheet circles with a 14±0.2 cm diameter and 2.0±0.02 mm thickness. The masa
149
circles were placed on a hotplate (C-MAG HS 10, IKA Industrie-und Kraftfahrzeugausrüstung
150
GmbH, Königswinter, Germany) at 350 °C for 1 minute, then turned and placed for further 30
151
seconds and flipped again for 15 seconds. Tortillas were left to cool down for 5 min, and a portion
152
of them was used immediately for analyses, while the rest were individually packed into poly-
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ethylene bags and stored at 4 °C for up to 4 days. Stored tortillas were withdrawn from the plastic
154
bags, and allowed to achieve the temperature at which specific analyses were carried out (Rendon-
155
Villalobos et al., 2002). For simplicity in notation, masa and tortillas were tagged as Mx and Tx,
156
where the symbol “x” denotes the amount of added yeast. In addition, two controls for masa and
157
tortilla were considered to contrast the results: a) The first control consisted of forming the basic
158
masa recipe without yeast and without subjecting it to the incubation step at 38 oC (CM1), and to
159
form tortillas (CT1). b) The second control consisted of forming the basic masa recipe without
160
yeast, but subjecting it to the heating process (2 h, 38 °C) (CM2), and to form tortillas (CT2).
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161 2.5. Rheological properties of masa variations
163
Oscillatory measurements of masa variations were performed with a Physica MCR 300 rheometer
164
(Physica Messtechnik GmbH, Stuttgart, Germany), and cone and plate geometry was selected, with
165
a rotating cone 50 mm in diameter, cone angle of 2°, and a gap of 0.05 mm. Each sample (~5.0 g)
166
was placed in the measuring system, and left to stabilize for 5 min at 25 °C for structure recovery.
167
To prevent dehydration, mineral oil was used to cover the edges between geometry and masa.
168
Temperature maintenance was maintained with a Physica TEK 150P temperature control system.
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Amplitude sweeps were carried out at 1 Hz and a strain range of 0.01-100%.
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2.6. Moisture content of masa and tortillas
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The moisture content of masa and tortilla variations was obtained by the AOAC-2000 vacuum oven
173
drying method 930.15. All samples were vacuum oven dried at 60 °C for 24 hours.
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2.7. ATR-FTIR of masa and tortilla variations
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Masa and tortilla variations were characterized by ATR-FTIR. The spectra were recorded on a
177
Perkin Elmer spectrophotometer (Spectrum 100-Perkin Elmer, Waltham, MA, USA) equipped with
178
a crystal diamond universal ATR sampling accessory. Each spectrum represented an average of four
179
scans. All spectra were deconvoluted using Gaussian and Lorentzian functions. In this case, the
180
assumed line shape was Lorentzian with a half-width of 15 cm-1. The resolution enhancement factor
181
was chosen as 1.5.
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2.8. Tortilla hardness
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Hardness of tortilla variations was measured with a Brookfield CT3-4500 texturometer (AMETEK,
185
Middleborough, MA, USA) equipped with a spherical probe TA18 (1.27 cm diameter). A whole
186
piece of each tortilla variation was placed on the texturometer plate with a square pierced hole (2.54 6
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cm diameter). Samples were compressed (1 cycle) at a rate of 1 mm·sec-1, until 50% penetration
188
was achieved.
189 2.9. Apparent amylose content
191
The method proposed by Zhu et al. (2008) was followed, with slight modifications. Masa samples
192
(500.0±0.1 mg) were transferred into an l00 mL volumetric flask, and 1 mL of ethanol was added.
193
The samples were dispersed in 10 mL of KOH (1N) for 1 h for complete dissolution. The volume
194
was adjusted to 100 mL in a volumetric flask. Then 2 mL of the diluted solution were taken and 50
195
mL of distilled water and 100 µL of alcoholic phenolphthalein solution (1% w/v) were added.
196
Hydrochloric acid (0.1N) was added dropwise until a slight pink color was obtained. Afterwards 2
197
mL of iodine solution (2.0 g of potassium iodide and 0.2 g of iodine in 100 mL of distilled water)
198
was added to the neutralized solution and then adjusted to 100 mL in a volumetric flask. The
199
solution was allowed to rest for 30 min to fully color development. The apparent amylose content
200
was calculated from the ratio of absorbance measurements at 620/510 nm. Scans were performed by
201
UV-Visible Spectrophotometer (Spectronic Genesys 2, Thermo Electron Corporation, Madison,
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WI, USA).
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203 2.10. Total sugars
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Total sugar determination of the masa variations was done using the phenol-sulfuric method
206
(Dubois et al., 1956) with some modifications. Samples of masa variations (0.5 g) were dispersed in
207
25 mL of water, an aliquot of 0.625 mL was taken and put into screw cap tubes (13×100 mm) and
208
0.625 mL of a phenol solution (5 % w/w) were added. The mixture was vortex-stirred and placed in
209
an ice bath ice for 5 min. Then, 2.5 mL of concentrated sulfuric acid was carefully added. The tubes
210
were then vortex-stirred again for 5 s and incubated for 5 min at boiling temperature. The tubes
211
were allowed to cool down to room temperature before reading the absorbance at 490 nm in UV-
212
Visible Spectrophotometer (Spectronic Genesys 2, Thermo Electron Corporation, Madison, WI,
213
USA). A glucose calibration curve was used as standard.
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2.11. In vitro starch digestibility
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About 100 mg of each tortilla variation were treated with a 2M KOH to disperse all starch fractions,
217
and then hydrolyzed by incubation with α-amylase (A-3176, 10-30 units.mg-1 solid, Sigma
218
Chemical Co. St Louis, MO, USA) and amyloglucosidase (A-7420, 14 units.mg-1, Sigma Chemical
219
Co. St Louis, MO, USA). Resistant starch (RS) was measured using the AACC-2000 method 32-40.
220
The tortilla samples were incubated with pancreatic α-amylase and amiloglucosidase during 16 h to 7
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hydrolyze the non-RS. The reaction was stopped by the addition of ethanol. Afterwards, the
222
samples were centrifuged to collect the RS precipitate. The pellet was then treated with 2M KOH
223
and hydrolyzed by incubation with AMG. For these determinations, the TS and RS kit analysis
224
from Megazyme International Ireland Ltd. (Bray Business Park, Bray, Co., Wicklow, Ireland) was
225
used. Available starch was calculated as the difference between TS and RS. The in vitro digestion
226
of the tortilla variations was performed using the Englyst et al. (1992) method. Starch classification
227
was based on the rate of hydrolysis, RDS (digested within 20 min) and SDS (digested between 20-
228
120 min). Values were adjusted at the available starch content previously determined.
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229 2.12. Data analysis
231
Data were analyzed using a one-way analysis of variance (ANOVA) and a Tukey’s test for a
232
statistical significance of p<0.05, using the Statgraphics 7 software (Statistical Graphics Corp.
233
Manugistics Inc., Cambridge, MA). The data were reported as means ± S.D. All experiments were
234
done in triplicate.
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235 3. Results and discussion
237
3.1. Amylolytic activity
238
The S. cerevisiae strain should display amylolytic activity for guaranteeing the reduction of amylose
239
chain fractions in the masa variations bulk. The kinetics exhibited a sigmoidal-like behavior with
240
lag phase lasting about 2 h (Figure 1). In this phase, the hydrolysis was relatively slow since starch
241
granules are compactly packed and relatively free of water. For longer times, water hydrates the
242
starch granules, facilitating the action of the amylolytic enzymes produced by S. cerevisiae. The
243
micrograph corresponding to hydrolysis time of ∼3.5 h, shows that the starch granule morphology
244
was characterized for displaying a central line known as the hilum or Maltese cross, where the
245
beginning of a fracture can be discerned. In the micrograph corresponding to hydrolysis time of ∼ 6
246
h, and fractures in the central region or hilum of the starch granule resulted evident, which cause the
247
Maltese cross to deform. As hydrolysis proceeded to ∼ 8 h, the starch grain was broken into several
248
fragments (the Maltese cross pattern disappeared), and at longer hydrolysis times (∼12 h) the starch
249
fragments were disrupted into smaller entities, which are more susceptible for amylolytic attack.
250
Overall, the results exhibited in Figure 1 show that the baking yeast used for masa treatment had
251
strong amylolytic activity.
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3.2. Moisture and total starch content of masa
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Non-significant different moisture contents were exhibited among the masa variations after thermal
255
incubation. Moisture content varied between from 69.8±0.6 g·100 g-1 for M0.25 to 70.6±0.7 g·100
256
g-1 for CM1. This was expected as the masa variations were put into poly-ethylene bags during
257
incubation. The total starch of masa after incubation was also quantified. The starch contents of
258
CM1 (68.4±0.6 g·100 g d.b.) and CM2 (68.3±0.5 g·100 g d.b.) and were non-significantly different
259
between themselves, but significantly different from those of the masa variations containing yeast.
260
The starch content for M0.25, M0.5 and M1.0 was of 66.3±0.5, 64.1±0.5 and 63.6±0.6 g·100 g d.b.,
261
respectively, and the reduction of the total starch content was ascribed to the amylolytic activity
262
induced by the yeast addition.
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263 3.3. Sugar and amylose contents of masa
265
NMF contains about 1.0 g total sugars 100 g-1. These sugars are used by the S. cerevisiae strain
266
producing carbon dioxide and water via invertase enzyme. The two masa controls, CM1 and CM2,
267
contained non-significantly different total sugar content (Figure 2.a). In contrast, the masa
268
variations containing S. cerevisiae showed a significant decrease in total sugars with respect to both
269
controls, and the reduction in total sugar contents was significantly higher the higher was the S.
270
cerevisiae concentration used. On the other hand, Figure 2.b shows that CM1 and CM2 had a non-
271
significantly different amylose content of about 32-34%. Upon nixtamalization the native maize
272
starch granules (which have an amylose content of about 25%), undergo partial gelatinization which
273
mainly takes place at the periphery of the grain, where fractionation of starch chains, including
274
debranching of amylopectin molecules, can occur. In turn, debranching increases the content of
275
apparent amylose (Lobato-Calleros et al., 2015). The masa variations showed a significant lower
276
amylose content with respect to CM1 and CM2. These results pinpoint that the amylolytic activity
277
of S. cerevisiae decomposed starch chains into simple glucose units via glucoamylase enzymes.
278
Gelatinized starch chains dispersed in the masa variations bulk are more susceptible to enzymatic
279
attack, producing in this way an important reduction in the apparent amylose content. The amylose
280
content was of about 28% for M0.25, 25% for M0.50 and 24% for M1.0. In subsequent reactions,
281
the produced sugars are used by gasification reactions catalyzed by invertase enzymes. The overall
282
effect was reflected as important reductions of total sugar and apparent amylose contents. The
283
reduction in amylose content in the masa variations with respect to CM1 and CM2 ranged between
284
12.5-29.4, depending on the baking yeast concentration used in masa variations.
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3.4. Viscoelasticity of masa
289
The viscoelastic properties are important for assessing the malleability of masa, which ultimately
290
affects its lamination, cutting and shaping, and has an impact on tortilla making and quality.
291
Figure 3.a exhibits the storage (G’) and loss (G’’) moduli for CM1 and CM2, as well as for the
292
yeast-treated masa variations M0.25, M0.5, and M1.0. For all cases, the storage modulus
293
displayed a monotonous decreasing trend, with a power-law decay γ
294
γ > 10% . This indicates that masa microstructure underwent rearrangements that reduced the
295
capacity of mechanical energy storage. G’ was lower for the masa variations incorporating yeast
296
than for CM1 and CM2, meaning that the storage modulus was reduced by the action of the yeast
297
treatment. Figure 3.b presents the behavior of G’ at γ = 1.0 % for the different masa variations.
298
CM1 and CM2 exhibited similar values, suggesting that the 2 h thermal treatment did not induce
299
appreciable changes in the masa microstructure. However, the storage modulus decreased
300
continuously with the amount of added yeast. It is apparent that yeast activity modified the
301
components of the masa, possibly the fraction of leached amylose dispersed in the continuous
302
phase of masa variations. On the other hand, the loss modulus in Figure 2.a (open symbols)
303
exhibited an interesting behavior reflected by a non-monotonous pattern. In fact, the loss modulus
304
decreased for small strain values ( γ < 0.3 %). However, increasing loss modulus was observed
305
for strain values in the range from 1.0% to 10%. This behavior could be attributed to the
306
aggregation of particles dispersed in the masa, producing a jamming-like pattern (Brown and
307
Jaeger, 2014). For higher strain values, the loss modulus decreased continuously with power-law
308
behavior γ
309
suggesting that the additional thermal treatment induced poor rearrangement of the starch fraction
310
dispersed in the masa. In contrast, the addition of yeast produced a significant reduction in the
311
loss modulus, being of about 35% for M1.0. The cause of such reduction can be attributed to
312
hydrolysis of amylose present in the periphery of starch granules induced possibly by the
313
amylolytic activity of the baking yeast colonies, reflected as a significant reduction of amylose
314
(Figure 2.a).
for strain values
. CM2 exhibited significant higher loss modulus than its CM1 counterpart,
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3.5. FTIR of masa
317
The FTIR spectra for the masa controls and the yeast-treated masa variations showed similar
318
characteristic bands (Figure 4.a). For all cases, a pronounced broad band was displayed at 3000-
319
3700 cm-1, resulting from the overlapping combination of -OH stretching (~3500 cm-1) linked to
320
hydration effects, and NH stretching (3300 cm-1) attributed to Amide A groups of proteins (Kong 10
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and Yu, 2007). These peaks were unchanged by yeast treatment. The peak at ~2930 cm-1 can be
322
related to CH stretching and can be attributed to free fatty acids and amylose-fatty acid inclusion
323
complexes (Flores-Morales et al., 2012). The formation of these complexes was an expected result
324
since nixtamalized maize flour contains ~4-5 % lipids, which can form amylose-lipid inclusion
325
complexes during the nixtamalization process. The broad band at 1700-1600 cm-1 with a large peak
326
at ~1640 cm-1 can be ascribed to C=O stretching and is linked to the Amide I group of maize
327
proteins (Flores-Morales et al., 2012). The bands at 1540, 1418, 1364 and 1242 cm-1 can be related
328
to conjugated carbonyl and carboxyl groups and C-O vibrations. The broad band at ~1080-950 cm-1
329
reflects C-O, C-C and C-H stretching and C-O-H bending vibrations, with starch corresponding to
330
the major fraction in maize flour. This wide band has been considered as a fingerprint of starch
331
structure (van Soest et al., 1995), finding that the band close to 1022 cm−1 increased for more
332
amorphous samples, while the bands at close to 995 and 1047 cm−1 were more prominent in more
333
crystalline samples. The ratio 1047/1022 was proposed as indicator of ordered structures in starch
334
(van Soest et al., 1995). The band at 995 cm−1is mainly due to COH bending vibrations, and is
335
sensitive to water-starch interactions, including hydrogen bonding (van Soest et al., 1995). Figure
336
4.b illustrates the deconvoluted FTIR spectra in the starch fingerprint region, showing three main
337
peaks at 1044, 1021 and 995 cm-1. The first two peaks are close to the nominal peaks 1047 and 1022
338
cm-1, a shift that could be caused by the limited amount of water in masa formulations. To be in line
339
with the terminology by van Soest et al. (1995), the ratio 1047/1022 was estimated by taking the
340
closest peaks to 1047 and 1022 cm-1. CM1 and CM2 exhibited similar ratio (Figure 4.c), indicating
341
that the heating of masa for 2 h at 38 oC (CM2) did not affect the short-range ordering of starch
342
chains. However, the ratio 1047/1022 increased with the yeast treatment, suggesting that yeast
343
activity reduced the amorphous starch components dispersed in the tortilla bulk. The increase of the
344
ratio 1047/1022 was higher as the amount of yeast added to masa was increased.
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3.6. Weight and thickness of tortillas
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The weight of tortillas was 27.2±0.5 g for CT1, and 26.9±0.4 g for CT2. On the other hand, tortillas
348
added with yeast showed similar weight, with variations from 27.1±0.4 g for T0.25 to 27.3±0.5 g
349
for T1.0. This means that the addition of yeast did not have a significant effect on the weight of
350
fresh tortillas. Similar effects were observed for tortilla thickness as the parameter varied from
351
1.82±0.2 mm for CT1 to 1.84±0.2 mm for T1.0.
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3.7. Moisture content and hardness of tortillas
356
The moisture content of tortillas is an important parameter since it has determinant effects in the
357
staling phenomenon. Figure 5.a shows that the moisture content of all the tortilla variations
358
decreased significantly between 2-3 % over the 4 days of storage. In general terms, the moisture
359
content of the tortilla variations was from lower to higher during storage as follows: CT1 < CT2 <
360
T0.25 < T0.5 < T1.0. These results indicate that the thermal treatment (2 h, 38 °C) to which CM2
361
was subjected induced a higher water retention respect to CT1, which was obtained from non-
362
thermally treated masa (CM1). The yeast-treated masa variations produced tortillas with improved
363
water retention with respect to the non-yeast treated controls CT1 and CT2. The higher the yeast
364
concentration the higher the moisture content of the tortilla variations. T1.0 exhibited an increase of
365
about 10% moisture content respect that of CT1, throughout storage time. A possible explanation
366
for the improved moisture content exhibited by the yeast-treated tortillas is that the cell wall of
367
yeast (S. cerevisae) represents about 50-65% of β-glucan, a branched polysaccharide that has a high
368
water holding capacity (Borchani et al., 2016), and to the increased yeast activity which produced
369
an increase in cells population in the masa bulk. Similar effect was reported for wheat bread added
370
with different hydrocolloids (sodium alginate, xanthan, κ-carrageenan and hydroxypropyl-
371
methycellulose). In fact, breads containing hydrocolloids showed lower loss of moisture content
372
(Clubbs et al et al., 2008).
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Loss of water is commonly accompanied by an increase in tortillas hardness. Fresh tortillas CT1
374
and CT2 exhibited the highest values of hardness (Figure 5.b), and became increasingly harder with
375
storage time. In contrast, the yeast-treated tortillas exhibited significantly lower hardness values
376
than the control tortillas throughout the storage time. The higher the yeast content, the lower was
377
the tortilla variation hardness.
378
significant hardness change with storage time, suggesting that yeast cells and in situ produced
379
polysaccharides contributed to practically maintain without change the tortillas hardness values.
380
Roman-Brito et al. (2007) reported that the addition of xanthan gum to maize tortillas decreased
381
hardness and retarded staling effects.
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3.8. FTIR of tortillas
384
Storage of tortillas is accompanied by retrogradation effects, which are originated by loss of
385
moisture content (Figure 5.a), increased hardness (Figure 5.b) and an increase in the organization of
386
the structure. In fact, Flores-Morales et al. (2012) detected that the band close to 1047 cm-1
387
increased with tortilla staling. As done for masa, deconvolution of tortilla variations was carried out
388
to detect changes in the ratio R1047/1022. The results are presented in Figure 5.c and show that the 12
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ratio R1047/1022 was larger for the yeast-treated tortillas than for the non-yeast treated tortillas. This
390
result is in line with results in Figure 4.c for masa variations. Amorphous structures are more
391
susceptible to enzymatic attack by baking yeast, leaving more ordered molecular structures (double
392
helical crystalline states) that are identified as resistant starch (Tester et al., 2004). Storage
393
promoted the formation of ordered molecular structures as reflected by the increased values of the
394
ratio R1047/1022. Besides, the rate of increase of such ratio was higher for tortillas made with masa
395
treated with baking yeast. In principle, the increased value of R1047/1022 accompanied with the
396
reduction of moisture content should lead to tortillas with increased hardness (i.e., staling).
397
However, this effect was not observed since the yeast-treated tortillas exhibited lower hardness
398
values than the non-yeast treated tortilla controls. The metabolic activity of the baking yeast should
399
play an important role in the modification of the tortilla properties. As mentioned above, increased
400
yeast cell population and the production of exo-polysaccharides acting as in situ hydrocolloid could
401
be behind the hindering of the staling effects in treated tortillas.
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402 3.9. Digestibility
404
TC1 exhibited about 79.21% RDS, 5.77% SDS and 15.23% RS (Figure 6.a), and these contents are
405
in line with those results reported by Bello-Perez et al. (2014) for tortillas made with commercial
406
nixtamalized flour. The heat treatment (38 oC by 2 h) did not affect the in vitro digestibility
407
fractions of TC2, which presented non-significant different fractions values similar to TC1.
408
However, the baking yeast treatment had an important effect on the in vitro digestibility. In fact, the
409
RDS fraction decreased and the RS fraction increased with the baking yeast content. Specifically,
410
the starch fractions for treated tortilla T1.0 were 70.75% RDS, 2.48% SDS and 27.52% RS.
411
Following the results described above, the increase of the RS fraction and the decrease of the RDS
412
fraction was likely induced by the metabolic activity of the baking yeast. These results are in line
413
with the decreases in total sugars and apparent amylose reported above. It can be postulated that
414
baking yeast consumes, via amylolytic activity, short-length starch chains that otherwise are easily
415
accessible by enzymatic activity. Storage time can modify the digestibility of tortillas by the effect
416
of starch retrogradation and moisture loss and re-distribution (Rendon-Villalobos et al., 2006;
417
Bello-Perez et al., 2014). Figure 6.b displays the starch fractions for the tortilla variations after four
418
days of storage time. The starch fractions for TC1 were 56.91% RDS, 19.02% SDS and 29.07% RS,
419
results that are similar to those reported by Bello-Perez et al. (2014) for tortillas made with
420
commercial nixtamalized maize flour. After four days of storage, T1.0 exhibited significant
421
increases of SDS and RS at the expenses of significant decreases in RDS. In fact, tortilla made with
422
masa treated with baking yeast exhibited 42.23% RDS, 23.54% SDS and 34.23% RS.
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Reorganization of starch molecules within complex semi-crystalline structures are linked to the
424
increase of the SDS and RS fractions in tortillas made with treated masa. Since the metabolic
425
activity of baking yeast is prompted to reduce the fraction of easily accessible starch chains (e.g.,
426
short-length chains), reorganized starch molecules in storage become conformed by preferably by
427
long-length chains, offering increased resistance for amylolytic action.
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428 4. Conclusions
430
The treatment of nixtamalized maize masa with baking yeast altered the in vitro digestibility of
431
freshly made and stored (4 days) tortillas with respect to that exhibited by the non-yeast treated
432
controls. Yeast-treated fresh tortillas exhibited lower RDS and SDS, but higher RS fractions than
433
the untreated controls. In the yeast-treated tortillas stored for four days, the beneficial changes on in
434
vitro digestibility were more pronounced, as the RDS fraction decreased, but the SDS and RS
435
fractions increased significantly with respect to that of the controls. The effect can be explained
436
from the amylolytic activity of the baking yeast, leading to reduced contents of total sugars and
437
apparent amylose. In all cases the hardness of the yeast-treated tortillas was lower than for the
438
untreated tortilla controls, and this was attributed to the increased yeast population and in situ
439
polysaccharides formation that acted as hydrocolloids.
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440 References
442
Arámbula-Villa, G., Saavedra-Rosiles, G., Rendón-Villalobos, R., Rodríguez-González, F.,
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Figueroa-Cárdenas, J.D., Méndez-Albores, J.A., 2017. Structural, thermal and rheological
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properties of nixtamalized maize masa obtained from varying the concentration of calcium
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hydroxide and cooking time. Bulgarian J. Agric. Sci. 234, 653–663.
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Bello-Perez, L.A., Flores-Silva, P.C., Agama-Acevedo, E., de Dios Figueroa-Cardenas, J., Lopez-
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Valenzuela, J. A., Campanella, O.H., 2014. Effect of the nixtamalization with calcium
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Borchani, C., Fonteyn, F., Jamin, G., Paquot, M., Thonart, P., Blecker, C., 2016. Physical,
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functional and structural characterization of the cell wall fractions of baker`s yeast
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Bressani, R., 1990. Chemistry, technology, and nutritive value of maize tortillas. Food Rev. Int. 6, 225-264. Brown, E., Jaeger, H.M., 2014. Shear thickening in concentrated suspensions: phenomenology, mechanisms and relations to jamming. Rep. Prog. Phys. 77, 046602. 14
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Bressani, R., 1990. Chemistry, technology, and nutritive value of maize tortillas. Food Rev. Int. 62, 225-264. Butterworth, P.J., Warren, F.J., Ellis, P.R., 2011. Human α-amylase and starch digestion: An interesting marriage. Starch/Stärke 63, 395-405. Camelo-Méndez, G.A., Agama-Acevedo, E., Tovar, J., Bello-Pérez, L.A., 2017. Functional study of
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Clubbs, E.A., Vittadini, E., Shellhammer, T.H., Vodovotz, Y., 2008. Effects of storage on the
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metabolic syndrome in an urban Mexican population. J. Nutr. 140, 1855-1863.
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Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28, 350-356. Englyst, H.N., Kingman, S.M., Cummings, J.H., 1992, Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46, S33–S50. Flores-Morales, A., Jiménez-Estrada, M., Mora-Escobedo, R., 2012. Determination of the structural
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changes by FT-IR, Raman, and CP/MAS 13C NMR spectroscopy on retrograded starch of
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maize tortillas. Carbohydr. Polym. 87, 61-68.
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García, S., Heredia, N. 2006. Mycotoxins in Mexico: Epidemiology, management, and control strategies. Mycopathologia 162, 255-264.
Hernández‐Uribe, J. P., Agama‐Acevedo, E., Islas‐Hernández, J. J., Tovar, J., Bello‐Pérez, L. A.
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2007. Chemical composition and in vitro starch digestibility of pigmented corn tortilla. J. Sci.
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Kong, J., Yu, S., 2007. Fourier transform infrared spectroscopic analysis of protein secondary
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structures. Acta Biochim. Biophys. Sin. 39, 549-559.
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Alvarez-ramirez, J., Vernon-Carter, E.J., 2015. Effect of lime concentration on gelatinized
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maize starch dispersions properties. Food Chem. 172, 353-360.
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Pappa, M.R., de Palomo, P. P., Bressani, R., 2010. Effect of lime and wood ash on the
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nixtamalization of maize and tortilla chemical and nutritional characteristics. Plant Foods
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Hum. Nutr. 65, 130-135.
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Peña-Reyes, R.A., Ramírez-Romero, G.A., Fernández-Perrino, F.J., Cruz-Guerrero, A.E., 2017.
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Effect of nixtamalization processing temperature on maize hydration and the textural
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properties of masa and tortillas. J. Food Process. Pres. 41, e13136. Rendon-Villalobos, R., Bello-Pérez, L.A., Osorio-Díaz, P., Tovar, J., Paredes-López, O., 2002.
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Effect of storage time on in vitro digestibility and resistant starch content of nixtamal, masa,
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and tortilla. Cereal Chem.79, 340-344.
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Rendón-Villalobos, R., Agama-Acevedo, E., Islas-Hernández, J.J., Sánchez-Muñoz, J., Bello-Perez,
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L.A., 2006. In vitro starch bioavailability of corn tortillas with hydrocolloids. Food Chem.
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97, 631-636.
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Román-Brito, J.A., Agama-Acevedo, E., Méndez-Montealvo, G., Bello-Pérez, L.A., 2007. Textural studies of stored corn tortillas with added xanthan gum. Cereal Chem. 84, 502-505. Saldana, G., Brown, H.E., 1984. Nutritional composition of corn and flour tortillas. J. Food Sci. 49, 1202-1203.
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Reynoso-Camacho, R., Ramos-Gómez, M., Gaytán-Martínez, M., Morales-Sánchez, E.,
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2015. Resistant starch formation in tortillas from an ecological nixtamalization
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process. Cereal Chem. 92, 185-192.
Sáyago-Ayerdi, S.G., Tovar, J., Osorio-Diaz, P., Paredes-López, O., Bello-Pérez, L.A., 2005. In
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vitro starch digestibility and predicted glycemic index of corn tortilla, black beans, and
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tortilla-bean mixture: effect of cold storage. J. Agric. Food Chem. 53, 1281-1285.
510 511
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Tester, R.F., Karkalas, J., Xin, Q., 2004. Starch-composition, fine structure and architecture. J. Cereal Sci. 39, 151-165.
van Soest, J.J.G., Tournois, H., de Wit, D., Vliegenthart, J.F.G., 1995. Short-range structure in
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(partially) crystalline potato starch determined with attenuated total reflectance Fourier-
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transform IR spectroscopy. Carbohydr. Res. 279, 201-214.
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Zhu, T., Jackson, D. S., Wehling, R. L., & Geera, B. (2008). Comparison of amylose determination
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methods and the development of a dual wavelength iodine binding technique. Cereal
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Chem. 85, 51-58.
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Figure 1. Hydrolysis advance of native maize starch subjected to the metabolic action of
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the yeast strain. The inset images illustrate the morphology of the starch granules for
11
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different hydrolysis times.
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0.9 b 0.8 0.7
c
0.6 0.5 CM1
CM2
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a
d
M0.5
M1.0
c
c
M0.5
M1.0
(b)
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Apparent Amylose (g/100 g d.b.)
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Total Sugars (g/100 g d.b)
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CM1
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Figure 2. (a) Total sugars and (b) apparent amylose contained in the different masa
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variations. Small letters above the bars denote significant statistical differences (p < 0.05).
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(a)
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G' and G'' (Pa)
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4
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0.1
1
4
a
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b
4
5x10
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MC1 4
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c
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6x10
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a
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Strain (%)
8x10
1.4x10
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(c)
ab c
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Figure 3. (a) Viscoelasticity moduli of the different masa variations. Closed and open
26
circles correspond to storage (G’) and loss (G’’) moduli, respectively. Squares – MC1,
27
circles – MC2, upper triangle – M0.25, sided triangle – M0.5 and star – M1.0. (b) Storage
28
modulus for strain 1.0% and the five different masa variations. (b) Loss modulus for strain
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1.0% and the five different masa variations. Small letters above bars indicate significant
30
different values (p < 0.05).
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- 931
-1155 - 1081
- 1242
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Amide I
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Starch
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Absorbance (a.u.)
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1600 1400 1200 1000 -1
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Absorbance (a.u.)
Wavenumber (cm )
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Wavenumber (cm )
(c)
a b
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d
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0.34
c cd
0.28
CM1
CM2
M0.25
M0.5
M1.0
36 37
Figure 4. (a) FTIR spectra of masa formulations (solid – CM1, dash – CM2, dot – M0.25,
38
dash-dot – M0.5 and short dash – M1.0). (b) Example of deconvolution of the starch
39
fingerprint region. (c) Ratio R1047/1022 for the different masa variations. Letter above bars
40
indicate statistical significant differences (p < 0.05).
41 4
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(a)
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3
4
3
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(b)
2.0 1.8 1.6 1.4
0.32
2
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(c)
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Hardness (N)
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0.28 0.26
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Moisture Content (g/100 g)
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0.24 0.22
0
1
2
Time (days)
47 48
Figure 5. (a) Evolution of the moisture content with storage time for tortilla variations. (b)
49
Hardness of tortilla variations as function of storage time. (c) FTIR ratio R1047/1022 as
50
function of storage time. For the three panels, the legends are as follows: Squares – TC1,
51
circles – TC2, upper triangle – T0.25, sided triangle – T0.5 and star – T1.0.
52
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(a)
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80 60 40 20 0 60
TC1
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Starch Fraction (%)
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TC2
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T0.5
T1.0
T0.5
T21.0
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Starch Fraction (%)
(b)
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59 60
TC2
T0.25
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61
Figure 6. Starch digestibility fractions for the different tortilla variations: (a) Fresh tortillas,
62
and (b) tortillas stored for 4 days. Unfilled bar – RDS; Oblique-fill bar – SDS; Vertical-fill
63
bar – RS.
64 65 66
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Highlights Baking yeast treatment of maize masa reduced sugars and apparent amylose content
•
Tortillas made with treated masa exhibited reduced contents of RDS and increased content of RS Baking yeast treatment led to tortillas with reduced hardness
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