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Development of a third-generation snack with type 4 resistant sorghum starch: Physicochemical and sensorial properties
Journal Pre-proof Development of a third-generation snack with type 4 resistant sorghum starch: Physicochemical and sensorial properties Alberto Escobar-Puentes, Susana Rincón, Adriana García-Gurrola, Alejandro Zepeda, Amira Daniela Calvo-López, Fernando Martínez-Bustos PII:
S2212-4292(18)30849-6
DOI:
https://doi.org/10.1016/j.fbio.2019.100474
Reference:
FBIO 100474
To appear in:
Food Bioscience
Received Date: 5 September 2018 Revised Date:
28 September 2019
Accepted Date: 28 September 2019
Please cite this article as: Escobar-Puentes A., Rincón S., García-Gurrola A., Zepeda A., Calvo-López A.D. & Martínez-Bustos F., Development of a third-generation snack with type 4 resistant sorghum starch: Physicochemical and sensorial properties, Food Bioscience (2019), doi: https://doi.org/10.1016/ j.fbio.2019.100474. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
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Development of a third-generation snack with type 4 resistant sorghum starch:
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Physicochemical and sensorial properties
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Running title: Extruded enriched-snack with resistant sorghum starch
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Alberto Escobar-Puentes1, Susana Rincón1, Adriana García-Gurrola1, Alejandro Zepeda2,
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Amira Daniela Calvo-López3, Fernando Martínez-Bustos3*.
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1
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Mérida, Mérida, Yucatán, 97118, México.
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2
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97203, México.
Departamento de Ingeniería Química-Bioquímica, Tecnológico Nacional de México/I.T.
Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán,
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3
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del Instituto Politécnico Nacional, Unidad Querétaro, Querétaro, 76230, México.
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*Corresponding author at: Departamento de Materiales Orgánicos, Centro de Investigación
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y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Querétaro,
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Libramiento Norponiente, No. 2000., Real de Juriquilla, 76230, Santiago de Querétaro,
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Querétaro, México. Email-address: [email protected]. Tel.:+52 442 2119905; fax:
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+52 442 2119938.
Departamento de Materiales Orgánicos, Centro de Investigación y de Estudios Avanzados
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Abstract
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Extruded third-generation fiber enriched snacks can be made using type 4 resistant starch.
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The present study aimed to evaluate and optimize some extrusion conditions, namely the
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transition or intermediate barrel temperature and the feed moisture in the direct formation
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of third-generation snacks with type 4 resistant starch using the phosphorylation of
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sorghum starch. The physicochemical and sensory properties of the optimal snack were
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investigated. An increase in barrel temperature resulted in an increase in the resistant starch
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(13.1 ± 0.5 g/100 g) and the expansion index values, as long as the feed moisture was
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maintained above 28%. The optimal conditions with a desirability value of 0.932 were a
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barrel temperature of 136°C and a feed moisture of 28.5%. The optimal third-generation
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snack had 17.1 ± 0.2 g/100 g of resistant starch and a desirable expansion index of 5.7 ±
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0.4, as well as general sensory acceptability (“I like moderately”). Phosphorylated resistant
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starch of the optimal snack showed a degree of substitution and a percentage of phosphorus
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(0.02 ± 0.00 and 0.35 ± 0.15%, respectively) within the limits allowed in foods. This study
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showed that using optimal extrusion conditions, phosphorylation of sorghum starch was
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possible to produce resistant starch and simultaneously to obtain an extruded snack with
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acceptable physical and sensorial properties. Additionally, it showed that sorghum is a good
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alternative source of starch for the formulation of extruded snacks.
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Keywords: Enriched-snacks, extrusion, Sorghum bicolor (L.) Moench, sorghum starch,
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type-4 resistant starch.
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Abbreviations:
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%P: phosphorus percentage
2
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2G-S: second-generation snacks
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3G-S: third-generation snacks
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3G-RS4: third-generation snacks with type-4 resistant starch
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BT: barrel temperature
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C-3G: control snacks without type-4 resistant starch
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DS: degree of substitution
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FM: feed moisture
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FT-IR: Fourier transform infrared spectroscopy
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RS: resistant starch
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RS4: type-4 resistant starch
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RSM: response surface methodology
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57
58
59
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3
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1. Introduction
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Extruded snacks are often termed “junk food” because they are typically fried and low in
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dietary fiber. However, their consumption is increasing, especially in developing countries,
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and the consumption of these unhealthy snacks can result in various metabolic diseases
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(Espinoza-Moreno et al., 2016). Therefore, the current trend in the snacks industry is the
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development of healthy foods (Tumuluru, 2016) using the incorporation of active
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compounds such as proteins, dietary fibers, and antioxidant compounds (Chávez et al.,
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2017; Cortés et al., 2014; Espinoza-Moreno et al., 2016)
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Resistant starch (RS) is a non-digestible polysaccharide, since it resists enzymatic digestion
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in the small intestine and is able to reach the large intestine of humans, where it can be
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fermented by the gut microbiota, providing protective effects against colorectal cancer and
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aiding in controlling metabolic diseases (Englyst et al., 1992; Fuentes-Zaragoza et al.,
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2011). Due to their physiological benefits, RS have been successfully used as a fiber-
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enriching ingredient in a range of food systems, such as cookies (Giuberti et al., 2017),
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pasta (Foschia et al., 2017), and breakfast cereals (Miller et al., 2011). Type-4 resistant
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starch (RS4) is produced using a chemical cross-linking reaction between hydroxyl (–OH)
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groups of starch and food-grade chemical agents, which form atypical linkages and block
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the access of enzymes, preventing their digestion (Fuentes-Zaragoza et al., 2011; Landerito
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and Wang, 2005). Chemical cross-linking using phosphorylation is the earliest and most
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preferred method for starch modification and RS4 production (Miller et al., 2011), and the
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phosphate salts mainly used are sodium tripolyphosphate alone or in a mixture with sodium
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trimetaphosphate. Phosphorylated starch has phosphate groups esterified with two –OH
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groups,
very
often
from
two
neighboring 4
starch
molecules.
Conventionally,
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phosphorylation is carried out in a batch system over long time periods using high
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concentrations of reagents (Moad, 2011), which may cause environmental contamination
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from unreacted chemicals and which can be
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chemicals is considered (Landerito and Wang, 2005). To overcome these problems, the
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phosphorylation reaction needed to produce RS4 can also be induced using reactive
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extrusion, a continuous and short-time high-temperature process, with the absence of
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effluents (Landerito and Wang, 2005; Manoi and Rizvi, 2010; Moad, 2011). Moreover,
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extrusion technology is widely used in the food industry (breakfast cereals, snack foods,
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and other similar products) due to its low cost, versatility, high productivity, and energy
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efficiency (Tovar-Jiménez et al., 2015). It is possible to obtain two types of extruded snack
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foods, such as second-generation snacks (2G-S) or ready to eat products (directly expanded
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through the extrusion exit) and third-generation snacks (3G-S), which are non-expanded
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pellets or glassy half-products formed by extruding starchy materials (Aguilar-Palazuelos et
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al., 2006) that are not expanded directly through the extrusion exit and need to be expanded
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to obtain a consumer-ready final product using additional processes, such as frying or
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microwave heating (Tumuluru, 2016), the latter being a fat-free method. Different studies
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have focused on the development of enriched 3G-S using proteins, anthocyanins, winter-
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squash flours, and orange fibers (Aguilar-Palazuelos et al., 2006; Camacho-Hernández et
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al., 2014; Delgado-Nieblas et al., 2012; Tovar-Jiménez et al., 2015). On the other hand, a
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recent study done by Calvo-López and Martínez-Bustos, (2017) showed that it is possible
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to develop 2G-S with RS4 using esterification of potato starch in a single extrusion step.
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However, no information is currently available concerning the development of 3G-S with
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type-4 resistant starch (3G-RS4) in a single extrusion step and based on sorghum starch.
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costly when removal of the unreacted
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Generally, extruded snacks are made from corn or potato starch, and optionally using other
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components, such as vegetable, cereal or legume flours. However, starch should be
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considered as the main component. In the present study, sorghum starch was used for the
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production of 3G-S. Sorghum (Sorghum bicolor) is the world’s fifth cereal crop and is
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drought-tolerant, grows with various soil conditions (Jafari et al., 2017) and is an
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economical alternative with desirable agro-technological characteristics. It can be regarded
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as a substitute for the common cereals, since it contains 70% starch similar to corn (Singh
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and Singh, 1995), besides being a naturally gluten-free cereal (Chávez et al., 2017). For
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these reason, several reports have focused on the development of different food systems
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such as pasta, breakfast cereals, and extruded snacks based on sorghum flours (Khan et al.,
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2013; Mkandawire et al., 2015; Wu et al., 2018).
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Studying the development of 3G-RS4 snacks using a single extrusion step based on
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sorghum starch and evaluating its physicochemical and sensory properties would be
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potentially beneficial. Therefore, the aims of this study were as follows: 1) to optimize the
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extrusion conditions (intermediate barrel temperature and feed moisture) and evaluate their
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effects on the expansion index values and the formation of RS4 using phosphorylation of
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sorghum starch, and 2) to analyze the physicochemical and sensorial properties of the
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optimal snack.
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2. Materials and Methods
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2.1 Materials 6
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2.1.1 Sample
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White sorghum (Sorghum bicolor (L.) Moench) “Fortuna” genotype harvested in June
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2015 in Guanjuato, Mexico (20°34'48.1"N 100°49'11.9"W) was identified and provided by
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Dr. Victor Pecina Quintero of the Experimental Station of the National Research Institute
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for Forestry, Agriculture and Livestock. The sorghum grains were received in a single
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batch in sealed kraft paper bags with an internal polyethylene liner and cleaned manually.
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Subsequently, sorghum grains were placed in polyethylene bags (200 x 300 mm, Aranda
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Bolsas de Polietileno, Queretaro, Queretaro, Mexico) and stored (4°C) at the Laboratory of
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Organic Materials of Research and Advanced Studies Center of the National Polytechnic
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Institute, Campus Queretaro, Mexico, for a maximum of 3 wk before the experiments.
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2.1.2 Chemicals
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Sodium trimetaphosphate, sodium tripolyphosphate, ammonium molybdate, ammonium
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vanadate, zinc acetate, HNO3, CuSO4, K2SO4, KH2PO4 and KBr were purchased from
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Sigma Aldrich Co., (St. Louis, MO, USA). Ethanol (99.7%), distilled water, CH3COOH,
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KOH, NaHSO3, 1 N NaOH volumetric solution and NaOH pellets were purchased from J.T.
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Baker (Phillipsburg, NJ, USA). The resistant starch assay kit was purchased from
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Megazyme International Ireland Ltd. (Wicklow, Ireland).
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2.2 Methods
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2.2.1 Sorghum starch isolation
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Starch was isolated from sorghum grains using the wet-milling method reported by Sira and
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Amaiz (2004) with some modifications. Briefly, 3 kg of sorghum grains were suspended in
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a container using 0.25% NaOH solution to cover twice the volume of the grains. After 18
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hr, the grain mass was washed exhaustively using tap water until reaching neutral pH. After
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steeping, grains were ready for wet milling. The pretreated grains were milled in a stone
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mill (Model M100, Fumasa, Queretaro, Mexico) to break the testa and expose the starch. A
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solution of 0.1 N NaHSO3 (~50 mL) was added during grinding. The ground slurry was
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screened using standard sieves (841, 595, 420, 250, 177, 149, 74, and 62.5 µm) (Mont Inox,
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Mexico City, Mexico) to separate the starch and the fiber fractions. The residue on the
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sieves was discarded and the screened solution was recovered and allowed to stand ~16 hr
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at room temperature (25-30°C). The starch was then recovered in the sediment, which was
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washed using an ethanol:water solution (50:50 v/v), centrifuged at 5,450 x g for 15 min at
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5°C (5,000 rpm in a 220.70 V06 rotor, Model Z513K centrifuge, Hermle Labortechnik
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GmbH Co., Wehingen, Germany) and dried in a convection oven at 40°C for 48 hr.
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2.2.2 Chemical composition
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Proximate analysis was determinated in triplicates using the AOAC Official Methods
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(1999). Moisture was determinated using standard method 925.09 based on sample weight
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loss after oven drying at 105°C for 2 hr. Crude protein was determined using the Kjeldahl
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nitrogen method 979.09 using a micro-kjeldahl digestor (Model Vapodest 200, Gerhardt 8
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GmbH & Co., Königswinter, Germany) and CuSO4 and K2SO4 as catalysts. A conversion
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factor of 6.25 was used to calculate crude protein. Fat cotent was estimated after 3 hr
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extraction using petroleum ether using the Soxhlet principle (AOAC method 923.05), and
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ash was calculated from sample weight after incineration in a muffle furnace (Model FE-
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341, Felisa, Guadalajara, Jalisco, Mexico) at 550°C for 2 hr (AOAC method 923.03).
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Isolated sorghum starch had 10 ± 1% moisture, 0.98 ± 0.01% crude protein, 1.4 ± 0.3%
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lipid, and 0.32 ± 0.04% ash.
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2.2.3 Extrusion
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The effect of extrusion conditions such as the barrel temperature (BT) and the feed
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moisture (FM) on the expansion index (EI) and the RS4 of 3G-S was studied using
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response surface methodology (RSM), using a two-factor central composite experimental
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design, which consisted of 13 assays with 5 center points (Table 1). Starch samples were
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extruded at different BT in the transition or intermediate zone (93.8–136°C) and at different
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FM (23.5-33.4%). Experimental limits that were defined using preliminary tests.
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Prior to the extrusion, sorghum starch (200 g) was conditioned with moisture and
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phosphate salts (4 g/100 g starch dry weight) using the dry-mixing procedure described by
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Landerito and Wang (2005). Briefly, the water necessary to achieve the FM values of the
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experimental design (Table 1) was used to dissolve a mixture of sodium trimetaphosphate
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and sodium tripolyphosphate (at a ratio of 99:1 w/w) and this was adjusted to pH 8.5 using
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a 1 N NaOH standard solution. The solution was stirred vigorously using a magnetic stirrer
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(Model FE-310, Felisa, Guadalajara, Jalisco, Mexico) until the chemical agents were
9
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completely dissolved (~10 min). Later, the solution was spray-atomized into starch and was
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mixed using a laboratory spatula. An additional starch sample was conditioned using only
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28.5% FM (optimal condition) without the phosphate salts and the catalyst NaOH, to obtain
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a control snack without RS4 (C-3G).
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Extrusion trials were done using a single screw extruder designed and manufactured by the
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Laboratory of Organic Materials of Research and Advanced Studies Center of the National
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Polytechnic Institute, Campus Queretaro, Mexico. The extruder was equipped with a screw
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(428 mm long and 19 mm diameter), a 1:1 compression ratio, a 40-rpm screw speed, a
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rectangular die (1.5 × 20 × 100 mm) and three heating zones. The BT in the intermediate
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zone varied according to the experimental design (Table 1), while the BT in the feed and
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high-pressure die zones was maintained constant at 60 and 70°C, respectively. The
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resulting non-expanded pellets were cut (2.5 x 2.5 cm) using scissors and dried in a forced
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air oven (Model FE293A, Felisa, Guadalajara, Jalisco, Mexico) at 40 ± 1°C until the
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moisture reached 10 ± 1%.
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2.2.4 Microwave-expansion
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Extruded pellets were expanded using heating (30 s) in a conventional microwave oven
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(Model MW843WA, 1200 W and 60 Hz, Samsung Electronics Co., Seoul, Korea). The EI
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was calculated as the average transversal area of the expanded product divided by the
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average transversal area of the non-expanded pellet (Aguilar-Palazuelos et al., 2006). The
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EI was measured in 15 samples. The measurements were determined using a Vernier
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calliper (Model Mitutoyo, Tokyo, Japan) having 0.05 mm accuracy.
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2.2.5 Resistant starch
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Microwave-expanded pellets were milled using a hammer mill equipped with a 0.25 mm
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sieve (Model 200, Pulvex, Mexico City, Mexico) to obtain flour, and the RS4 was then
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quantified using a RS assay kit (Cat. no. RSTAR 11/02, Megazyme International Ireland,
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Ltd.) based on AACC standard method 32-40.01 (AACC, 2010) which was briefly
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described by Foschia et al. (2017) as follows: “samples were incubated with pancreatic α-
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amylase and amyloglucosidase (AMG) at 37°C for 16 hr, during which time non-resistant
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starch was solubilized and hydrolysed to D-glucose by the combined action of the two
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enzymes. The reaction was terminated by the addition of 99.7% ethanol and the RS was
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recovered as a pellet on centrifugation at 1500 × g for 10 min in capped 50 mL centrifuge
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tubes (3,000 rpm in a 220.80 V02 rotor, Hermel Labortechnik GmbH Co.,). The residue
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was washed twice by suspension in aqueous 50% (v/v) ethanol, followed by centrifugation.
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Excess liquid was removed using decantation. RS in the pellet was dissolved in KOH; this
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solution was neutralized using acetate buffer (pH 3.8) and the starch was quantitatively
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hydrolysed to glucose using AMG. D-Glucose was measured using glucose
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oxidase/peroxidase reagent (GOPOD) and this was the measure of the RS”. Absorbance at
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510 nm was measured using a spectrophotometer (Model Spectronic 200, Thermo Fisher
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Scientific, Pittsburgh, PA, USA). RS was calculated from the absorbance value using the
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Equation (1) supplied from the kit: RS = ΔE x F x
100 1 100 162 (1) 0.1 1000 180
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where, ∆E = absorbance (of the reaction) read against the reagent blank, F = 100 (µg of D-
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glucose)/absorbance of 100 µg of glucose, 100/0.1 = correction of the volume, 1/1000 = 11
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factor of conversion of µg to mg, 100/w = factor that expresses the starch as a percentage of
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the weight of flour, w = weight in mg of sample measured, and 162/180 = conversion factor
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of free D-glucose to D-anhydrous.
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2.2.6 Optimization
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The numerical function of desirability was used to optimize and determine the best
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combination of the extrusion variables using the method of Espinoza-Moreno et al. (2016).
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The responses of EI and RS4 from the 13 assays were fitted to a second-order polynomial
247
equation. The backward regression procedure was used to improve the models, and non-
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significant terms (p>0.05) were deleted from the second-order polynomial. A reduced
249
quadratic model and reduced cubic model were used for goodness-of-fit of EI and RS4,
250
respectively. Model adequacies were checked in terms of R2 values, adequate precision
251
values, lack of fit, and the significance of the model’s p value. Optimization, response
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surface plots, and data were obtained using Design Expert (Version 7.0.0, Stat-Ease Inc.,
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Minneapolis, MN, USA).
254
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2.2.7 Determination of phosphorus, degree of substitution and Fourier transform
256
infrared spectroscopy (FT-IR) analysis
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The phosphorus (%P) was measured using the method described by Smith and Caruso
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(1964). Briefly, starch samples (10 g) in a glass dish were treated with 10 mL of zinc
259
acetate solution (10%, w/w), and the mixture was evaporated to dryness sequentially in a
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steam bath and a hot plate. The dried material was ashed at 550°C in the muffle furnace 12
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(Model FE-341, Felisa) for 3 hr. After cooling, the ash was washed using 10 mL 29%
262
(w/w) nitric acid and 15 mL water. Then, this solution was diluted using 10 mL 0.25%
263
(w/w) ammonium vanadate, and 10 mL 5% (w/w) ammonium molybdate. After 10 min, the
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absorbance was measured at 420 nm against a sample blank. A calibration curve (0–40 µg
265
phosphorus/mL; y = 0.0654x + 0.0075; R2 = 0.99) was made using KH2PO4. The degree of
266
phosphate substitution (DS) was calculated using Equation 2:
DS =
162 x %P (2) 3,100 − (306 x %P)
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where DS = degree of substitution, %P = phosphorus percentage of the phosphorylated
268
starch (dw), 162 = the molar mass of the anhydroglucose unit, 3100 = the atomic weight of
269
phosphorus multiplied by 100 and 306 = the molar mass of the phosphate substituent.
270
FT-IR spectra were obtained using a spectrometer (Model Spectrum GX, Perkin Elmer Co.,
271
Boston, MA, USA) equipped with an EasiDiff diffuse reflectance accessory (Model Pike
272
Technology, Madison, WI, USA). Samples were prepared by mixing 4 mg of starch with
273
98 mg of anhydrous KBr and measured using the diffuse reflectance mode over the
274
wavenumber range between 4000 and 400 cm-1 in 24 scans at a resolution of 4 cm-1.
275
Previously, KBr was used as a spectrophotometric blank and the pure KBr background
276
spectrum was subtracted from the sample spectrum using the software that came with the
277
instrument. Spectra were baseline-corrected by drawing a straight line between 4000 and
278
400 cm-1 (Capron et al., 2007) using Fityk software (Version 0.9.8, Fityk, Oxfordshire,
279
UK).
280
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2.2.8 Sensory analysis 13
282
A sensory analysis was done with 33 semi-trained panelists that were recruited from the
283
Tecnológico Nacional de México/I.T. Mérida (Merida, Yucatan, Mexico), 25 students, 3
284
administrative staff and 5 professors, 48% male and 52% female, and with an age range of
285
23 to 48 yr, who are habitual snack consumers and voluntarily agreed to participate in the
286
sensory evaluation. Before the test, a training session was done to provide attribute
287
definitions to panellists. Textural characteristics, flavor and appearance attributes for an
288
extruded and expanded snack were discussed according to Philipp et al. (2017). Later, using
289
normal light and room temperature, panelists were monadically asked to evaluate 5 sensory
290
attributes (color, texture, crispness, flavor and overall acceptance) using a 9-point hedonic
291
scale, where 1 represented ‘dislike extremely’, 2 represented “dislike very much”, 3
292
represented “dislike moderately”, 4 represented “dislike slightly”, 5 represented “neither
293
like nor dislike”, 6 represented “like slightly”, 7 represented “like moderately”, 8
294
represented “like very much” and 9 represented ‘like extremely’. Two containers of snacks
295
(5 pieces/container) labelled with a 3-digit random code were given to each of the panelists:
296
one containing 3G-RS4, and the other, C-3G snacks. All samples were expectorated and
297
cold tap water (flavor free) was available for rinsing.
298
299
2.2.9 Statistical analysis
300
Two-way analysis of variance (ANOVA) was done to assess the statistical significance of
301
the model. An alpha of p<0.05 was used to determine significance using the statistical
302
software Design Expert. For the sensory data, differences between groups were compared
303
using Student´s t-test using the statistical program GraphPad Prism (Version 6.01,
304
GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant at 14
305
p<0.05, although the authors have also chosen to use p<0.01 for some of the data to
306
indicate the greater significance of the differences. Results were reported as the mean ±
307
standard deviation.
308
309
3. Results and Discussion
310
311
3.1 Effect of independent factors on EI and RS4
312
The extrusion conditions, such as BT and FM, exerted a significant influence on the
313
chemical reactions and physical phenomena observed in the final extruded product. The
314
experimental values of EI and RS4 in the 3G-S ranged from 2.53-6.6 and from 4.9-13.1
315
g/100 g, respectively, (Table 1). Results from the ANOVA (Table 2) showed that the
316
models were significant for EI and RS4 (p<0.01); additionally, adeq precision values >4, R2
317
values >0.825 and non-significant lack of fit for both responses suggested that the models
318
were sufficiently reliable. Equations 3 and 4 for EI and RS4, respectively, in terms of the
319
uncoded factors were:
320
Y = 53.9 − 0.94(BT) + 4.38e$% (3)
321
Y&'( = 1176 − 11(BT) − 70(FM) + 0.56(BT ∗ FM) + 0.01(BT), + 1.21(FM), −
322
9.85e$% (BT ∗ FM),
323
EI is a physical property that is highly related to the sensory acceptance of extruded snacks
324
(Philipp et al., 2007) and is influenced by the different extrusion conditions, such as FM
325
and BT (Tovar-Jiménez et al., 2015). In the current work, BT showed a significant effect
(4)
15
326
(p<0.01) in its linear and quadratic terms (Table 2) on the EI response. According to the
327
data, EI values tended to increase as BT increased at >127°C, while FM remained below
328
29% (Fig. 1a). The maximal EI of 6.6 ± 0.1 was observed at 136°C for BT and at 28.5% for
329
FM. At this temperature, the starch granules were opened, allowing the entrance of water
330
molecules (i.e., gelatinization) and they were removed as water vapor with pressure during
331
microwave heating (Cortés et al., 2014; Delgado-Nieblas et al., 2012; Tovar-Jiménez et al.,
332
2015). On the other hand, EI tended to decrease as FM increased because values of >30%
333
exerted a cooling effect inside the extruder, facilitating the material flow, decreasing the
334
temperature and the shear rate and inhibiting: (1) the opening of starch granules, (2) water
335
absorption and the gelatinization phenomenon, and subsequently, (3) expansion during
336
microwave heating. The values reported in the present study were higher than the 4.75 EI
337
reported for a commercially expanded snack (Camacho-Hernández et al., 2004) and similar
338
to the EI of 7 observed by Delgado-Nieblas et al. (2012) at BT >135°C and FM <24% for
339
3G-S. Delgado-Nieblas et al. (2012) mentioned that expansion resulted from events such as
340
the structural transformations of biopolymers, transitions, and phase transformations,
341
leading to the formation and collapse of air bubbles.
342
For the RS4 response, both BT and FM significantly influenced all terms (Table 2). A low
343
RS4 of 4.9 ± 0.1 g/100 g was observed at 115°C and 28.5% of FM; however, a high RS4
344
was observed at the highest extrusion temperature (Table 1). According to the RSM plots
345
(Fig. 1b), the RS4 increased up to 13.1 ± 0.5 g/100 g when BT increased up to 136°C and
346
FM was maintained at ~28.5%,
347
pressures and high temperatures, led to depolymerization of the starch molecule and the
348
intra-molecular and inter-molecular -OH starch groups were exposed. Subsequently, the
that corresponded to assay 6 (Table 1.) High shear
16
349
cross-linking reaction with the phosphate salts would occur (Manoi and Rizvi, 2010; Moad,
350
2011). Previous studies have obtained phosphorylated starch using extrusion in the form of
351
directly expanded or 2G-S (Landerito and Wang, 2005; Manoi and Rizvi, 2010; Moad,
352
2011) and not in the form of non-expanded or intermediate pellets such as 3G-S. Calvo-
353
López and Martínez-Bustos (2017) developed 2G-S based on potato starch with esterified
354
RS4 ranging from 23.1-55.8 g/100 g, higher values as compared with those obtained in this
355
study. The differences could be due to the different extrusion conditions necessary for the
356
production of 2G-S, such as a lower moisture (16%) and a higher BT (>150°C), which
357
could favor the chemical cross-linking reaction. On the other hand, high moisture values
358
(>25%) are necessary for the development of 3G-S. However, this high moisture could act
359
as a coolant inside the extruder barrel, decreasing the barrel temperature and leading to less
360
chemical cross-linking, and consequently lower RS4 (<20 g/100 g). Therefore, both BT and
361
FM were important for carrying out the cross-linked reaction and RS4 formation. These
362
results are similar to those reported by Kahraman et al. (2015), who mentioned that the
363
increase in the reaction temperature of 38 to 70°C increased RS4 from 15.9 to 49.3 g/100 g,
364
using
365
parameter for the formation of RS4 using reactive extrusion is the pH value; in this study,
366
the pH was adjusted to 8.5. According to Moad (2011), to form a starch diester or a cross-
367
linking reaction between -OH groups of adjacent starch molecules, a pH value of around 8
368
was necessary.
the conventional batch method with a 3 hr reaction time. Another important
369
370
3.2 Optimization
17
371
The extrusion conditions were optimized using the numerical method of desirability, and
372
the desirability value was 0.932 (Fig. 2). Espinoza-Moreno et al. (2016) reported a
373
desirability of 1 and noted that desirability values within the range of 0.7-1.0 provide a
374
good and acceptable product. The predicted values of EI and RS4 were 6.34 and 12.9
375
g/100 g, respectively, and optimal conditions were a BT of 136°C and an FM of 28.5%.
376
These optimal conditions were used to obtain an optimal snack. The optimal extruded snack
377
had RS4 and EI of 17.0 ± 0.2 g/100 g and 5.7 ± 0.3, respectively. A commercial portion of
378
~50 g of 3G-RS4 snacks provides 8.57 g RS4. Currently, a recommended daily intake of
379
RS has not been established; however, Murphy et al. (2008), Fuentes-Zaragoza et al.
380
(2011), and Birt et al. (2013) suggested a daily minimal intake of 5-6 g to observe a
381
reduction in the response to insulin and beneficial effects for health. Therefore, this snack
382
product could be considered a good source of resistant starch as a fiber ingredient.
383
384
3.3 Degree of substitution and FT-IR spectroscopy
385
DS and %P for 3G-RS4 snacks were 0.02 ± 0.00 and 0.35 ± 0.15%, respectively. This
386
phosphorus fell within the permissible limits, since the US Food and Drug Administration
387
(FDA) (2013) limits the phosphorus in modified food starches to 0.4%. Landerito and
388
Wang (2005), who cross-linked waxy maize starch using extrusion, reported a %P of 1.63,
389
a higher value compared to those obtained in the present study, which could be attributed
390
to different extrusion conditions and intrinsic starch-type factors.
391
Figure 3 shows the FT-IR spectra of sorghum starch, phosphorylated RS4 from 3G-RS4
392
snacks, and starch from C-3G snacks. The spectrum corresponding to sorghum starch
18
393
showed typical band signals at 1157, 1080 and 1018 cm-1, corresponding to the C-O
394
stretching bonds; bands at 929, 861, 765 and 575 cm-1 correspond to stretching of the
395
anhydroglucose ring. The signal at 2923 cm-1 corresponds to the vibration of C-H bonds
396
from methyl groups, and signals at 3421 and 1645 cm-1 correspond to the stretching of the
397
hydroxyl groups of the starch molecule and linked water molecules, respectively (Rivera-
398
Corona et al., 2014).
399
Regarding the phosphorylated starch from optimal 3G-RS4 snacks, new signals were
400
observed in comparison with sorghum starch and starch from C-3G snacks. The stretching
401
of the P3O9 ring of sodium trimetaphosphate can be observed at 520 cm-1 (Curry et al.,
402
2008). The new weak signals at 1259, 1298, and 1319 cm-1 are characteristics of P=O bonds
403
in cross-linked polysaccharides and confirms that -OH groups of starch were esterified with
404
phosphate salts (Ashwar et al., 2017; Ren et al., 2012;). Additionally, a new characteristic
405
absorption band at 1088 cm-1 was attributed to P-O-C stretching (Singh and Nath, 2012).
406
The low cross-linking and substitution degree led to weak spectroscopic signals (Gao et al.,
407
2014; Li et al., 2009; Ren et al., 2012). On the other hand, for C-3G starch, new signal
408
bands were seen at 2950 and 2890 cm-1. According to Acquistucci (2016), stretching bands
409
between 3000 and 2800 cm-1 are assigned to intrinsical lipids in the starch molecule, that
410
could be exposed by the several extrusion conditions.
411
412
3.4 Sensory acceptance of optimal expanded snacks
413
Figure 4 shows the sensory results of the 3G-RS4 and C-3G snacks. The 3G-RS4 snacks
414
had average scores of "neither like-nor dislike” for color, “slightly like" for texture and
19
415
flavor, and "like moderately" for crispness and general acceptance. Highest scores were
416
obtainded for 3G-RS4 snacks and 66% of the panelists gave a rating higher than 7 (“I like
417
moderately”) in terms of general acceptance. However, statistical analysis showed that
418
there were no significant differences (p>0.05) in the sensory properties measured between
419
3G-RS4 and C-3G snacks. The phosphorylation of sorghum starch may have no effect on
420
the texture and crispness of 3G-RS4, implying that RS4 did not have any negative influence
421
in terms of general acceptance. This effect was probably due to fiber type (RS4), in that
422
other fibers are known to reduce expansion volumes, making harder extrudate products
423
with less crispiness (Cortés et al., 2014; Wang et al., 2017). For example, in extruded
424
breakfast cereals from whole-grain sorghum flours, Mkandawire et al. (2015) observed that
425
the high fiber in the flours limited the expansion of the extrudates. In the present study,
426
expansion using microwave-heating did not negatively influence their sensorial
427
acceptability, compared with the expansion using a frying method (BahramParvar et al.,
428
2014). These results are similar to those reported by Aigster et al. (2011) and Maziarz et al.
429
(2013), who added commercial RS to muffins, focaccia bread, chicken curry, and cereal
430
bars, and reported that partial replacement of flour with RS increased these sensory
431
characteristics without affecting overall acceptability.
432
433
4. Conclusions
434
The phosphorylation of isolated sorghum starch using optimal extrusion conditions had the
435
potential to induce the formation of RS4 and simultaneously allowed the development of
436
3G-S with general sensory acceptability. The intermediate barrel temperature had a positive
437
influence on the formation of RS4 and the EI. Degree of substitution and FT-IR studies 20
438
confirmed that phosphorylation of sorghum starch using sodium trimetaphosphate and
439
sodium tripolyphosphate was within the allowable limits of the FDA. RS4 and microwave-
440
expansion did not affect the sensorial acceptability of the optimal snack. Isolated sorghum
441
starch was an efficient raw material to produce extruded snacks; therefore, may be possible
442
to use in the snack-food industry.
443
444
Acknowledgements
445
The first author thanks CONACYT for the MSc degree scholarship (No. 386579). The
446
authors thank MSc Verónica Flores-Casamayor, MSc Reina Aracely Mauricio Sánchez,
447
MSc José Juan Véles-Medina and BSc Carlos Alberto Avila Herrera from Cinvestav-
448
Querétaro for their technical support.
449
450
Conflict of Interest Statement
451
The authors declare that they have no conflict of interest with respect to the study described
452
in this manuscript.
453
454
455 456
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Table 1. Experimental design with coded and actual values, and experimental results of EI and RS4. Independent variables Experimental results Coded Actual Dependent responses Run X1 X2 BTa (°C) FMb (%) EI c RS4d (g/100 g) -1 -1 100 25 2.8 ± 0.1 10.1 ± 0.3 1 1 -1 130 25 5.4 ± 0.1 8.495 ± 0.152 2 -1 1 100 32 3.02 ± 0.04 8.9 ± 0.1 3 1 1 130 32 4.152 ± 0.051 8.9 ± 0.4 4 -1.414 0 93.8 28.5 3.96 ± 0.03 9.2 ± 0.4 5 1.414 0 136 28.5 6.6 ± 0.1 13.1 ± 0.5 6 0 -1.414 115 23.5 2.68 ± 0.04 7.7 ± 0.3 7 0 1.414 115 33.4 4.2 ± 0.1 6.8 ± 0.1 8 0 0 115 28.5 2.5 ± 0.1 5.5 ± 0.2 9 0 0 115 28.5 3.33 ± 0.3 5.0 ± 0.2 10 0 0 115 28.5 3.1 ± 0.1 5.23 ± 0.23 11 0 0 115 28.5 3.0 ± 0.1 5.3 ± 0.3 12 0 0 115 28.5 3.23 ± 0.32 4.9 ± 0.1 13 a b Means ± standard deviation (n = 3). BT and FM represents the transition barrel temperature and the feed moisture, respectively. cEI and dRS4 represents the expansion index and the type 4 resistant starch, respectively.
602 603 604 605 606 607
28
608 609 610 611 612 613 614 615
Table 2. ANOVA for RS4 and EI responsesa.
616 617
Factor RS4a EIb 5.27 3.09 Intercept Barrel temperature (Linear term) 1.39* 0.94* Feed moisture (Linear term) -0.24* ND Barrel temperature-Feed moisture (Interaction term) 0.36* ND Barrel temperature2 (Quadratic term) 2.92* 0.99* 2 Feed moisture (Quadratic term) 0.97* ND Barrel temperature * Feed moisuture2 (Cubic term) -1.81* ND 0.998 0.825 R2 <0.01 <0.01 p-value for model 0.345ns 0.0852ns p-value for lack of fit 69.44 12.65 Adeq precision a RS4 and bEI represents type 4 resistant starch and expansion index responses, respectively. ns Not significant at p>0.05; * Significant at p<0.01; ND Not determinated.
618 619 620 621 622 623 624 625 626 627 29
628 629 630 631 632 633
Figure legends
634
Fig. 1 Effect of transition barrel temperature and feed moisture on (a) expansion index and
635
(b) type-4 resistant starch (X1 and X2 corresponds to intermediate barrel temperature and
636
feed moisture, respectively)
637
Fig. 2 Overall desirability for expansion index and type-4 resistant starch responses to
638
optimize the extrusion
639
temperature and feed moisture, respectively)
640
Fig. 3 FT-IR spectra of (a) native sorghum starch, (b) optimal third-generation snack whit
641
type 4 resistant starch and (c) control third-generation snack
642
Fig. 4 Sensory evaluation of optimal third-generation snacks with 17 g/100 g of type-4
643
resistant starch (3G-RS4) and control third-generation snacks (C-3G). Figure shows the
644
score for each attribute. Mean values are shown with error bars of standard deviations (n =
645
33). A p<0.05 value was considered significant.
conditions (X1 and X2 corresponds to intermediate barrel
646 647
30
S2212-4292(18)30849-6
DOI:
https://doi.org/10.1016/j.fbio.2019.100474
Reference:
FBIO 100474
To appear in:
Food Bioscience
Received Date: 5 September 2018 Revised Date:
28 September 2019
Accepted Date: 28 September 2019
Please cite this article as: Escobar-Puentes A., Rincón S., García-Gurrola A., Zepeda A., Calvo-López A.D. & Martínez-Bustos F., Development of a third-generation snack with type 4 resistant sorghum starch: Physicochemical and sensorial properties, Food Bioscience (2019), doi: https://doi.org/10.1016/ j.fbio.2019.100474. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
1
Development of a third-generation snack with type 4 resistant sorghum starch:
2
Physicochemical and sensorial properties
3
Running title: Extruded enriched-snack with resistant sorghum starch
4
Alberto Escobar-Puentes1, Susana Rincón1, Adriana García-Gurrola1, Alejandro Zepeda2,
5
Amira Daniela Calvo-López3, Fernando Martínez-Bustos3*.
6
1
7
Mérida, Mérida, Yucatán, 97118, México.
8
2
9
97203, México.
Departamento de Ingeniería Química-Bioquímica, Tecnológico Nacional de México/I.T.
Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán,
10
3
11
del Instituto Politécnico Nacional, Unidad Querétaro, Querétaro, 76230, México.
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*Corresponding author at: Departamento de Materiales Orgánicos, Centro de Investigación
13
y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Querétaro,
14
Libramiento Norponiente, No. 2000., Real de Juriquilla, 76230, Santiago de Querétaro,
15
Querétaro, México. Email-address: [email protected]. Tel.:+52 442 2119905; fax:
16
+52 442 2119938.
Departamento de Materiales Orgánicos, Centro de Investigación y de Estudios Avanzados
17 18 19 20 21
1
22
Abstract
23
Extruded third-generation fiber enriched snacks can be made using type 4 resistant starch.
24
The present study aimed to evaluate and optimize some extrusion conditions, namely the
25
transition or intermediate barrel temperature and the feed moisture in the direct formation
26
of third-generation snacks with type 4 resistant starch using the phosphorylation of
27
sorghum starch. The physicochemical and sensory properties of the optimal snack were
28
investigated. An increase in barrel temperature resulted in an increase in the resistant starch
29
(13.1 ± 0.5 g/100 g) and the expansion index values, as long as the feed moisture was
30
maintained above 28%. The optimal conditions with a desirability value of 0.932 were a
31
barrel temperature of 136°C and a feed moisture of 28.5%. The optimal third-generation
32
snack had 17.1 ± 0.2 g/100 g of resistant starch and a desirable expansion index of 5.7 ±
33
0.4, as well as general sensory acceptability (“I like moderately”). Phosphorylated resistant
34
starch of the optimal snack showed a degree of substitution and a percentage of phosphorus
35
(0.02 ± 0.00 and 0.35 ± 0.15%, respectively) within the limits allowed in foods. This study
36
showed that using optimal extrusion conditions, phosphorylation of sorghum starch was
37
possible to produce resistant starch and simultaneously to obtain an extruded snack with
38
acceptable physical and sensorial properties. Additionally, it showed that sorghum is a good
39
alternative source of starch for the formulation of extruded snacks.
40
Keywords: Enriched-snacks, extrusion, Sorghum bicolor (L.) Moench, sorghum starch,
41
type-4 resistant starch.
42
Abbreviations:
43
%P: phosphorus percentage
2
44
2G-S: second-generation snacks
45
3G-S: third-generation snacks
46
3G-RS4: third-generation snacks with type-4 resistant starch
47
BT: barrel temperature
48
C-3G: control snacks without type-4 resistant starch
49
DS: degree of substitution
50
FM: feed moisture
51
FT-IR: Fourier transform infrared spectroscopy
52
RS: resistant starch
53
RS4: type-4 resistant starch
54
RSM: response surface methodology
55
56
57
58
59
60
61
3
62
1. Introduction
63
Extruded snacks are often termed “junk food” because they are typically fried and low in
64
dietary fiber. However, their consumption is increasing, especially in developing countries,
65
and the consumption of these unhealthy snacks can result in various metabolic diseases
66
(Espinoza-Moreno et al., 2016). Therefore, the current trend in the snacks industry is the
67
development of healthy foods (Tumuluru, 2016) using the incorporation of active
68
compounds such as proteins, dietary fibers, and antioxidant compounds (Chávez et al.,
69
2017; Cortés et al., 2014; Espinoza-Moreno et al., 2016)
70
Resistant starch (RS) is a non-digestible polysaccharide, since it resists enzymatic digestion
71
in the small intestine and is able to reach the large intestine of humans, where it can be
72
fermented by the gut microbiota, providing protective effects against colorectal cancer and
73
aiding in controlling metabolic diseases (Englyst et al., 1992; Fuentes-Zaragoza et al.,
74
2011). Due to their physiological benefits, RS have been successfully used as a fiber-
75
enriching ingredient in a range of food systems, such as cookies (Giuberti et al., 2017),
76
pasta (Foschia et al., 2017), and breakfast cereals (Miller et al., 2011). Type-4 resistant
77
starch (RS4) is produced using a chemical cross-linking reaction between hydroxyl (–OH)
78
groups of starch and food-grade chemical agents, which form atypical linkages and block
79
the access of enzymes, preventing their digestion (Fuentes-Zaragoza et al., 2011; Landerito
80
and Wang, 2005). Chemical cross-linking using phosphorylation is the earliest and most
81
preferred method for starch modification and RS4 production (Miller et al., 2011), and the
82
phosphate salts mainly used are sodium tripolyphosphate alone or in a mixture with sodium
83
trimetaphosphate. Phosphorylated starch has phosphate groups esterified with two –OH
84
groups,
very
often
from
two
neighboring 4
starch
molecules.
Conventionally,
85
phosphorylation is carried out in a batch system over long time periods using high
86
concentrations of reagents (Moad, 2011), which may cause environmental contamination
87
from unreacted chemicals and which can be
88
chemicals is considered (Landerito and Wang, 2005). To overcome these problems, the
89
phosphorylation reaction needed to produce RS4 can also be induced using reactive
90
extrusion, a continuous and short-time high-temperature process, with the absence of
91
effluents (Landerito and Wang, 2005; Manoi and Rizvi, 2010; Moad, 2011). Moreover,
92
extrusion technology is widely used in the food industry (breakfast cereals, snack foods,
93
and other similar products) due to its low cost, versatility, high productivity, and energy
94
efficiency (Tovar-Jiménez et al., 2015). It is possible to obtain two types of extruded snack
95
foods, such as second-generation snacks (2G-S) or ready to eat products (directly expanded
96
through the extrusion exit) and third-generation snacks (3G-S), which are non-expanded
97
pellets or glassy half-products formed by extruding starchy materials (Aguilar-Palazuelos et
98
al., 2006) that are not expanded directly through the extrusion exit and need to be expanded
99
to obtain a consumer-ready final product using additional processes, such as frying or
100
microwave heating (Tumuluru, 2016), the latter being a fat-free method. Different studies
101
have focused on the development of enriched 3G-S using proteins, anthocyanins, winter-
102
squash flours, and orange fibers (Aguilar-Palazuelos et al., 2006; Camacho-Hernández et
103
al., 2014; Delgado-Nieblas et al., 2012; Tovar-Jiménez et al., 2015). On the other hand, a
104
recent study done by Calvo-López and Martínez-Bustos, (2017) showed that it is possible
105
to develop 2G-S with RS4 using esterification of potato starch in a single extrusion step.
106
However, no information is currently available concerning the development of 3G-S with
107
type-4 resistant starch (3G-RS4) in a single extrusion step and based on sorghum starch.
5
costly when removal of the unreacted
108
Generally, extruded snacks are made from corn or potato starch, and optionally using other
109
components, such as vegetable, cereal or legume flours. However, starch should be
110
considered as the main component. In the present study, sorghum starch was used for the
111
production of 3G-S. Sorghum (Sorghum bicolor) is the world’s fifth cereal crop and is
112
drought-tolerant, grows with various soil conditions (Jafari et al., 2017) and is an
113
economical alternative with desirable agro-technological characteristics. It can be regarded
114
as a substitute for the common cereals, since it contains 70% starch similar to corn (Singh
115
and Singh, 1995), besides being a naturally gluten-free cereal (Chávez et al., 2017). For
116
these reason, several reports have focused on the development of different food systems
117
such as pasta, breakfast cereals, and extruded snacks based on sorghum flours (Khan et al.,
118
2013; Mkandawire et al., 2015; Wu et al., 2018).
119
Studying the development of 3G-RS4 snacks using a single extrusion step based on
120
sorghum starch and evaluating its physicochemical and sensory properties would be
121
potentially beneficial. Therefore, the aims of this study were as follows: 1) to optimize the
122
extrusion conditions (intermediate barrel temperature and feed moisture) and evaluate their
123
effects on the expansion index values and the formation of RS4 using phosphorylation of
124
sorghum starch, and 2) to analyze the physicochemical and sensorial properties of the
125
optimal snack.
126
127
2. Materials and Methods
128
129
2.1 Materials 6
130
131
2.1.1 Sample
132
White sorghum (Sorghum bicolor (L.) Moench) “Fortuna” genotype harvested in June
133
2015 in Guanjuato, Mexico (20°34'48.1"N 100°49'11.9"W) was identified and provided by
134
Dr. Victor Pecina Quintero of the Experimental Station of the National Research Institute
135
for Forestry, Agriculture and Livestock. The sorghum grains were received in a single
136
batch in sealed kraft paper bags with an internal polyethylene liner and cleaned manually.
137
Subsequently, sorghum grains were placed in polyethylene bags (200 x 300 mm, Aranda
138
Bolsas de Polietileno, Queretaro, Queretaro, Mexico) and stored (4°C) at the Laboratory of
139
Organic Materials of Research and Advanced Studies Center of the National Polytechnic
140
Institute, Campus Queretaro, Mexico, for a maximum of 3 wk before the experiments.
141
142
2.1.2 Chemicals
143
Sodium trimetaphosphate, sodium tripolyphosphate, ammonium molybdate, ammonium
144
vanadate, zinc acetate, HNO3, CuSO4, K2SO4, KH2PO4 and KBr were purchased from
145
Sigma Aldrich Co., (St. Louis, MO, USA). Ethanol (99.7%), distilled water, CH3COOH,
146
KOH, NaHSO3, 1 N NaOH volumetric solution and NaOH pellets were purchased from J.T.
147
Baker (Phillipsburg, NJ, USA). The resistant starch assay kit was purchased from
148
Megazyme International Ireland Ltd. (Wicklow, Ireland).
149
150
2.2 Methods
7
151
152
2.2.1 Sorghum starch isolation
153
Starch was isolated from sorghum grains using the wet-milling method reported by Sira and
154
Amaiz (2004) with some modifications. Briefly, 3 kg of sorghum grains were suspended in
155
a container using 0.25% NaOH solution to cover twice the volume of the grains. After 18
156
hr, the grain mass was washed exhaustively using tap water until reaching neutral pH. After
157
steeping, grains were ready for wet milling. The pretreated grains were milled in a stone
158
mill (Model M100, Fumasa, Queretaro, Mexico) to break the testa and expose the starch. A
159
solution of 0.1 N NaHSO3 (~50 mL) was added during grinding. The ground slurry was
160
screened using standard sieves (841, 595, 420, 250, 177, 149, 74, and 62.5 µm) (Mont Inox,
161
Mexico City, Mexico) to separate the starch and the fiber fractions. The residue on the
162
sieves was discarded and the screened solution was recovered and allowed to stand ~16 hr
163
at room temperature (25-30°C). The starch was then recovered in the sediment, which was
164
washed using an ethanol:water solution (50:50 v/v), centrifuged at 5,450 x g for 15 min at
165
5°C (5,000 rpm in a 220.70 V06 rotor, Model Z513K centrifuge, Hermle Labortechnik
166
GmbH Co., Wehingen, Germany) and dried in a convection oven at 40°C for 48 hr.
167
168
2.2.2 Chemical composition
169
Proximate analysis was determinated in triplicates using the AOAC Official Methods
170
(1999). Moisture was determinated using standard method 925.09 based on sample weight
171
loss after oven drying at 105°C for 2 hr. Crude protein was determined using the Kjeldahl
172
nitrogen method 979.09 using a micro-kjeldahl digestor (Model Vapodest 200, Gerhardt 8
173
GmbH & Co., Königswinter, Germany) and CuSO4 and K2SO4 as catalysts. A conversion
174
factor of 6.25 was used to calculate crude protein. Fat cotent was estimated after 3 hr
175
extraction using petroleum ether using the Soxhlet principle (AOAC method 923.05), and
176
ash was calculated from sample weight after incineration in a muffle furnace (Model FE-
177
341, Felisa, Guadalajara, Jalisco, Mexico) at 550°C for 2 hr (AOAC method 923.03).
178
Isolated sorghum starch had 10 ± 1% moisture, 0.98 ± 0.01% crude protein, 1.4 ± 0.3%
179
lipid, and 0.32 ± 0.04% ash.
180
181
2.2.3 Extrusion
182
The effect of extrusion conditions such as the barrel temperature (BT) and the feed
183
moisture (FM) on the expansion index (EI) and the RS4 of 3G-S was studied using
184
response surface methodology (RSM), using a two-factor central composite experimental
185
design, which consisted of 13 assays with 5 center points (Table 1). Starch samples were
186
extruded at different BT in the transition or intermediate zone (93.8–136°C) and at different
187
FM (23.5-33.4%). Experimental limits that were defined using preliminary tests.
188
Prior to the extrusion, sorghum starch (200 g) was conditioned with moisture and
189
phosphate salts (4 g/100 g starch dry weight) using the dry-mixing procedure described by
190
Landerito and Wang (2005). Briefly, the water necessary to achieve the FM values of the
191
experimental design (Table 1) was used to dissolve a mixture of sodium trimetaphosphate
192
and sodium tripolyphosphate (at a ratio of 99:1 w/w) and this was adjusted to pH 8.5 using
193
a 1 N NaOH standard solution. The solution was stirred vigorously using a magnetic stirrer
194
(Model FE-310, Felisa, Guadalajara, Jalisco, Mexico) until the chemical agents were
9
195
completely dissolved (~10 min). Later, the solution was spray-atomized into starch and was
196
mixed using a laboratory spatula. An additional starch sample was conditioned using only
197
28.5% FM (optimal condition) without the phosphate salts and the catalyst NaOH, to obtain
198
a control snack without RS4 (C-3G).
199
Extrusion trials were done using a single screw extruder designed and manufactured by the
200
Laboratory of Organic Materials of Research and Advanced Studies Center of the National
201
Polytechnic Institute, Campus Queretaro, Mexico. The extruder was equipped with a screw
202
(428 mm long and 19 mm diameter), a 1:1 compression ratio, a 40-rpm screw speed, a
203
rectangular die (1.5 × 20 × 100 mm) and three heating zones. The BT in the intermediate
204
zone varied according to the experimental design (Table 1), while the BT in the feed and
205
high-pressure die zones was maintained constant at 60 and 70°C, respectively. The
206
resulting non-expanded pellets were cut (2.5 x 2.5 cm) using scissors and dried in a forced
207
air oven (Model FE293A, Felisa, Guadalajara, Jalisco, Mexico) at 40 ± 1°C until the
208
moisture reached 10 ± 1%.
209
210
2.2.4 Microwave-expansion
211
Extruded pellets were expanded using heating (30 s) in a conventional microwave oven
212
(Model MW843WA, 1200 W and 60 Hz, Samsung Electronics Co., Seoul, Korea). The EI
213
was calculated as the average transversal area of the expanded product divided by the
214
average transversal area of the non-expanded pellet (Aguilar-Palazuelos et al., 2006). The
215
EI was measured in 15 samples. The measurements were determined using a Vernier
216
calliper (Model Mitutoyo, Tokyo, Japan) having 0.05 mm accuracy.
10
217
218
2.2.5 Resistant starch
219
Microwave-expanded pellets were milled using a hammer mill equipped with a 0.25 mm
220
sieve (Model 200, Pulvex, Mexico City, Mexico) to obtain flour, and the RS4 was then
221
quantified using a RS assay kit (Cat. no. RSTAR 11/02, Megazyme International Ireland,
222
Ltd.) based on AACC standard method 32-40.01 (AACC, 2010) which was briefly
223
described by Foschia et al. (2017) as follows: “samples were incubated with pancreatic α-
224
amylase and amyloglucosidase (AMG) at 37°C for 16 hr, during which time non-resistant
225
starch was solubilized and hydrolysed to D-glucose by the combined action of the two
226
enzymes. The reaction was terminated by the addition of 99.7% ethanol and the RS was
227
recovered as a pellet on centrifugation at 1500 × g for 10 min in capped 50 mL centrifuge
228
tubes (3,000 rpm in a 220.80 V02 rotor, Hermel Labortechnik GmbH Co.,). The residue
229
was washed twice by suspension in aqueous 50% (v/v) ethanol, followed by centrifugation.
230
Excess liquid was removed using decantation. RS in the pellet was dissolved in KOH; this
231
solution was neutralized using acetate buffer (pH 3.8) and the starch was quantitatively
232
hydrolysed to glucose using AMG. D-Glucose was measured using glucose
233
oxidase/peroxidase reagent (GOPOD) and this was the measure of the RS”. Absorbance at
234
510 nm was measured using a spectrophotometer (Model Spectronic 200, Thermo Fisher
235
Scientific, Pittsburgh, PA, USA). RS was calculated from the absorbance value using the
236
Equation (1) supplied from the kit: RS = ΔE x F x
100 1 100 162 (1) 0.1 1000 180
237
where, ∆E = absorbance (of the reaction) read against the reagent blank, F = 100 (µg of D-
238
glucose)/absorbance of 100 µg of glucose, 100/0.1 = correction of the volume, 1/1000 = 11
239
factor of conversion of µg to mg, 100/w = factor that expresses the starch as a percentage of
240
the weight of flour, w = weight in mg of sample measured, and 162/180 = conversion factor
241
of free D-glucose to D-anhydrous.
242
243
2.2.6 Optimization
244
The numerical function of desirability was used to optimize and determine the best
245
combination of the extrusion variables using the method of Espinoza-Moreno et al. (2016).
246
The responses of EI and RS4 from the 13 assays were fitted to a second-order polynomial
247
equation. The backward regression procedure was used to improve the models, and non-
248
significant terms (p>0.05) were deleted from the second-order polynomial. A reduced
249
quadratic model and reduced cubic model were used for goodness-of-fit of EI and RS4,
250
respectively. Model adequacies were checked in terms of R2 values, adequate precision
251
values, lack of fit, and the significance of the model’s p value. Optimization, response
252
surface plots, and data were obtained using Design Expert (Version 7.0.0, Stat-Ease Inc.,
253
Minneapolis, MN, USA).
254
255
2.2.7 Determination of phosphorus, degree of substitution and Fourier transform
256
infrared spectroscopy (FT-IR) analysis
257
The phosphorus (%P) was measured using the method described by Smith and Caruso
258
(1964). Briefly, starch samples (10 g) in a glass dish were treated with 10 mL of zinc
259
acetate solution (10%, w/w), and the mixture was evaporated to dryness sequentially in a
260
steam bath and a hot plate. The dried material was ashed at 550°C in the muffle furnace 12
261
(Model FE-341, Felisa) for 3 hr. After cooling, the ash was washed using 10 mL 29%
262
(w/w) nitric acid and 15 mL water. Then, this solution was diluted using 10 mL 0.25%
263
(w/w) ammonium vanadate, and 10 mL 5% (w/w) ammonium molybdate. After 10 min, the
264
absorbance was measured at 420 nm against a sample blank. A calibration curve (0–40 µg
265
phosphorus/mL; y = 0.0654x + 0.0075; R2 = 0.99) was made using KH2PO4. The degree of
266
phosphate substitution (DS) was calculated using Equation 2:
DS =
162 x %P (2) 3,100 − (306 x %P)
267
where DS = degree of substitution, %P = phosphorus percentage of the phosphorylated
268
starch (dw), 162 = the molar mass of the anhydroglucose unit, 3100 = the atomic weight of
269
phosphorus multiplied by 100 and 306 = the molar mass of the phosphate substituent.
270
FT-IR spectra were obtained using a spectrometer (Model Spectrum GX, Perkin Elmer Co.,
271
Boston, MA, USA) equipped with an EasiDiff diffuse reflectance accessory (Model Pike
272
Technology, Madison, WI, USA). Samples were prepared by mixing 4 mg of starch with
273
98 mg of anhydrous KBr and measured using the diffuse reflectance mode over the
274
wavenumber range between 4000 and 400 cm-1 in 24 scans at a resolution of 4 cm-1.
275
Previously, KBr was used as a spectrophotometric blank and the pure KBr background
276
spectrum was subtracted from the sample spectrum using the software that came with the
277
instrument. Spectra were baseline-corrected by drawing a straight line between 4000 and
278
400 cm-1 (Capron et al., 2007) using Fityk software (Version 0.9.8, Fityk, Oxfordshire,
279
UK).
280
281
2.2.8 Sensory analysis 13
282
A sensory analysis was done with 33 semi-trained panelists that were recruited from the
283
Tecnológico Nacional de México/I.T. Mérida (Merida, Yucatan, Mexico), 25 students, 3
284
administrative staff and 5 professors, 48% male and 52% female, and with an age range of
285
23 to 48 yr, who are habitual snack consumers and voluntarily agreed to participate in the
286
sensory evaluation. Before the test, a training session was done to provide attribute
287
definitions to panellists. Textural characteristics, flavor and appearance attributes for an
288
extruded and expanded snack were discussed according to Philipp et al. (2017). Later, using
289
normal light and room temperature, panelists were monadically asked to evaluate 5 sensory
290
attributes (color, texture, crispness, flavor and overall acceptance) using a 9-point hedonic
291
scale, where 1 represented ‘dislike extremely’, 2 represented “dislike very much”, 3
292
represented “dislike moderately”, 4 represented “dislike slightly”, 5 represented “neither
293
like nor dislike”, 6 represented “like slightly”, 7 represented “like moderately”, 8
294
represented “like very much” and 9 represented ‘like extremely’. Two containers of snacks
295
(5 pieces/container) labelled with a 3-digit random code were given to each of the panelists:
296
one containing 3G-RS4, and the other, C-3G snacks. All samples were expectorated and
297
cold tap water (flavor free) was available for rinsing.
298
299
2.2.9 Statistical analysis
300
Two-way analysis of variance (ANOVA) was done to assess the statistical significance of
301
the model. An alpha of p<0.05 was used to determine significance using the statistical
302
software Design Expert. For the sensory data, differences between groups were compared
303
using Student´s t-test using the statistical program GraphPad Prism (Version 6.01,
304
GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant at 14
305
p<0.05, although the authors have also chosen to use p<0.01 for some of the data to
306
indicate the greater significance of the differences. Results were reported as the mean ±
307
standard deviation.
308
309
3. Results and Discussion
310
311
3.1 Effect of independent factors on EI and RS4
312
The extrusion conditions, such as BT and FM, exerted a significant influence on the
313
chemical reactions and physical phenomena observed in the final extruded product. The
314
experimental values of EI and RS4 in the 3G-S ranged from 2.53-6.6 and from 4.9-13.1
315
g/100 g, respectively, (Table 1). Results from the ANOVA (Table 2) showed that the
316
models were significant for EI and RS4 (p<0.01); additionally, adeq precision values >4, R2
317
values >0.825 and non-significant lack of fit for both responses suggested that the models
318
were sufficiently reliable. Equations 3 and 4 for EI and RS4, respectively, in terms of the
319
uncoded factors were:
320
Y = 53.9 − 0.94(BT) + 4.38e$% (3)
321
Y&'( = 1176 − 11(BT) − 70(FM) + 0.56(BT ∗ FM) + 0.01(BT), + 1.21(FM), −
322
9.85e$% (BT ∗ FM),
323
EI is a physical property that is highly related to the sensory acceptance of extruded snacks
324
(Philipp et al., 2007) and is influenced by the different extrusion conditions, such as FM
325
and BT (Tovar-Jiménez et al., 2015). In the current work, BT showed a significant effect
(4)
15
326
(p<0.01) in its linear and quadratic terms (Table 2) on the EI response. According to the
327
data, EI values tended to increase as BT increased at >127°C, while FM remained below
328
29% (Fig. 1a). The maximal EI of 6.6 ± 0.1 was observed at 136°C for BT and at 28.5% for
329
FM. At this temperature, the starch granules were opened, allowing the entrance of water
330
molecules (i.e., gelatinization) and they were removed as water vapor with pressure during
331
microwave heating (Cortés et al., 2014; Delgado-Nieblas et al., 2012; Tovar-Jiménez et al.,
332
2015). On the other hand, EI tended to decrease as FM increased because values of >30%
333
exerted a cooling effect inside the extruder, facilitating the material flow, decreasing the
334
temperature and the shear rate and inhibiting: (1) the opening of starch granules, (2) water
335
absorption and the gelatinization phenomenon, and subsequently, (3) expansion during
336
microwave heating. The values reported in the present study were higher than the 4.75 EI
337
reported for a commercially expanded snack (Camacho-Hernández et al., 2004) and similar
338
to the EI of 7 observed by Delgado-Nieblas et al. (2012) at BT >135°C and FM <24% for
339
3G-S. Delgado-Nieblas et al. (2012) mentioned that expansion resulted from events such as
340
the structural transformations of biopolymers, transitions, and phase transformations,
341
leading to the formation and collapse of air bubbles.
342
For the RS4 response, both BT and FM significantly influenced all terms (Table 2). A low
343
RS4 of 4.9 ± 0.1 g/100 g was observed at 115°C and 28.5% of FM; however, a high RS4
344
was observed at the highest extrusion temperature (Table 1). According to the RSM plots
345
(Fig. 1b), the RS4 increased up to 13.1 ± 0.5 g/100 g when BT increased up to 136°C and
346
FM was maintained at ~28.5%,
347
pressures and high temperatures, led to depolymerization of the starch molecule and the
348
intra-molecular and inter-molecular -OH starch groups were exposed. Subsequently, the
that corresponded to assay 6 (Table 1.) High shear
16
349
cross-linking reaction with the phosphate salts would occur (Manoi and Rizvi, 2010; Moad,
350
2011). Previous studies have obtained phosphorylated starch using extrusion in the form of
351
directly expanded or 2G-S (Landerito and Wang, 2005; Manoi and Rizvi, 2010; Moad,
352
2011) and not in the form of non-expanded or intermediate pellets such as 3G-S. Calvo-
353
López and Martínez-Bustos (2017) developed 2G-S based on potato starch with esterified
354
RS4 ranging from 23.1-55.8 g/100 g, higher values as compared with those obtained in this
355
study. The differences could be due to the different extrusion conditions necessary for the
356
production of 2G-S, such as a lower moisture (16%) and a higher BT (>150°C), which
357
could favor the chemical cross-linking reaction. On the other hand, high moisture values
358
(>25%) are necessary for the development of 3G-S. However, this high moisture could act
359
as a coolant inside the extruder barrel, decreasing the barrel temperature and leading to less
360
chemical cross-linking, and consequently lower RS4 (<20 g/100 g). Therefore, both BT and
361
FM were important for carrying out the cross-linked reaction and RS4 formation. These
362
results are similar to those reported by Kahraman et al. (2015), who mentioned that the
363
increase in the reaction temperature of 38 to 70°C increased RS4 from 15.9 to 49.3 g/100 g,
364
using
365
parameter for the formation of RS4 using reactive extrusion is the pH value; in this study,
366
the pH was adjusted to 8.5. According to Moad (2011), to form a starch diester or a cross-
367
linking reaction between -OH groups of adjacent starch molecules, a pH value of around 8
368
was necessary.
the conventional batch method with a 3 hr reaction time. Another important
369
370
3.2 Optimization
17
371
The extrusion conditions were optimized using the numerical method of desirability, and
372
the desirability value was 0.932 (Fig. 2). Espinoza-Moreno et al. (2016) reported a
373
desirability of 1 and noted that desirability values within the range of 0.7-1.0 provide a
374
good and acceptable product. The predicted values of EI and RS4 were 6.34 and 12.9
375
g/100 g, respectively, and optimal conditions were a BT of 136°C and an FM of 28.5%.
376
These optimal conditions were used to obtain an optimal snack. The optimal extruded snack
377
had RS4 and EI of 17.0 ± 0.2 g/100 g and 5.7 ± 0.3, respectively. A commercial portion of
378
~50 g of 3G-RS4 snacks provides 8.57 g RS4. Currently, a recommended daily intake of
379
RS has not been established; however, Murphy et al. (2008), Fuentes-Zaragoza et al.
380
(2011), and Birt et al. (2013) suggested a daily minimal intake of 5-6 g to observe a
381
reduction in the response to insulin and beneficial effects for health. Therefore, this snack
382
product could be considered a good source of resistant starch as a fiber ingredient.
383
384
3.3 Degree of substitution and FT-IR spectroscopy
385
DS and %P for 3G-RS4 snacks were 0.02 ± 0.00 and 0.35 ± 0.15%, respectively. This
386
phosphorus fell within the permissible limits, since the US Food and Drug Administration
387
(FDA) (2013) limits the phosphorus in modified food starches to 0.4%. Landerito and
388
Wang (2005), who cross-linked waxy maize starch using extrusion, reported a %P of 1.63,
389
a higher value compared to those obtained in the present study, which could be attributed
390
to different extrusion conditions and intrinsic starch-type factors.
391
Figure 3 shows the FT-IR spectra of sorghum starch, phosphorylated RS4 from 3G-RS4
392
snacks, and starch from C-3G snacks. The spectrum corresponding to sorghum starch
18
393
showed typical band signals at 1157, 1080 and 1018 cm-1, corresponding to the C-O
394
stretching bonds; bands at 929, 861, 765 and 575 cm-1 correspond to stretching of the
395
anhydroglucose ring. The signal at 2923 cm-1 corresponds to the vibration of C-H bonds
396
from methyl groups, and signals at 3421 and 1645 cm-1 correspond to the stretching of the
397
hydroxyl groups of the starch molecule and linked water molecules, respectively (Rivera-
398
Corona et al., 2014).
399
Regarding the phosphorylated starch from optimal 3G-RS4 snacks, new signals were
400
observed in comparison with sorghum starch and starch from C-3G snacks. The stretching
401
of the P3O9 ring of sodium trimetaphosphate can be observed at 520 cm-1 (Curry et al.,
402
2008). The new weak signals at 1259, 1298, and 1319 cm-1 are characteristics of P=O bonds
403
in cross-linked polysaccharides and confirms that -OH groups of starch were esterified with
404
phosphate salts (Ashwar et al., 2017; Ren et al., 2012;). Additionally, a new characteristic
405
absorption band at 1088 cm-1 was attributed to P-O-C stretching (Singh and Nath, 2012).
406
The low cross-linking and substitution degree led to weak spectroscopic signals (Gao et al.,
407
2014; Li et al., 2009; Ren et al., 2012). On the other hand, for C-3G starch, new signal
408
bands were seen at 2950 and 2890 cm-1. According to Acquistucci (2016), stretching bands
409
between 3000 and 2800 cm-1 are assigned to intrinsical lipids in the starch molecule, that
410
could be exposed by the several extrusion conditions.
411
412
3.4 Sensory acceptance of optimal expanded snacks
413
Figure 4 shows the sensory results of the 3G-RS4 and C-3G snacks. The 3G-RS4 snacks
414
had average scores of "neither like-nor dislike” for color, “slightly like" for texture and
19
415
flavor, and "like moderately" for crispness and general acceptance. Highest scores were
416
obtainded for 3G-RS4 snacks and 66% of the panelists gave a rating higher than 7 (“I like
417
moderately”) in terms of general acceptance. However, statistical analysis showed that
418
there were no significant differences (p>0.05) in the sensory properties measured between
419
3G-RS4 and C-3G snacks. The phosphorylation of sorghum starch may have no effect on
420
the texture and crispness of 3G-RS4, implying that RS4 did not have any negative influence
421
in terms of general acceptance. This effect was probably due to fiber type (RS4), in that
422
other fibers are known to reduce expansion volumes, making harder extrudate products
423
with less crispiness (Cortés et al., 2014; Wang et al., 2017). For example, in extruded
424
breakfast cereals from whole-grain sorghum flours, Mkandawire et al. (2015) observed that
425
the high fiber in the flours limited the expansion of the extrudates. In the present study,
426
expansion using microwave-heating did not negatively influence their sensorial
427
acceptability, compared with the expansion using a frying method (BahramParvar et al.,
428
2014). These results are similar to those reported by Aigster et al. (2011) and Maziarz et al.
429
(2013), who added commercial RS to muffins, focaccia bread, chicken curry, and cereal
430
bars, and reported that partial replacement of flour with RS increased these sensory
431
characteristics without affecting overall acceptability.
432
433
4. Conclusions
434
The phosphorylation of isolated sorghum starch using optimal extrusion conditions had the
435
potential to induce the formation of RS4 and simultaneously allowed the development of
436
3G-S with general sensory acceptability. The intermediate barrel temperature had a positive
437
influence on the formation of RS4 and the EI. Degree of substitution and FT-IR studies 20
438
confirmed that phosphorylation of sorghum starch using sodium trimetaphosphate and
439
sodium tripolyphosphate was within the allowable limits of the FDA. RS4 and microwave-
440
expansion did not affect the sensorial acceptability of the optimal snack. Isolated sorghum
441
starch was an efficient raw material to produce extruded snacks; therefore, may be possible
442
to use in the snack-food industry.
443
444
Acknowledgements
445
The first author thanks CONACYT for the MSc degree scholarship (No. 386579). The
446
authors thank MSc Verónica Flores-Casamayor, MSc Reina Aracely Mauricio Sánchez,
447
MSc José Juan Véles-Medina and BSc Carlos Alberto Avila Herrera from Cinvestav-
448
Querétaro for their technical support.
449
450
Conflict of Interest Statement
451
The authors declare that they have no conflict of interest with respect to the study described
452
in this manuscript.
453
454
455 456
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Table 1. Experimental design with coded and actual values, and experimental results of EI and RS4. Independent variables Experimental results Coded Actual Dependent responses Run X1 X2 BTa (°C) FMb (%) EI c RS4d (g/100 g) -1 -1 100 25 2.8 ± 0.1 10.1 ± 0.3 1 1 -1 130 25 5.4 ± 0.1 8.495 ± 0.152 2 -1 1 100 32 3.02 ± 0.04 8.9 ± 0.1 3 1 1 130 32 4.152 ± 0.051 8.9 ± 0.4 4 -1.414 0 93.8 28.5 3.96 ± 0.03 9.2 ± 0.4 5 1.414 0 136 28.5 6.6 ± 0.1 13.1 ± 0.5 6 0 -1.414 115 23.5 2.68 ± 0.04 7.7 ± 0.3 7 0 1.414 115 33.4 4.2 ± 0.1 6.8 ± 0.1 8 0 0 115 28.5 2.5 ± 0.1 5.5 ± 0.2 9 0 0 115 28.5 3.33 ± 0.3 5.0 ± 0.2 10 0 0 115 28.5 3.1 ± 0.1 5.23 ± 0.23 11 0 0 115 28.5 3.0 ± 0.1 5.3 ± 0.3 12 0 0 115 28.5 3.23 ± 0.32 4.9 ± 0.1 13 a b Means ± standard deviation (n = 3). BT and FM represents the transition barrel temperature and the feed moisture, respectively. cEI and dRS4 represents the expansion index and the type 4 resistant starch, respectively.
602 603 604 605 606 607
28
608 609 610 611 612 613 614 615
Table 2. ANOVA for RS4 and EI responsesa.
616 617
Factor RS4a EIb 5.27 3.09 Intercept Barrel temperature (Linear term) 1.39* 0.94* Feed moisture (Linear term) -0.24* ND Barrel temperature-Feed moisture (Interaction term) 0.36* ND Barrel temperature2 (Quadratic term) 2.92* 0.99* 2 Feed moisture (Quadratic term) 0.97* ND Barrel temperature * Feed moisuture2 (Cubic term) -1.81* ND 0.998 0.825 R2 <0.01 <0.01 p-value for model 0.345ns 0.0852ns p-value for lack of fit 69.44 12.65 Adeq precision a RS4 and bEI represents type 4 resistant starch and expansion index responses, respectively. ns Not significant at p>0.05; * Significant at p<0.01; ND Not determinated.
618 619 620 621 622 623 624 625 626 627 29
628 629 630 631 632 633
Figure legends
634
Fig. 1 Effect of transition barrel temperature and feed moisture on (a) expansion index and
635
(b) type-4 resistant starch (X1 and X2 corresponds to intermediate barrel temperature and
636
feed moisture, respectively)
637
Fig. 2 Overall desirability for expansion index and type-4 resistant starch responses to
638
optimize the extrusion
639
temperature and feed moisture, respectively)
640
Fig. 3 FT-IR spectra of (a) native sorghum starch, (b) optimal third-generation snack whit
641
type 4 resistant starch and (c) control third-generation snack
642
Fig. 4 Sensory evaluation of optimal third-generation snacks with 17 g/100 g of type-4
643
resistant starch (3G-RS4) and control third-generation snacks (C-3G). Figure shows the
644
score for each attribute. Mean values are shown with error bars of standard deviations (n =
645
33). A p<0.05 value was considered significant.
conditions (X1 and X2 corresponds to intermediate barrel
646 647
30